IN PARTIAL FULFILLMENTS FOR THE DEGREE OF...
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ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
UPDATE FROM PAKISTAN
SUBMITTED BY:
MISBAH SULTANA
(REG. # DBC
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
(Ph.D) IN BIOCHEMISTRY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
THE UNIVERSITY OF LAHORE
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
UPDATE FROM PAKISTAN
SUBMITTED BY:
MISBAH SULTANA
(REG. # DBC-021230
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
(Ph.D) IN BIOCHEMISTRY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
THE UNIVERSITY OF LAHORE
2012-2017
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
UPDATE FROM PAKISTAN
SUBMITTED BY:
MISBAH SULTANA
02123009)
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
(Ph.D) IN BIOCHEMISTRY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
THE UNIVERSITY OF LAHORE
2017
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
(Ph.D) IN BIOCHEMISTRY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
THE UNIVERSITY OF LAHORE
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
IN PARTIAL FULFILLMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY
EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN
In the name of Allah the most gracious the most mercifulIn the name of Allah the most gracious the most merciful
"Bismillah al rahman al rahim"
In the name of Allah the most gracious the most merciful
i
"Bismillah al rahman al rahim"
In the name of Allah the most gracious the most merciful
"Bismillah al rahman al rahim"
In the name of Allah the most gracious the most merciful
"Bismillah al rahman al rahim"
In the name of Allah the most gracious the most merciful
"Bismillah al rahman al rahim"
In the name of Allah the most gracious the most mercifulIn the name of Allah the most gracious the most merciful
ii
SUPERVISORY COMMITTEE
We, the Supervisory Committee, certify that the contents and form of thesis submitted by MISBAH SULTANA (DBC-02123009) have been found satisfactory after receiving all the evaluation reports (External/Internal) and recommended it for the award of the degree of Doctor of Philosophy (PhD) in Biochemistry under title “ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS AND EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN PUNJAB. AN UPDATE FROM PAKISTAN”.
SUPERVISOR: PROF. DR. ARIF MALIK ___________________
Institute of Molecular Biology and Biotechnology (IMBB/
Center for Research in Molecular Medicine (CRiMM)
The University of Lahore.
CO-SUPERVISOR: PROF. DR. M. H. QAZI____________________
Institute of Molecular Biology and Biotechnology (IMBB/
Center for Research in Molecular Medicine (CRiMM)
The University of Lahore.
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EXAMINATION COMMITTEE
The thesis viva MISBAH SULTANA (REG.# DBC-02123009) was held on 00-00-2015 at the Institute
of Molecular Biology and Biotechnology/ Center for Research in Molecular Medicine (CRiMM), The
University of Lahore. The Supervisor/Co-Supervisor and the examiner gave satisfactory remarks on thesis
and viva and were approved for the award of the degree of Doctor of Philosophy (PhD) in Biochemistry
under title “ASSESSMENT OF CIRCULATING BIOCHEMICAL MARKERS ANTIOXIDATIVE STATUS
AND EXTRAPOLATIVE FACTORS IN PRETERM INFANTS SUFFERING FROM ANEMIA IN
PUNJAB. AN UPDATE FROM PAKISTAN”.
______________________________________ ______________________________________
EXTERNAL EXAMINER INTERNAL EXAMINER
PROF. DR. ARIF MALIK
INSTITUTE OF MOLECULAR BIOLOGY AND
BIOTECHNOLOGY (IMBB/CENTER FOR
RESEARCH IN MOLECULAR MEDICINE (CRIMM).
____________________________________________
PROF. DR. M. H. QAZI
DIRECTOR,
Institute of Molecular Biology and Biotechnology (IMBB/Center for research in molecular medicine (CRiMM)
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ACKNOWLEDGEMENTS
ALL PRAISES FOR ALLAH, WHO GUIDES US FROM DARKNESS TO LIGHT AND HELP
US IN ALL HARDSHIPS, AND ALL MY REVERENCE IS FOR HIS HOLY PROPHET
MOHAMMAD (P.B.U.H) WHO ENABLES US TO RECOGNIZE OUR CREATOR. I AM GRATEFUL
TO THAT SPIRITUAL POWER WHO HELPS ME AT EVERY STAGE OF MY LIFE. GREAT
PERSONALITIES HAVE BROUGHT IMPECCABLE TRANSFORMATION AND AMAZING
CHANGES IN THE USUAL ROUTINES. WHETHER IT IS POLITICS OR ARTS OR SCIENCE,
WHETHER IT IS RELIGIOUS OR PHILOSOPHICAL, WHETHER IT IS BIG OR SMALL, THE
IMPACT MADE BY PEOPLE MADE THEM FAMOUS PERSONALITIES. PROF. DR. ARIF MALIK
“INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY/CENTER FOR RESEARCH
IN MOLECULAR MEDICINE” MY RESPECTED SUPERVISOR; HIS IDEAS HAVE MANAGED TO
COME THROUGH AND HAVE REVOLUTIONIZED THE FACE OF MODERN SCIENCE. IT IS MY
FOREMOST DUTY TO EXPRESS MY HEARTIEST AND SINCEREST GRATITUDE TO MY
RESPECTED CO-SUPERVISOR PROF. DR. M.H. QAZI, DIRECTOR, INSTITUTE OF
MOLECULAR BIOLOGY AND BIOTECHNOLOGY FOR THEIR KIND GUIDANCE DURING MY
STUDIES. I AM MUCH OBLIGED TO HIM FOR PRODUCTIVE GUIDANCE.
MISBAH SULTANA
REG.# DBC-02123009
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DECLARATION OF ORIGINALITY
NAME OF STUDENT: MISBAH SULTANA
REG.#: (REG.# DBC-02123009)
DECLARATION
1-understand what plagiarism is and I am aware of the University’s policy in this regard.
2-I declare the thesis is my own original work. Where other people’s work has been used (Either from a
printed source, Internet or any sources), this has properly acknowledged and referenced in accordance
with departmental requirements.
3-I have not used work previously produced by another student or any other person to hand in as my own.
4-I have not allowed, and will not allow, anyone to copy my work with the intention of passing it off as
his or her own work.
SIMILARITY INDEX ( %) ON DAY (0-0-2017 At 0:00 PM)
SIGNATURE OF STUDENT: -----------------------------------------------------------------------------
SIGNATURE OF SUPERVISOR: ------------------------------------------------------------------------
SIGNATURE OF CO-SUPERVISOR: ------------------------------------------------------------------
SIGNATURE OF LIBRARIAN: --------------------------------------------------------------------------
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DEDICATION
Dedicated
To
MY LOVELY FATHER, Whose Prayers and Constant Encouragement Enabled Me to
Complete this Task of Learning
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LIST OF ABBREVIATIONS
TRH Thyrotropin releasing hormone
TSH Thyroid stimulating hormone
VIP Vasoactive inhibitory peptide
PAF Prolactin activating factor
PIF Prolactin inhibitory factor
MATI/II Methionine adenosyltransferase
SAM S-adenosyl methionine
GNMT Glycine N-methyltransferase
SAH S- adenosylhomocysteine
SAHH S- adenosylhomocysteine hydrolase
CSβ Cystathionine beta
MS Methionine synthase
MSR Methionine synthase reductase
THF Tetrahydrofolate
MTHFR Methlene tetrahydrofolate reductase
CH2-THF 5, 10-Methlenetetrahydrofolate
CH3-THF 5-methltetrahydrofolate
PLP Pyridoxal Phosphate
SHMT Serine methylhydroxytransferase
VitB6 Vitamin B6 (pyridoxine)
VitB9 Vitamin B9 (Folic acid)
VitB12 Vitamin B12 (Cobalamine)
NAC N-acetylcysteine
Glu Glutamate
r-Glu-Cys Gamma-Glutamate-Cysteine
Glu-Cys ligase Glutamate-Cysteine ligase
GSH synthase Glutathione synthase
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NADPH Nicotinamide adenine dinucleotide Phosphate
FADPH Flavin adenine dinucleotide Phosphate
8-oxodG 8-oxo- 7, 8 dihydro-2’-deoxyguanosine
8-oxoGuo 8-oxo- 7, 8 dihydroguanosine
I- Iodide ion
I2 Iodine molecule
MIT Mono-iodotyrosine
DIT Di-iodotyrosine
RT3 Reverse tri-iodothyronine
T3 Tri-iodothyronine
T4 Tetra-iodothyronine
ALT Alanine aminotransferase
AST Aspartate aminotransferase
BUN Blood urea nitrogen
CAT Catalase
GSH Glutathione
GSH Glutathione
GSSH Glutathione disulphide
GSHr Glutathione reductase
GSHPx Glutathione peroxidase
CAT Catalase
O2- Superoxide free radical
H2O2- Hydrogen peroxide free radical
SOD Superoxide dismutase
MDA Malondialdehyde
ONOO- Peroxynitrate
TPP Total Plasma Peroxidases
NO Nitric oxide
NOS Nitric oxide synthase
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LPO Lipid peroxidation
ROS Reactive oxygen specie
SOD Superoxide dismutase
TBARS Thiobarbituric acid reactive substances
TNF Tumor necrosis factor
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LIST OF SYMBOLS
SYMBOLS NAME
°C Degree centigrade
> Greater than
< Less than
rmp Rotation per minute
µl Micro liter
% Percentage
nm Nano meter
mg/dl Mille gram per deci liter
mg/kg Mille gram per killo gram
g/dl Gram per deci liter
IU/L International units per liter
"Bismillah al rahman al rahim"
SUPERVISORY COMMITTEE
EXAMINATION COMMITTEE
ACKNOWLEDGEMENT
DEDICATION
LIST OF ABBREVIATIONS
LIST OF SYMBOLS
CHAPTERS
CHAPTER ONE
CHAPTER TWO
CHAPTER THREE
CHAPTER FOUR
CHAPTER FIVE
CHAPTER SIX
CHAPTER SEVEN
"Bismillah al rahman al rahim"
SUPERVISORY COMMITTEE
EXAMINATION COMMITTEE
ACKNOWLEDGEMENT
DEDICATION
LIST OF ABBREVIATIONS
LIST OF SYMBOLS
CHAPTERS
CHAPTER ONE
CHAPTER TWO
CHAPTER THREE
CHAPTER FOUR
CHAPTER FIVE
CHAPTER SIX
CHAPTER SEVEN
"Bismillah al rahman al rahim"
SUPERVISORY COMMITTEE
EXAMINATION COMMITTEE
ACKNOWLEDGEMENT
LIST OF ABBREVIATIONS
LIST OF SYMBOLS
INTRODUCTION
REVIEW OF LITERATURE
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
CONCLUSION
LITERATURE CITED
APPENDIX
TABLE OF CONTENTS
INTRODUCTION
REVIEW OF LITERATURE
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
CONCLUSION
LITERATURE CITED
APPENDIX
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TABLE OF CONTENTS
DESCRIPTION
INTRODUCTION
REVIEW OF LITERATURE
MATERIALS AND METHODS
DISCUSSION
CONCLUSION
LITERATURE CITED
TABLE OF CONTENTS
DESCRIPTION
REVIEW OF LITERATURE
MATERIALS AND METHODS
LITERATURE CITED
MATERIALS AND METHODS MATERIALS AND METHODS
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PAGE #
1
11
23
59
131
146
90
92
120
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TABLE OF CONTENTS
S.# DESCRIPTION PAGE. #
1.0 INTRODUCTION 01
2.0 REVIEW OF LITERATURE 11
2.1 OXIDATIVE STRESS MARKERS 11
2.2 INFLAMMATION IN UMBILICAL CORD IN
BLOOD
11
2.3 NON-PROTEIN BOUND IRON AS
PREDICTIVE MARKER OF NEONATAL
BRAIN DAMAGE
12
2.4 HUMAN MILK ENHANCES ANTIOXIDANTS 12
2.5 LEUKOCYTOSIS IN PRETERM INFANTS
WITH INTRAVENTRICULAR
HEMORRHAGE
13
2.6 INFLAMMATION AT BIRTH 13
2.7 NITRIC OXIDE THERAPY IN PREMATURE
INFANTS
14
2.8 NPBI AND F2-ISOPROSTANES IN NEWBORN 14
2.9 MELATONIN PROTECTS AGAINST
OXIDATIVE DAMAGE
14
2.10 MATERNAL ANEMIA 15
2.11 IRON STATUS AT BIRTH 15
2.12 MORTALITY OF PRETERM BIRTHS 15
2.13 IRON DEFICIENCY ANEMIA AND SEVERE
PREGNANCY OUTCOME
15
2.14 PREGNANCY OUTCOME ASSOCIATED
WITH ANEMIA AND IRON
16
2.15 IRON IN PREGNANCY 16
2.16 NEGATIVE EFFECT OF OXIDATIVE STRESS
IN FETUS AND NEWBORN
18
2.17 HYPOXIA AND NITRIC OXIDE 20
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2.18 PARTURITION AND OXIDATIVE STRESS 20
2.19 OXIDATIVE STRESS AND DIABETIC
PREGNANCY
21
2.20 ROLE OF OXIDATIVE STRESS IN
MENOPAUSE AND AGE RELATED
FERTILITY DECLINE
21
2.21 STRATEGIES TO OVERCOME OXIDATIVE
STRESS IN ASSISTED REPRODUCTION
21
2.22 PHYSIOLOGICAL STATUS OF MOTHERS 22
2.23 ROLE OF OXIDATIVE STRESS AND LIPID
PEROXIDATION IN PRETERM DELIVERY
23
3.0 MATERIALS AND
METHODS
31
3.0 MATERIALS AND METHODS 32
3.1 INCLUSION CRITERIA 32
3.2 EXCLUSION CRITERIA 32
3.3 CHEMICALS 33
3.4 BIOCHEMICAL ASSAYS 33
3.4.1 ESTIMATION OF GLUTATHIONE (GSH) 33
3.4.2 ESTIMATION OF CATALASE (CAT) 33
3.4.3 ESTIMATION OF THIOBARBITURIC ACID
REACTIVE SUBSTANCES (TBARS)
33
3.4.4 ESTIMATION OF SUPER OXIDE
DISMUTASE (SOD)
34
3.4.5 ESTIMATION OF GLUTATHIONE
REDUCTASE (GR)
34
3.4.6 ESTIMATION OF VITAMIN C 34
3.4.7 DETERMINATION OF ADVANCED
OXIDATIVE PROTEIN PRODUCTS (AOPPs)
35
3.4.8 PREPARATION AND DETERMINATION OF
(AGEs)
35
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3.4.9 DETERMINATION OF GAMMA
GLUTAMYL TRANSFERASE (GGT)
35
3.4.10 DETERMINATION OF NITRIC OXIDE 36
3.4.11 DETERMINATION OF TUMOR NECROSIS
FACTOR BY ABCAM’s TNF ALPHA
HUMAN ELISA KIT
36
3.4.12 ESTIMATION OF VITAMIN-E 36
3.4.13 DETERMINATION OF VITAMIN A 37
3.4.14 DETERMINATION OF INTERLEUKIN-6 “IL-
6” LEVELS
37
3.4.15 ESTIMATION OF C-REACTIVE PROTEIN 38
3.4.16 DETERMINATION OF GPx 38
3.4.17 DETERMINATION OF TOTAL
CHOLESTEROL (TCh)
38
3.4.18 DETERMINATION OF TRIGLYCERIDES
(TG)
38
3.4.19 ESTIMATION OF LOW DENSITY
LIPOPROTEIN (LDL)
39
3.4.20 DETERMINATION OF HIGH DENSITY
LIPOPROTEINS (HDL)
39
3.4.21 ESTIMATION OF TOTAL BILIRUBIN 39
3.4.22 DETERMINATION OF
MYELOPEROXIDASE (MPO)
39
3.4.23 ESTIMATION OF SODIUM AND POTASSIUM 40
3.4.24 DETERMINATION OF NEUTROPHILS 40
3.4.25 DETERMINATION OF MAGNESIUM AND
CALCIUM
40
3.4.26 DETERMINATION OF COMPLETE BLOOD
COUNT (CBC) IN THE SERUM
40
3.4.27 ESTIMATION OF SERUM FOLATE
(VITAMIN B9) AND VITAMIN B12
57
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3.4.28 DETERMINATION OF TOTAL IRON
BINDING CAPACITY
41
3.4.28 DETERMINATION OF RENAL FUNCTION
TESTS (RFTs)
41
3.4.29 ESTIMATION OF VITAMIN D (Vit D) IN THE
SERUM OF PRETERM INFANTS
41
4.0 RESULTS 42
4.1 LEVEL OF HEMOLYTIC CELLS IN PRETERM INFANTS (PTI)
43
4.2 LEVEL OF RFT and LFT IN PRETERM INFANTS (PTI)
44
4.3 LEVEL OF LIPID PROFILE IN PRETERM INFANTS (PTI)
44
4.4 LEVEL OF STRESS BIOCHEMICAL MARKERS IN PRETERM INFANTS (PTI)
45
4.5 LEVEL OF DIFFERERENT TYPES OF VITAMINS (B12, A, C, E AND D) IN PRETERM INFANTS (PTI)
45
4.6 EFFECT OF PHYSICAL MARKERS ON PRETERM INFANTS (PTI)
46
4.7 LEVEL OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) IN PRETERM INFANTS (PTI)
46
4.8 LEVEL OF ELECTROLYTES (HCO3, Ca, Na, K, Mg, CL-, Zn, Cu AND P) IN PRETERM INFANTS (PTI)
46
4.9 LEVEL OF INFLAMMATORY BIOMARKERS IN PRETERM INFANTS (PTI)
47
5.0 DISCUSSION 49
6.0 SUMMARY 65
7.0 CONCLUSION 69
8.0 PART II 71
9.0 INTRODUCTION 72
10.0 REVIEW OF LITERATURE 79
10.1 SELENIUM ROLE IN PRETERM LABOR 81
10.2 VITAMIN D AND SPONTANEOUS PRETERM
BIRTH
82
10.3 ROLE OF THYROID HORMONE IN 84
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PRETERM DELIVERY: ANATOMY AND
PHYSIOLOGY OF THYROID GLAND
10.4 THYROID FUNCTION DURING PREGNANCY 86
10.5 MATERNAL THYROID FUNCTION AND
FETAL BRAIN DEVELOPMENT
87
10.6 IMPACT OF THYROID AUTOANTIBODIES 87
10.7 PRETERM PREMATURE RUPTURE OF
MEMBRANES (PPROM)
88
10.8 COMMON BIOLOGICAL PATHWAYS TO
PRETERM BIRTH
89
10.9 INTRAUTERINE INFLAMMATION AND
INFECTION PATHWAY
89
10.10 EXTRACELLULAR MATRIX DEGRADATION PATHWAY
90
10.11 FETAL STRESS PATHWAY 91
10.12 FETAL ANOMALIES PATHWAY 92
10.13 ESTROGEN METABOLISM PATHWAY 92
11.0 MATERIALS AND
METHODS
94
11.0 MATERIALS AND METHODS 95
11.1 SOURCE OF DATA 95
11.2 INCLUSION CRITERIA 95
11.3 EXCLUSION CRITERIA 95
11.4 CHEMICALS 95
11.5 FOLLOWING PARAMETERS WILL BE
ESTIMATED
95
11.5.1 ESTIMATION OF THYROID PROFILE IN
SERUM SAMPLES
96
11.5.2 ESTIMATION OF T3 (Tri-iodothyronine)
ELISA
96
11.5.3 ESTIMATION OF T4 (Thyroxine) ELISA 96
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11.5.4 ESTIMATION OF TSH (Thyroid stimulating
hormone) ELISA
96
11.5.5 ESTIMATION OF TPO (Thyroid peroxidase)
IgG ELISA
96
11.5.6 ESTIMATION OF Thyroglobulin IgG ELISA 96
11.5.7 ESTIMATION OF STRESS BIOMARKERS IN
SERUM SAMPLES
96
11.5.8 ESTIMATION OF THIOBARBITURIC ACID
REACTIVE SUBSTANCES (TBARS)
96
11.5.9 ESTIMATION OF SUPER OXIDE
DISMUTASE (SOD)
96
11.5.10 ESTIMATION OF GLUTATHIONE (GSH) 97
11.5.11 ESTIMATION OF CATALASE (CAT) 97
11.5.12 DETERMINATION OF NITRIC OXIDE 97
11.5.12.1 PRINCIPLE 97
11.5.12.2 CHEMICALS REQUIRED 98
11.5.12.3 PROTOCOL 98
11.5.13 ESTIMATION OF INTERLEUKIN-6 (IL-6) (BY
ELISA KIT)
98
11.5.13.1 PROCEDURE 98
11.5.14 DETERMINATION OF TUMOR NECROSIS
FACTOR BY ABCAM’s TNF ALPHA HUMAN
ELISA KIT
99
11.5.15 DETERMINATION OF GLUTATHION
PEROXIDASE (GPx)
100
11.5.15.1 PROCEDURE 101
11.5.16 ESTIMATION OF GLUTATHIONE
REDUCTASE (GR)
101
11.5.17 DETERMINATION OF AOPPs 101
11.5.18 DETERMINATION OF VITAMIN 102
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11.5.19 ESTIMATION OF VITAMIN-E IN SAMPLE 102
11.5.19.1 PRINCIPLE 102
11.5.19.2 REAGENTS 102
11.5.19.3 PROCEDURE 103
11.5.20 ESTIMATION OF VITAMIN-C IN LIVER
SAMPLE
103
11.5.20.1 PRINCIPLE 103
11.5.20.2 REAGENTS 103
11.5.21 EXTRACTION OF VITAMIN-C 103
11.5.21.1 PROCEDURE 104
12.0 RESULTS 105
12.1 THYROID BIOMARKERS PROFILE OF
PRETERM MOTHER PATIENTS Vs
CONTROL
106
12.2 CIRCULATING STRESS BIOMARKERS
PROFILE OF PRETERM MOTHERS VS
CONTROL
106
12.3 ANTIOXIDANT BIOMARKERS PROFILE OF
PRETERM MOTHER PATIENTS Vs
CONTROL
107
13.0 DISCUSSION 111
14.0 CONCLUSION 118
15.0 LITERATURE CITED 120
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LIST OF TABLES
S.# DESCRIPTION PAGE. #
TABLE-01 DEMOGRAPHIC DISTRIBUTION OF DATA 32
TABLE-02 VARIABLES OF MEDICAL IMPORTANCE IN THE PRETERM INFANTS (PTI) SUFFERING FROM ANEMIA
47
TABLE-03 THYROID BIOMARKERS PROFILE OF PRETERM DELIVERY
FEMALE PATIENTS Vs CONTROL
108
TABLE-04 STRESS BIOMARKERS PROFILE OF PRETERM DELIVERY
FEMALE PATIENTS Vs CONTROL
108
TABLE-05 ANTIOXIDANT BIOMARKERS PROFILE OF PRETERM
MOTHERS Vs CONTROL
109
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LIST OF FIGURES
S.# DESCRIPTION PAGE. #
FIGURE 01 PATHOGENESIS OF PRETERM INFANTS 62
FIGURE 02 MECHANISM OF DIFFERENT PARAMETERS INDUCING
PRETERM BIRTH
117
CHAPTER ONE INTRODUCTION MISBAH, 2017
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INTRODUCTION
CHAPTER ONE INTRODUCTION MISBAH, 2017
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1.0 INTRODUCTION
Infants born before completion of 37 weeks of pregnancy are termed as premature.
Almost 12% of deliveries in normal population are thought to be premature. ‘Preterm’ and
‘preemie’ are the terms used for premature babies. Short gestational period/premature birth is
responsible for underdeveloped organs in such babies, therefore they are at higher risk to develop
multiple problems, such as problems associated with respiratory and digestive system etc.
Additionally the development and growth of brain, lungs also remain improper. In premature
birth fetus will also be improperly developed. Immature development of lungs such as lungs will
not be able to make ‘surfactant’ also termed as respiratory distress syndrome (RDS). Normally
alveoli plays essential role in lungs surfactant as the level decreases it effects breathing whereas,
it is very difficult to reopen the alveolar sac. Such babies are aided with artificial surfactant.
Likewise, bronchopulmonary dysplasia is also one of the lungs disease can be seen in babies
born prematurely it is characterized by injured lungs hence, also known as chronic lung disease.
In 22-25 weeks of pregnancy the lungs are not properly formed. If baby is born in this condition
would not be able to survive outside the womb whereas, the respiratory problems can be
managed in the following ways
a) Proper inhalation of oxygen by the help of nasal cannulas, regulating air ways, b) Aided by
alveoli function as only oxygen cannot deliver continuous positive airway pressure (CPAP). c)
Addiitional support is delivered by intubation; in that case tube is being connected with the
aiding machine termed as ventilator. If breathing centre is not properly developed it may result in
breathing pauses (apnea). Apnea is a reason for reduction of heart rate and that apnea is
maintained by the help of nasal canula, ventilator, CPAP and other breathing aids. Essentially
after 38 weeks of pregnancy changes can be observed in the babies include change in their heart
and lungs whereas, these changes may not occur in the case of preterm babies. Hence, they may
suffer severe infectious state. Such babies may have low blood pressure, improperly developed
intestine and several other medical conditions that may be effectively treated with the
administration of proper medication only. So, minor quantities of feed may be provided to them
which may be even less than one teaspoon. In such condition breast feed (milk) is considered
best for the babies (especially premature babies) it may best nourish and lest exposed to
infections, fewer initial complications and improved growth. Mothers are hence boosted to breast
feed her baby.
CHAPTER ONE INTRODUCTION MISBAH, 2017
3
The prevalence of necrotizing enterocolitis (NEC) is higher in newborns and is believed to be
the reason for consequential problems. Therefore, such babies are fed with the help of tiny tubes
by the nose and mouth, blood vessels of immature babies are more fragile and can be ruptured
easily bleeding problems may be from minor to severe which may be the reason for lifetime
developmental issues. Retina is usually developed at 34th week of pregnancy hence in premature
babies they may have undeveloped retina, breathing centre and due to such issues they are kept
in oxygen tanks. Ophthalmologists regularly observe these preterm babies for the overgrowth of
the blood vessels of retina. Newborns are highly prone to infections; therefore, they are given
with antibiotics to accelerate immune system to overcome infections. There are numerous factors
involved in premature birth of which some directly affect mother and babies ultimately leads to
early labor. Maternally inherited causes remain preeclampisa (because of toxemia during
pregnancy), chronic diseases of heart, kidney or liver etc. Certain kind of infections (infections
caused by B streptococcus, urinary tract and vaginal infections, fetal/placental tissue infections),
Abuse of drugs (such as cocaine), abnormality in structure of the uterus, cervical inability
(failure of the cervix to remain closed during pregnancy), preterm birth history, factors
concerning the pregnancy, abnormally placental functioning, polyhydramnios, placental
abruption, placenta Previa as well as premature rupture of uterus membrane. These factors
influence the fetus, its behavior and contribute as unfavorable environment for uterus, and serve
as reason for several gestation (twins, triplets, etc.) and hereditary abnormalities.
Globally, in 2010 15 million preterm births were estimated of which 1 million died
(Blencowe et al., 2012). Premature birth remains second largest risk factor for death in babies
under the age of 5 years. Another set of studies show premature babies died in their early four
months of life (Liu et al., 2012). Premature babies are prone to several health issues throughout
their life span. In developed countries rate of premature birth is one major health issue whereas,
underdeveloped countries lack evidence about the preterm deliveries. Born Too Soon (report)
tells us about the preterm births country wide and the records shows that it occurrence is
increasing. The effects of premature birth usually spread from birth to whole life. Preterm babies
are not actually with properly developed organs hence, they should be provided with an extra
care and attention as they are more prone to undergo health issues such as, intellectual
deficiency, chronic diseases of kidneys, lungs and liver, impaired vision and hearing
(Anonymous, 2007).
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4
Special attention is required in this sector there should be proper gynecological diagnosis
and monitoring of NCDs, premature babies are at higher risk of hypertension and diabetes (Hovi
et al., 2007). There is a great relationship among preterm births and NCDs throughout the world.
About 9 million people below 60 years of age usually die because of NCDs, and the rate is
increasing day by day in Africa and other low-income counties (Anonymous, 2011). According
to WHO approximatly 135 million infants were delivered in 2010 of which 15 million almost
11.1% of deliveries were premature of which significant higher rates of premature births were
observed in Africa and Asia (Blencowe et al., 2012). According to the Millennium Development
Goal Regions in South Asia estimated highest rates of preterm deliveries to about 13.4% of all
live births. The rate is predominantly increased in Asia and states of Africa. Figures about
premature birth-related death are indefinite in relevant countries. In 88 countries the estimated
preterm delivery rate is less than 10%, at the same time in 11 countries the preterm birth rate is
15% or more. There are at least 10 countries including India, China, Nigeria, Pakistan, Indonesia,
United States, Bangladesh, the Philippines, Democratic Republic of the Congo and Brazil, which
have highest preterm birth rates. About 60% of all premature births belong to the upmentioned
countries. Death rates are higher in low gestational age (Qiu et al., 2011). 16% of total preterm
babies born were of less than 32 weeks of gestational age. Rate of death and disease are highest
in the premature babies. In high-income countries the developments of medical care enhanced
the rate of survival and coped with the outcomes of newborn infants efficiently (Saigal and
Doyle, 2008). In 1990, only two-third of premature born babies survived without any deficiency
(Mohangoo et al., 2011). Consesus in underdeveloped countries suggested that only 30% babies
were born in between 28-32 weeks of gestation but rate of mortatlity was increased in under-
developed countries as compared to developed ones.
On the chart of 65 countries about 10,000 living births are reported annually in Europe,
America and Australia. From the year 1999-2010 rates of preterm deliveries have increased
drastically, it is almost increased from 2 million to 2.2 million in year 2010. Rate of mortality in
newborns were reduced only in three countries that include Ecuador, Croatia and Estonia.
Preterm birth rates are constant in fourteen countries like less then 0.5%. In several under-
developed countries rate of preterm birth is either similar or in some cases it is bit higher. Late
and moderate preterm [LAMP] at 32 to 35 weeks, increase the percentage from previous reports
in low-income conteries (Davidoff et al., 2006). According to the previous reports in these
CHAPTER ONE INTRODUCTION MISBAH, 2017
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countries no change was observed in preterm births from 1990 to 2010. The data obtained from
many little- and medium-income countries rate of preterm should be carefully understood as
there was substantial data gap and the obtained information was not enough to show the
characteristics of relevant inhabitants. Rate of preterm birth in low and medium income states
such as China and South Asia have increased like other countries.
The NICU is an intensive care unit, arranged for the special care of preterm babies or
who have complications soon after birth. The NICU staff includes the following, Neonatologists,
Pediatricians, Neonatal nurse practitioners (NNPs), Nurses and respiratory therapists, Dietitians,
Physical therapists, and includes many other health care professionals for the care of a premature
baby. NICU babies heart rate, breathing rate, and oxygen levels monitored continuously. Most
premature babies are placed in incubator after birth, which provides babies with warm moist air.
Anemia has been defined as lower number of RBC’s than normal. Main function of RBC’s is to
deliver oxygen to tissues in the body. At the time of preterm birth, major cause of anemia is
declination in the level of erythropoietin (EPO) because of the low pressure of oxygen
(Stockman et al., 1984; Brown et al., 1984). In pregnant women iron deficiency anemia is
common public health problem in developing countries (Pasricha et al., 2008). Two third of
expecting women are affected and have low birth weight, high maternal morbidity and mortality
rate (Baig-Ansari et al., 2008). Mutually low socio economical and educational statuses are the
main reasons for high occurrence of anemia (Mahe et al., 2004). Twenty to fifty (20-50%)
percent of people in the world suffer from iron deficiency anemia. Iron deficiency is the most
significant risk factor of anemia in pregnant females (Breymann, 2006). Deficiency not only
affects mother but it also affects infant particularly his/her intellectual and psychomotor
functions and leads to anemia (Kilbride et al., 1992). According to WHO, anemia is a basic
health problem in countries with occurrence of anemia higher than 40% (Anonymous, 2001).
Anemia is defined by WHO as Hemoglobin less than 11gm in pregnancy, and it is further
divided into three categories:
I. Mild (9.0 - 10.9gm %),
II. Moderate (7.0-8.9gm) and
III. Severe degree (<7.0 gm%) (Marahatta R, 2007).
Anemia is a complication common in young, poor, pregnant women of racial minority
(Scholl et al., 1992). Early diagnosis and management of anemia is significant in direction to
CHAPTER ONE INTRODUCTION MISBAH, 2017
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prevent the maternal and prenatal morbidity (Aimaku and Olayemi, 2003). The incidence,
etiology and degree of severity of anemia differ in diverse populations worldwide. Almost 35%
women without pregnancy and 51% of women on whole suffer anemia. The rate might vary in
different regions as the occurence of anemia in females is 3 to 4 time higher in under developing
or low-income regions (Adiba et al., 2007). The frequency of anemia in pregnant women is as
much as 65% in South Asia (Schultink et al., 1993). In order to decrease anemia in pregnant
women an effective care should be taken at primary level. Sufficient supply of drugs to remove
the causes of anemia should be provided to improve the levels of Hb in pregnant women it may
also contribute in the plan to reduce the chances of anemia in infants (Massawe et al., 1999).
Vast number of pregnant women visited and got admitted in gynecological units with multiple
complications as the current study aims to rule out the causes and estimate the regularity of
anemic cases. Normally there are three main causes of anemia, First is the insufficient production
of RBC’s, Physiologic anemia is the example of less production of RBC’s because of growth of
the babies and Anemia is common in premature babies than full-term babies. Second, Break
down of red blood cells are so fast i.e., it is problematic when the mother and baby have different
blood types (Rh/ABO incompatibility). These babies usually suffer from jaundice
(hyperbilirubinemia) or Infections or genetic disorders can also cause this problem. And finally,
internal bleeding in premature babies can transfer blood from the baby to the mother in the
womb. Symptoms of anemia includes palness of skin, lethargy because of low energy, feeding is
not proper as baby gets tired, rapid heart rate and quick breathing when resting etc.
Tests for the diagnosis of anemia include estimation of following factors in the blood od
the patients;
• Hemoglobin - oxygen carrying protein in red blood cells
• Hematocrit - % of blood comprises red blood cells
• Reticulocyte count - % of immature red blood cells present in the blood. It measures new cells
in the blood which is produced in the body.
In the first few weeks of life, preterm infants may suffer from anemia due to the
following reasons, (a) Because of rapid growth in first months of life needs sufficient amount of
blood to maintain or stablize the levels of hemoglobin (Collard, 2009), (b) In new born babies
the formation of epo primarily produced from liver have no ability to generate the red blood cells
due to hypoxic condition (Ziajani et al., 1981 and Eckardt et al., 1992) subsequently the liver
CHAPTER ONE INTRODUCTION MISBAH, 2017
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have less ability regarding to anemia because it is the major factor for the development of
preterm infants (c) Erythropoiesis is the rate limiting enzyme which is expressed instantaneously
at the time of birth because it inhibit the transcription factor of hypoxia like hypoxia-inducible
factors (HIF) (Kling et al., 1996), (d) there are number of of iatrogenic phlebotomies in preterm
infants to cause the anemia as in such conditions the levels of red blood cells decreases due to
proginator cells (Strauss, 1995), (e) anemia can occur by certain pathological circumstances that
can cause prematurity (Carroll and Widness, 2012).
The severe type of anemia may be caused by lower gestational period. According to the
recent reports gestational period have positive correlation with the concentration of hemoglobin
in first 4 weeks of life because desired levels of hemoglobin have significant role in biological
system (Jopling et al., 2009). In first 28 days of life infants with gestational period of 4 to 5
months have significantly decreased levels of Hb as compared to infants with gestational period
of 35-42 weeks (Jopling et al., 2009). In premature infants deterioration of hemoglobins and
decreased potential to carry oxygen contributes in the occurrence of anemia. During the first 3
month of gestation the levels of hemoglobin were increased in fetus. During the gestational
period the levels of hemoglobin F type progressively moves to the type of adult hemoglobin (Hb
A). At 40 weeks gestational age, the Hb F and Hb A present in the same quantity in flow
(Stockman and Pochedly, 1988), but in premature infant the circulation of Hb F type is higher.
The potential of oxygen affinity is higher in Hb F type as compare to Hb A type and this
potential is differ due to the action of 2,3-diphosphoglycerate (Nathan and Oski, 1993).
Subsequently the binding affinity of oxygen were high in the F type hemoglobin as compare to
Hb A in this results the delivery of oxygen is low and cause the tissue damage and hypoxia.
According to the result of previous work the newborn babies get inferior due to the higher
concentration of 2,3-diphosphoglycerate (Delivori et al., 1971). The desire levels of hemoglobin
have the significantly role to carry the oxygen from lungs to tissue for proper function.
Experimental trials in future can deliver complete info about prematurity.
Multiple iatrogenic phlebotomies contribute to the anemia in first few weeks of life in
premature babies (Widness, 2008). According to one study during the first month of preterm
infant life conducted that the phleobotomies average were held in reserve. The physicians
collected the blood from the veins of newborns to determine the parameters that includes; type of
blood, concentration of hemoglobin, arterial blood gases, level of lactic acid, level of antibioties
CHAPTER ONE INTRODUCTION MISBAH, 2017
8
in blood, calcium ionization, blood culture, level of glucose, complete blood count, level of
phosphorus, level of theophylline, platelet count, thyroid perameters, concentration of bilirubin
and electrolytes. Levels of phlebotomies were high in newborn babies analyzed from drawn
blood and were compared with normal infants (Freise et al., 2010).
Generally for the therapy of premature anemic infants five main approaches are
employed, treatment involves iron supplementation, delayed cord clamping, low blood count,
transfusion of red blood cells. Moreover, it is nutritionally insensitive in poor infants (DeAlarcon
and Werner, 2005). Iron intake incareses the activity in blood (Ohle, 1999, Maier et al., 1996).
Therefore sufficient iron is supplemented to preterm infants in common practice by clinicians.
For clamping of umbilical cord (a part through which baby and mother are attached) two clamps
are joined and cord will be cut down. An observation shows postponement (i.e., 30-180 seconds)
in clamping of umbilical cord significantly increased the Hb levels (McDonald and Middleton,
2008). Delaying in the clamping of umbilical cord also resulted in iron over load and reduction
in the RBC production (Rabe et al., 2008). In contrast there are various studies that indicate
delay in the clamping of umbilical cord have no effect on the reduction of RBCs (Strauss et al.,
2008). There are some risks associated with the clamping of cord still unclear. Another side
effect associated with the delayed cord clamping is polycythemia, such effects can be deffered
efficiently by clamping in time. Nicholl et al. determined delaying in cord clamping has more
beneficial than risky effects in a moderate condition (Nicholl et al., 2010). Mechansim of
delaying cord clamping reduces iatogenic blood loss which is valuable approach in removing the
anemic prematurity (Strauss, 1995; Widness et al., 1996; Kling et al., 1997 and Ohls, 2002).
A link has been discussed in the studies where blood transfusion estimation of
phlebotomy infants during initial weeks ranging from 11 to 22 Ml/KG resembles 15 to 30 % of
total blood count in the infants (Hume, 1999; Widness, 2008). Estimation of blood volume with
the help of blood analyzer signifies decreased volume of blood in premature infants (Alves-
Dunkerson et al., 2002). Therefore monitors are used to corrext the performace of many trsts by
blood nalayzer so the complete volume reduces and has been significantly reduced.In a clinical
trials of blood monitors decvice performed by 93 infants (birth weight <1000g) to regulate the
possible phlebotomy diminiation (Widness et al., 2005). Clinical trials show 33% reduction in
the count of RBCs through transfusion. Furthermore, blood clinical monitoring is used to analyze
the clinical trials in preterm infants for first two weeks (birth weight>1000g) proved 46%
CHAPTER ONE INTRODUCTION MISBAH, 2017
9
reduction with 30% production in phlebotomics (Madan et al., 2005). If the blood monitors are
able to investigate glucose, bilirubin, blood urea nitrogen and creatinine will reduce the
iatrogenic phlebotomies. Almost about 80% theoretical phlebotomy reduction is possible with
blood monitors which would significantly result in reduced blood transfusions in preterm infants
(Alves-Dunkerson etal, 2002). Transfusions in infant increases toxic possible factors related to
circulatory overload (TACO) and Transfusion-related acute lung injury (TRALI). Reserchers
indicate transfusions in infants can be accomplished by overload activity in fluid, balanced
electrolyte and red blood cell transfusion (Ohlson and Aher, 2006).
CHAPTER TWO REVIEW OF LITERATURE MISBAH, 2017
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REVIEW OF LITERATURE
CHAPTER TWO REVIEW OF LITERATURE MISBAH, 2017
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2.0 REVIEW OF LITERATURE
2.1 OXIDATIVE STRESS MARKERS
Reactive oxygen species are responsible for the production of free radicals having
unpaired electron in their outermost shell. Basically they are the oxidizing agents that are
produced as consequences of oxygen metabolism. Free radicals due to the unpaired electron are
highly reactive molecules. These reactive oxygen species consist of wide range of radicals such
as superoxide ion (O2-), hydroxyl ion (OH-), nitric oxide (NO), lipid peroxyl (LOO) and peroxyl
(RO2) and from non-radicals molecules it contains hydrogen peroxide (H2O2), singlet oxygen
(O2), hypochloric acid (HOCL), ozone (O3) and lipid peroxide (LOOH). The free radicals of
oxygen and its derivatives have significant potential for the intiation of damaging process by
binding of free radicals with macromolecules that includes proteins, carbohydrates, DNA and
lipid molecules. Free radicals from reactive oxygen specis readily binds with polyunsaturated
fatty acids (PUFA) and results in lipid peroxidation and its products such as malondialdehyde
(MDA), MDA is also known as universal oxidative stress biomarker. Concentration of MDA
was measured in terms of TBARS. Pro-oxidants can also bind with protein molecules and lead to
the modification in secondary and tertiary structure. Oxidants can also directly damage proteins
like albumins and results in products such as advanced oxidative protein products (AOPPs) by
the activation of enzyme known as myeloperoxidase (MPO). As per some prior studies the levels
of antioxidants effects vaginal delivery (VD) and elective cesarean section (CS) on antioxidant
status (the level of glutathione (GSH) and ferric lowering the capacity of (FRAP)), the
antioxidant activities includes superoxide dismutase (SOD), glutathione peroxidase (GPx),
catalase (CAT), the concentration of lipid peroxidation (LP), protein and DNA damage in the
umbilical cord blood. Lipid peroxides and carbonyl proteins are crucial product of oxidative stess
mechanism. A need to protect DNA from oxidants is necessary as it will contribute in the
formation of DNA adducts and ultimately lead to DNA damage.
2.2 INFLAMMATION IN UMBILICAL CORD IN BLOOD
An enzyme system is present in RBCs, which prevents the cell from accumulation of
non-functional proteins and its fragments (Fagan, 1986). Malondialdehyde (MDA) was measured
in erythrocytes of maternal and cord blood to check the lipid peroxidation. Oxidative damage to
proteins was estimated by proteolytic degradation of and measuring lipid peroxidation end-
products. Levels of stress markers, reduced levels of glutathione reductase (GR), interleukin-6
CHAPTER TWO REVIEW OF LITERATURE MISBAH, 2017
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(IL-6) in preterm infants is responsible for the inflammation of umbilical cord and damage to
neighbouring tissues and such infants are provided with the appropriate dosages of antenatal
corticosteroids for the treatment and for accelerating maturation of fetal lungs (Miracle et al.,
2008). Increased oxidative stress, formation of oxygen radicals in pretermie’s is reason for the
tissue and organ damage as gestation is a physiological state in which tissue oxygen requirement
and metabolic demands are increased (Buonocore et al., 2002 and Saugstad, 2005). Increased
oxidative stress is defined as imbalance in the levels of oxidants and antioxidants in the body
increased stress and free radicals can cause damage to fetus and can suppress the antioxidant
defense system (Gitto et al., 2002). In relation to this aspect, it is necessary to rule out production
of intracellular antioxidant components (Davis and Auten, 2010). Two different comparative
analyses of antenatal corticosteroid (betamethasone) were conducted: the first compared the
results in between those who were administered and those not administered whereas, second
compared mothers who received the complete cycle with those given only a partial antenatal
corticosteroid cycle.
2.3 NON-PROTEIN BOUND IRON AS PREDICTIVE MARKER OF NEONATAL
BRAIN DAMAGE
Out of 130 million births worldwide each year four million are reported as asphyxia and
million will die. It is necessary to develop effective interventions for the proper understanding of
pathophysiological mechanisms and ultimately leads to brain injury. According to the set of
studies traditional and new markers for oxidative stress were studied to develop relationships in
neurodevelopmental stages and stress markers.
2.4 HUMAN MILK ENHANCES ANTIOXIDANTS
Preterm infants show immature activity of antioxidant defense mechasim. Hydroxyl
radiclas are very aggressive and they directly reduce levels of antioxidants. Practically by
products of DNA oxidation or phenylalanine in urine serve as the major marker for reliable
activity. Human milk contain is enriched with the large amounts of antioxidants (Ledo et al.,
2009). Oxidative stress may be related with the prematurity as discussed human milk is enriched
with antioxidants, therefore feeding with mother milk could diminish related risks and can serve
as best prognostic measure in such infants. Number of studies signifies antioxidative property of
human milk and its use in the case of premature infantsm (Friel et al., 2002) An observation in
the study confirms the human milk provided better antioxidant protection and less ascorbate as
CHAPTER TWO REVIEW OF LITERATURE MISBAH, 2017
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increased ascorbate is responsible for increased oxidants in the extracellular matrix (ECM). Not
only antioxidants, but human milk is also enriched with vitamin C that is essential antioxidant for
defense mechanism in neonates. Premature infants have lower elimination of radicals that
increases lipid peroxidation and oxidative stress which can only be reduced by consumption of
enough antioxidants (Silvestre et al., 2008).
2.5 LEUKOCYTOSIS IN PRETERM INFANTS WITH INTRAVENTRICULAR
HEMORRHAGE
The most common type of intracranial hemorrhage is intraventricular hemorrhage which
exists in premature newborn babies. IVH is common factor for increased morbidity and mortility
in infants with low birth weight. Most intracerebral hemorrhage may occur in first 3 days of birth
(Fanaroff et al., 2007; Fanaroff et al., 2003; Rezvanian, 2011). A relationship can be observed in
between intravenous hemmorage and leukocytes. For the development of relationship CBC was
done within 1 hour of such infants, it signified increased rate of leukocytes which increases the
risks of blood injury along with the increased oxidative stress. Moreover, leukocytosis may be
responsible for subclinical maternal or neonatal infection (Fanaroff et al., 2003 and Wilson-
Costello, 2007)
2.6 INFLAMMATION AT BIRTH
In the response of increased oxidative stress, systemic inflammation is increased. Levels
of pro-inflammatory cytokines were recorded and they were in higher concentration in the cord
blood or in the blood of neonatal which is responsible for neuronal disorders such as white
matter brain damage (WMD) (Duggan et al., 2001 and Minagawa et al., 2002). As per the
number of studies increased secretion of pro-inflammatory cytokines have direct relationship
with cerebral/brain damage. Hansen-Pupp et al., (2008) postulated that as the concentration of
pro-inflammatory cytokine increase, it increases adverse developmental outcomes after birth.
TNF-α is a type of cytokine responsible for the increased adverse effects after birth such as it
causes brain injury and is involved in both types of mechanisms including tolerance and
sensitization (Rosenzweig et al., 2007). TNF- α inhibits proliferation and migration of neuronal
precursor cells, triggers of apoptosis which in turn influence white matter and causes injury
(Downen et al., 1999; Ben-Hur et al., 2003 and Feldhaus et al., 2004). As these infants are
particularly deficient in antioxidants and in anti-inflammatory defenses, they are more prone to
have potentially harmful long-term effects on their developing lungs and brain (Yeung, 2006).
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2.7 NITRIC OXIDE THERAPY IN PREMATURE INFANTS
The rate of survival in premature infants can be efficiently improved with the
administration of appropriate ratio of nitric oxide (NO) and by inhalation therapies which are
responsible for the protection of bronchopulmonry dysplasia in premature infants (Philipp et al.,
2007). Administration of NO and inhalation therapy not only improved the antioxidant status but
NO was responsible for immature lungs vasoconstriction and influx of plasma air (Saugstad,
2003). Nitric oxide (NO) significantly improved secondary mechanisms such as airways
structure and alveolarization as well (McCurnin et al., 2005; Bland et al., 2005 and
Balasubramaniam et al., 2006).
2.8 NON-PROTEIN BOUND IRON (NPBI) AND F2-ISOPROSTANES IN NEWBORN
Non protein bound iron (NBI) and F2 Isoprostanes in premature infants are also
responsible for the increased oxidative stress and stimulates prooxidants in plasma (Gutteridge
and Halliwell, 2000; Thibeault, 2000). In perinatal time especially immature infants, low evel of
stored form of iron, decrased iron binding sites and low level of ceruplasmain may be associated
with the appearance of NBPI in aminotic fluid. F2 isoprostanes contain a series of prostaglandins
like ponds in vivo and in vitro by catalyzed peroxidation of phospholids bound archiodionic acid
(Morrow and Roberts, 1996 and Delanty et al., 1996). Signorini et al., (2008) confirms the
association of lipid peroxidation NPBI concentration.
2.9 MELATONIN PROTECTS AGAINST OXIDATIVE DAMAGE
Oxidative stress remains responsible for the pathogenesis of bronchopulmonary dysplasia
(BPD). Bronchopulmonary dysplasia (BPD) is a chronic pneumopthay observed in newborn
infants with prolonged respiratory tract. Melatonin detoxifies free radicals under the influence of
mechanisms directly and indirectly. Melatonin (MT) or N-acetyl-5-metoxy-tryptamine is the
indole synthesized from the pineal gland during night. The fetus receives MT from the mother,
followed by premature delivery and leads to prolonged (MT) deficiency. Defined deficiency may
be harmful because neurohormone has important functions. Blood MT influences the sleep-wake
cycle and changes the physiology from day time to night time in a well-coordinated manner and
aids in the synchronization of circadian rhythmicity in all tissues. Pan et al., (2009) investigated
the factors of affecting the MT or imbalancement of antioxidants and oxidants in the lung.
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2.10 MATERNAL ANEMIA
Pregnancy is often refered to number of diseases/complications such as anemia. Maternal
anemia is the most common disorder. Incidence of maternal anemia in developing countries such
as Pakistan is significantly increasing (Ahmad et al., 2011). Under the influence of oxidative
stress (OS) and scavenging activity of free radicals, physiological and metabolic functions are
altered. Pregnancy itself is a very stressful condition, levels of antioxidants decreases (Khanna et
al., 2010).
2.11 IRON STATUS AT BIRTH
Defined relationship in between low birth weight and the level of hemoglobin helps to
explain complications such as iron deficiency in infants (Salsbury, 2001). Almost ninty five
percent (95%) of premature infants (26-34 weeks of gestational age) show low birth weight, iron
deficiency and decreased growth. Maternal anemia is the reason for premature delivery of infants
lead to iron deficiency at the time of birth (Sichieri et al., 2006).
2.12 MORTALITY OF PRETERM BIRTHS
There are different factors involved in the very low birth weight (VLBW) in pretermies
including perinatal, neonatal, postnatal morbidity and mortality and is responsible for premature
complications and infections. Neonates with very low birth weight (VLBW) are 30 times at
greater risk of mortality (Kabir et al., 1995). Due to the advancement in neonatal and perinatal
cares rate of VLBW are significantly decreased (Chowdareddy et al., 2014). Anemia is the major
risk factor to cause preterm delivery. The rate of mortality has the negative relationship with
gestational age. However, obstetric and perinatal complications in this population of pregnant
women could be significantly reduced by providing better antenatal care and timely interventions
where needed.
2.13 IRON DEFICIENCY ANEMIA AND SEVERE PREGNANCY OUTCOME
Iron deficiency and anemia results in impaired transport of hemoglobin. Supply of
oxygen to uterus, placenta and fetus is also impaired that causes cellular dysfunction. Mechanism
can be explained by impaired myometrial contractility leads to atonic uterus and placental
dysfunction contributes to preterm birth and growth restriction in newborns. Impaired delivery of
oxygen contributes in impaired wound healing (Jaleel and Khan, 2008).
Erythropoietin (Epo) is a 30.4 kD glycoprotein hormone that contains 165 amino acids
and is produced by peritubular fibroblast cells that are located in the kidney. Erythropoetin (Epo)
CHAPTER TWO REVIEW OF LITERATURE MISBAH, 2017
16
is produced by the kidney in response of decreased circulating oxygen in the blood which is
sensed through hypoxia-inducible factor. The primary function of Epo is to produce erythrocytes
through erythropoiesis. Premature infants may develop anemia in their first few weeks of life and
results blood loss, shortened red blood cell lifespan, low plasma erythropoietin levels and
inadequate erythropoiesis.
2.14 PREGNANCY OUTCOME ASSOCIATED WITH ANEMIA AND IRON
Maternal anemia may be diagnosed before mid-pregnancy and it contributes as one of the
primary risk factor for the preterm delivery. Moreover, at the later stages particularly third
trimester high concentration of hemoglobin increases the process of hematocrit and serum
ferritin levels in pregnant females. These factors collectively are responsible for the preterm
delivery. Several studies established positive correlation in between maternal anemia and
preterm delivery (gestation age < 36 weeks). Anemic pregnant females required extra medical
care with special emphasis on the levels of hemoglobin. Physical paramters such as education,
ethnicity, gestational age, and smoking all collectively increase incidence of preterm delivery.
Studies explain concentration of Hematocrits ≥ 40% before 20 weeks of pregnancy, it
was also documented that 18% of women released vaginal bleeding at the time of the first
antenatal. Risks of preterm delivery increases upto five folds due to vaginal bleeding because of
iron deficiency and increases risks of preterm delivery. The prevlance of preterm delivery was
increased 1.6 times in such women those were having levels of hemoglobin in between 100 and
109 g/L. Improved levels of hemoglobin reduced the risks of preterm deliveries.
2.15 IRON IN PREGNANCY
As discussed maternal anemia is one of the common problems during pregnancy
therefore, iron supplementation is made necessary during the course of pregnancy. Iron
deficiency increases the risks of preterm delivery, fetal growth restriction leading to the cause of
preeclampsia to many folds (Scholl, 2005). Apart from that iron supplementation and increased
reserves of iron is found to increase some gestational complications i.e., gestational diabetes and
increased oxidative stress hence, iron supplementation to females who are not iron deficient may
increase their risk factors.
Among low-income minority women in their reproductive years are believed to be iron
deficient such as low level of hemoglobin or hematocrit. Women belonging to poor and ethnic
minorities are reported with that the adverse outcomes of pregnancy in respect to low levels of
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hemoglobin and increased storage of iron. However, desired levels of iron must be attained and
were significantly helpful in pregnant women. Most frequent relationship was in between
nutrients and ethnicicty whether females belong to developed or undeveloped countries.
Menstruation cycle is the major factor in woman for loss of iron in form of menstrual blood.
Prevalence of iron deficiency and IDA was increased in women belonging to group of
minorities, below poverty line and with less education. Iron deficiency was even higher in
women with more than 2 children (Looker et al., 1997). Another estimate shows fifty percent of
women were not having adequate iron stores during pregnancy (Yip et al., 2001; Perry et al.,
1995). During pregnancy (3-4mg/d) is substantial amount of iron required and risks of iron
deficiency and IDA increases with gestation. Women belonging to lower income class were
more prone to develop anemia as compared to those belonging to stable income (Perry et al.,
1995).
Anemia is also known as “sickness index” for the body (Hoffman et al., 2000). Anemia
includes pathologically abnormal levels of hemoglobin that may cause the thalassemia some
times, or may be because of abnormal beta chain of hemoglobin therefore, hemoglobin cannot
perform its pathological function and develops thalassemia or folate/B12 deficinency.
Additionally it may also contribute as the lead cause for other chronic diseases such as
cardiovascular diseases, cancer and some infectitious and chronic inflammatory disorders
(Hoffman et al., 2000). The levels of Hemoglobin and hematocrit were reduced throughout the1st
and 2nd trimesters and it reaches to the lowest point by second to early 3rd trimester and
ultimately rises up to the normal level (Anonymous, 1990) but in later pregnancy, it becomes
very hard to distinguish in between physiologic anemia by iron deficiency internally and
externally (Anonymous, 2002).
According to the previous studies it is suggested that during 1st trimester the meternal
anemia affected preterm delivery (Zhou et al., 1998). According to the study of Allen (2001), it
suggested that there are three main factors in regulation of potential mechanisms of maternal
IDA and may be the reason for preterm delivery. In the presence of oxidative stress it leads to
chronic hypoxia and is responsible for the release of CRH from placenta and stress hormone
from fetus. Excess production of free radicals and decreased levels of antioxidants lead to the
damage to maternal fetal unit and is the reason for preterm delivery. These types of free radicals
attain ability to damage organs, tissues and to even cells present in biological system (Halliwell
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and Gutteridge, 1999). Oxidative stress over time is now thought to be major factor of aging,
reason for the development of diseases such CVD, cancer etc. Iron overload remains associated
with the stress status that further contributes in the pathogenesis of hepatitis and diabetes
(Fernandez-Real et al., 2002; Jiang et al., 2004). Preliminary findings suggest that the
overloading of iron increases excretion of 8OHdG and MDA in the maternal-fetal unit.
2.16 NEGETIVE EFFECT OF OXIDATIVE STRESS IN FETUS AND NEWBORN
As the oxidative stress is increasesd in maternal fetus production of free radicals and
decreased levels of antioxidants contribute in the disruption of cell cycle, fibrinolysis and
signaling transduction (Wagenaar et al., 2004). Well-established fact reveals that reactive oxygen
species (ROS) are imperative for fertilization (Dennery, 2004). In moderate concentration of free
radicals and sufficient supply of antioxidant defense system they continuously maintain the
process of fetal growth and cell aerobic metabolisms whereas, during abnormalities endogenous
or exogenous production of free radicals increases attack to biological macromolecules such as
carbohydrates, lipids, proteins and DNA (Halliwell, 1994 and Buonocore and Perrone, 2006).
Fenton chemistry, Hypoxia, endothelial damage, inflammation, arachidonic acid cascades
are some other mechanisms that results in the synthesis of highly reactive products. Fenton
reaction is responsible for the DNA damage (such as fragmentation, apoptosis, base
modifications and strand breaks), protein, lipid (arachidonic acid), and polysaccharides oxidation
(Saugstad, 1996). Premature born infants have weak defense system therefore, are very sensitive
to oxidative stress under the influence of fenton reaction (FR). Initially at the time of birth
newborn has to compete with the environmental changes and with the maintainence of oxygen
pressure (PO2 100torr) as compare to intrauterine environment (20-25 torr) (Maulik et al., 1999).
Then with the antioxidant defense which helps to copeup with hyperoxic challenges. Finally
pretermies have increased sensitivity against inflammatory infections and increased levels of free
iron in their plasma and tissue (Ciccoli et al., 2003).
Oxidative stress in hypoxic fetuses and neonates initiates damaging processes such as
lipid peroxidation that results in the formation of fragmants of malondialdehyde (MDA) that in
term combines with the DNA and leads to formation of DNA adducts and DNA damage. MDA
further converts stress markers including serum total hydroperoxides, isoprostanes, non protein
bound iron (NBPI) and advanced oxidative protein products (AOPPs). Whereas, serum levels of
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antioxidants decreases in addition with red blood cells (Ciccoli et al., 2003; Buonocore et al.,
1998).
Isoprostanes belongs to the family of prostaglandins secreted by the oxidation of
arachidonic acid and its derivates by binding to free radicals generated in the condition of
oxidative stress (Morrow and Roberts, 1996). Concentration of isoprostanes can be measured by
the circulating plasma and can be later detected in the urinary excretions. Particularly, 8-iso-
PGF2, is a major isoform of isoprostane which is relatively more stable and can be estimated in
the biofluids, it serves as most reliable oxidative bio-marker (Pratico, 1999). Levels of F2-
isoprostanes were measured with the help of mass spectrophotometer and gas spectrophotometer
in newly born infants. Levels of F2-isoprostanes were significantly higher in preterm babies as
compared to healthy ones (Comporti et al., 2004). Significant inverse relation was observed in
between gestational age and isoprostanes in plasma. Whereas, significance in plasma levels of
isoprostanes in mothers before and after pregnancies were not observed. ROS was also found to
be involved in the damaging of amniotic epithelium and chorioamniotic collagen. Concentrations
of F2-isoprostanes were significantly increased in patients due to the breakdown of membrane as
compared to normal infants (Longini et al., 2006).
ROS induced rupture of membrane in pregnant state; it also abnormally binds free amino
acids and causes abnormal synthesis of proteins. Moreover, it also disturbs membrane integrityby
the oxidation of polyunsaturated fatty acids (PUFA) and leads to modification of chrioamnitic
biology and cell dysfunction (Masumoto et al., 1992 and Ogino et al., 2005). The most abundant
oxygen free radical is superoxide anion that was initially discovered during bacteriocidal activity
and was further involved in production of other oxide derivatives that includes hydrogen
peroxide (H2O2) and hydroxyl ions (OH-). Radicals were neutralized by the action of
antioxidants such as superoxide dismutase (SOD) and catalase (CAT) (Klebanoff, 1980).
Oxidative stress and its permeability of capillaries increases and promotes transportation of
certain cytokines whereas, mechanisms in between oxidative stress and inflammation are not
well-known. The production of cytokines during infection amplifies the induction of hypoxic-
ischemic effects. It was suggested that the production of cytokines were increased in state of
infections and inflammation (Dietrich et al., 2004).
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2.17 HYPOXIA AND NITRIC OXIDE
During the hypoxic condition the normal physiology of cells were altered by the over
production of Nitric oxide (NO) in the presences of eNO synthase (eNOS) in endothelial cells
(Meini et al., 2003). NO produced by the activation of eNOS in the presenaces of arginine and
oxygen. Once NO is produced it combines with superoxide radicals that converts other toxic free
radicals such as peroxynitrile, hydroxyl radicals, nitrogen dioxide. Cascade reactions of NO2+
activates series of enzymes (Beckman and Koppenol, 1996) as well as some other damaging
metabolites including nitryl chloride (NO2Cl), nitrogen dioxide radical (NO2), sysnthesized
through nitrite reaction. The reaction ends up to final metabolite which is termed as
hypochlorous acid (HOCl) with the help of neutophilic myeloperoxidase (MPO) activation
(Eiserich et al., 1996). There are three types of NOS yet established as follow: endothelia NOS
(eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) (Rothman et al., 1992; Cozzi et al.,
1990 and Kaur et al., 1999). As the hypoxic condition prevails it increased oxidative stress by
the up-regulation of NO and some other varities of cells such as (endothelial cells, macrophages,
neurons and astrocytes) (Dalkara and Moskowitz, 1997). According to the experimental studies
at initial stages NO has shown protective effect against vasoconstriction and it promotes
perfusion by the activation of NOS 3 (Iadecola et al., 1997). However, expression of NOS 1 and
NOS 2 were also increased after ischemic condition (Dalkara et al., 1994). Excess in the amount
of NO was responsible for the increased oxidative injury by change in Ca+ homeostasis that has
major role in cellular signal transduction and in regulation of several cellular processes like
proliferation (Meini et al., 2003).
Another well-known fact states that gene expression may be regulated by the cascade
pathway termed as MAPK pathway. Cascade is as follow Ras/Raf/ERK. All of the cascade
reactions are regulated under the normal concentrations of Ca2 and NO however, any major
fluctuations in these levels may result in cell cycle arrest (Mannick et al., 1999). Hence in the
light of knowledge negative relationship may be observed in between NO, and Ca+ for
regulation of cell cycle.
2.18 PARTURITION AND OXIDATIVE STRESS
Term labor stimulates compensatory defense mechanism that includes enzymatic and
non-enzymatic antioxidants in the compartment of fetal red blood cells (Buhimschi et al., 2003).
The given mechanism shows protection to neonate at the time of birth against hypoxic condition.
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Reduced levels of antioxidant in preterm neonates, and increased status of free radicals may
cause damage at cell and tissue level including bronchopulmonary dysplasia and retinopathy
(Saugstad, 2003 and Dani et al., 2004).
2.19 OXIDATIVE STRESS AND DIABETIC PREGNANCY
Complications of pregnancy contribute in overproduction of free radicals, lipid
peroxidation and reduced activity of antioxidants which can be measured in terms of levels of
MDA and other antioxidants such as glutathione (GSH), catalase (CAT) and superoxide
dismutase (SOD). Elevated concentraitons of MDA signified increased hemolysis of
erythrocytes in premature infants as compared to heathy ones (Djordjevic et al., 2004).
2.20 ROLE OF OXIDATIVE STRESS IN MENOPAUSE AND AGE RELATED
FERTILITY DECLINE
Reactive oxygen species (ROS) is found to affect the levels of sex hormones. ROS is
found to reduce the levels of estrogen with the passage of time by the over production of free
radicals. During premenopausal to menopausal period the scavenger activity of both glutathione
peroxides and superoxide dismutase were reduced in the ovary (Okatani et al., 1993).
2.21 STRATEGIES TO OVERCOME OXIDATIVE STRESS IN ASSISTED
REPRODUCTION
Under the influence of low oxygen development of embryo and fertilization is not proper
(Burton et al., 2003). Initially, such conditions were avoided that resulted in increase of free
radicals with supplementation of proper antioxidants the oxidative insult may be reduced
effectively (Catt and Henman, 2000). During cryopreservation hydrogen peroxide levels were
reduced with effective therapy of ascorbate (Lane et al., 2002). ROS has harmful effects at the
quality of embryo and oocyte in patients suffering with endometriosis. Levels of inflammatory
cytokines such as; TNF-α, ILs, MMPs significantly increases peritoneal fluids in patients with
endometeriosis and unexplained infertility (Saleh and Agarwal, 2002). Strategies involve sperm
preparation methods that affect ART outcomes. Preparation by centrifugation has been directly
linked with the formation of reactive oxygen species. It has been reported that seminal plasma
has high levels of antioxidants and protects the spermatozoa from DNA damage and lipid
peroxidation (Potts et al., 2000).
Whereas minimum amounts of reactive oxygen species (ROS) are necessary for the
physiological process of sperm’s functions. On the other hand, unbalanced levels of reactive
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oxygen species have negative impact on spermatozoa and fertility rate. Procreating is a natural
process in the life of couples that relies on special planning but not in intercession. The couples
are tried to be conceive but 15-25% is unable to cause pregnancy and they seek out medical
suggestion to improve the chance of successful fertilization and pregnancy. The guidelines of
World Health Organization suggested that male factors are responsible for infertility which
included variation in the concentration and motility rate of sperm. According to recent work,
oxidative stress is supposed to be a major and credible cause of male infertility. Generally
oxidative stress is a condition in which there is imbalancing of systemic radicals such as reactive
oxygen species and in biological system in which antioxidants acts as defense mechanisms.
According to the some perior studies production of ROS were increased by prolong (16-20
hours) co-incubation of sperm-oocyte as compare to some other prospective randomized
controlled studies to using a shorter time incubation of sperm-oocyte (Kattera and Chen, 2003;
Gianaroli et al., 1996) because the rate of fertilization and implantation will be improved at the
incubation of 1-2 hours which lead to significantly increased the quality of embryos (Dirnfeld et
al., 2003).
2.22 PHYSIOLOGIC STATUS OF MOTHERS
Those factors which are maternal physiological factors and are interested in the study are
as follow: gestational diabetes, Eclampsia, chronic hypertension, pre-pregnancy diabetes,
cervical cerclage, pregnancy weight gain and history of preterm birth. Nevertheless there are
other illness and complications that influence pregnancy results which are not recorded in the US
data base records, like; kidney or bladder (urinary tract) infection; severe nausea, vomiting,
dehydration and vaginal bleeding; cervix had to be sewn shut; hypertension also Pregnancy-
Induced Hypertension [PIH]), pre-Eclampsia; placental complications (such as placental
abruption, placenta previa), car accident and blood transfusion. Pre-pregnancy conditions that
may include diabetes mellitus, hypertension, pre-Eclampsia and asthma are also related to an
increased chance of developing preterm birth (Aly et al., 2011; Madan et al., 2010). About 30%,
the reasons for preterm births are either fetus or mother related; such as maternal diseases or
uterine fetal retardation (Gravett, Rubens, & Nunes, 2010). From 1989 to 2000, there has been an
increase of 55% in the incidence of preterm births in US (Ananth, Joseph, Oyelese, Demissie, &
Vintzileos, 2005). With the increase in the intensity of pregnancy induced hypertension there is
also in an increase in the risk of developing preterm births (Ye, Li, Ma, Ren, & Liu, 2010).There
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are many studies which support the concept that pregnant women with vaginal bleeding have
double the risk of developing preterm birth. In early pregnancy, vaginal bleeding is always
related to the risk factor of preterm birth (Hackney & Glantz, 2011; Ramaeker & Simhan, 2012).
Yet, Yang et al supported that in African women vaginal bleeding is not associated with preterm
birth (Yang et al., 2004).
2.23 ROLE OF OXIDATIVE STRESS AND LIPID PEROXIDATION IN PRETERM
DELIVERY
When the rate of the production of the free radicals becomes more than the removal rate
by the cellular defense mechanism, this state is known as Oxidative Stress (Burton & Jauniaux,
2010). There has been a strong relation between the oxidative stress and the preterm labor and
other diseases of pregnancy such as PROM, Early Deliveries, IUGR, Pre-Eclampsia and many
other diseases associated with preterm infants (Buhimschi et al., 2003; O’Donovan & Fernandes,
2004). Not only in placenta, fetus and newborns the numbers of cells divide rapidly, but there is
a rapid growth of division in the maternal cells compartments as well. One of many pregnancies
is a state where there is a need of an antioxidant which has to be present in the body for
combating this stress. (Kaiser and Allen, 2002) Free radicals are produce during pregnancy, also
the other oxidant molecules which exceeds the value of the antioxidant which are available as a
buffer in pregnant mothers and developing fetus. Cellular damage is the end result of the above
mentioned process, in which not only the preterm labor and early deliveries are associated, but
also it includes IUGR, PROM and Pre-Eclampsia and many other serious issues for mature
infants (Joshi et al., 2008). The excess of these free radicals attacks the endothelial lining of
vessels if these are not are not buffered and many more system also by acquiring the nucleic
acids of many lipids, carbohydrates and proteins and hence they denature the DNA of these cells
(Burton and Jauniaux, 2010). Normal placental growths are greatly affected by these free
radicals and may cause stillbirths OR miscarriages, also causing number of chromosomal
disorders at the same time (Stein et al., 2008).
Pre-Eclampsia can also occur by the disturbance in the vessels vasodilator mechanism
caused by the oxidative stress, resulting in the elevation of the maternal blood pressure, hence
placental blood flow is impaired (Buhimschi et al., 2003) Alteration in the genetic metabolic
detoxification is also associated with the oxidative stress by the cytochrome P450 1A1 gene and
many others (Agarwal et al., 2005). Cytotoxic damage to the proteins may be caused by the
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oxidative oxygen and nitrogen, DNA OR Lipids, until unless the non-enzymatic and enzymatic
oxidants equalize there harmful effects. There are many for free radicals to act, e.g. ROS formed
by the mediation of the iron which causes the Lipids and DNA damage is due to the fluctuation
of the iron molecules from their normal values; that is the transport of oxygen to the tissues,
results in the cellular damage due to the iron induced free radicals. Macrophages and Neutrophils
release free radicals because of the presence of the inflammation and infection in the body. Free
radicals of specific area are referred as Reactive Oxygen Species (ROS) because many free
radicals which biologically important are derived from oxygen (Saugstad, 1996).
Reactive Oxygen Species (ROS) is defined as mutual consideration about including not
only the superoxide anion and hydroxyl radicals but also including the other radicals such as
hydrogen peroxide, is a derivation of molecular oxygen. Reactive Oxygen Species (ROS) have
many different mechanism for its generation, like the normal movement of electron in the fatty
acid and the mitochondria, metabolism of the prostaglandin, ischemic re-perfusion, low levels of
oxygen at cellular level (HYPOXIA), state in which there is an excess of oxygen at cellular
levels (HYPEROXIA), activation of macrophages and neutrophils (INFLAMMATION),
hypoxanthine-xanthine oxidase system in the endothelial cells (ADINOSINE TRIPHOSPHATE
DEGRADATION), high levels of free circulating metals and Fenton reaction (FERROUS to
FERRIC ACID) (Shoji and Koletzko, 2007). Due to development and growing of placenta the
rate of the flow of the blood in the vessels increases and as gestational period advances in its
later life, if there will be a damage to the endothelial cells of the blood vessels then it would
cause a very harmful and particularly a very serious damage to the vessels. During vasodilatation
blood vessels usually uses nitric oxide (NO) signaling to initiate the process. There are near to
circumference of muscle cells that surrounds the wall of the blood vessels which dilate the blood
vessels itself in order to control the additional flow of the blood. The increased blood flow for
the growing fetus and placenta is provided by the vasodilation effect in the blood vessels
(Edemann and Schiffrin, 2004).
The process of vasodilatation effect in the blood vessels is disrupting by the amount lf
free radicals present in the blood. Hence, there is a low supply of blood to the fetus and placenta
due to vasoconstriction in the blood vessels, which result in IUGR, Pre-Eclampsia and Preterm
Labor (PTL) (Stein et al., 2008) Chromosomal disorders, may arise due to the presence of
oxidative stress that may lead to fetal death. There is also an effect of oxidative stress in a
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reversal manner that it may cause the disability of the body to detoxify the oxygen radicals
present in blood (Blondi et al., 2005). The Cytochrome P450 A1A gene (CYP1A1), Glutathione
S-transferases µ1 (GST m1), and 01 (GSTT1), they all can interfere with the detoxification
process. There is a cascade of fatty acid that is triggered by Reactive Oxidative Species (ROS)
such as arachidonic acid that lead to premature contractions, dilatation of the cervix and delivery
of baby before the 37th week of gestation (Joshi et al., 2008).The only management of this
process is to balance the free radicals of oxygen and nitrogen in the blood is by balancing the
values by giving external oxygen and nitrogen via supplementation. The enzyme Superoxide
Dismutase (SOB) is included in the defense system of anti-oxidants, glutathione and catalase.
Those infants who are premature in their births are at high risk for oxidative stress, it is due to
the both endogenous as well as acquired exogenous antioxidant defenses that do not activate in
maturation in the third trimester (Finer & Leone, 2009; Baba & McGrath, 2008).
Through the production of free radicals oxidative stress is a contributing factor in tissue
injury and by leading inflammatory cytokines by the reactive oxygen/nitrogen and end up in
preterm labor (PTL) (Pressman, 2011). There is a lot of evidence that is increasing about the
oxidative stress that it is the end point of a very complex process that either they are determined
genetically or they are activated by an in-utero stressor. Those new born infants which are born
premature or they are born preterm labor are highly at risk for induced oxidative stress tissue
damage. Oxygen therapy is the first choice of management in the premature infants and
HYPEROXIA which is known as high levels of oxygen and in intrauterine environment where
the oxygen is in much less in amount as compared to hyperoxic state results in exposing the
infants to a very high free radicals zone. (Maulik et al., 1999) Later, the mechanism of
antioxidant defense is induced by hyperoxia and it is comparatively damaged in infants who are
premature (Speer and Silverman, 1998). Thirdly, there is an increased risk for preterm infants to
be induced for inflammation and infection which causes the effect of oxidative stress (Saugstad,
1988). There is a presence of free iron in the tissues and plasma of premature infants as
compared to full term babies. There is a variety of disease which is caused by the oxidative stress
to an individual organ or sometime to a group of organs frequently in the neonates which include
retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), bronchus-pulmonary
dysplasia (BPD), periventricular leukomalacia (PVL) and intra-ventricular hemorrhage (IVH)
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(Clyman, 1989; Archer et al., 1989 and Sanderud, 1993).In spite of their timing preterm and full
term were considered to be similar process which follows a same and common pathway.
However, the basic reason and the initial process of preterm births is still unclear,
Romero et al (2006) used the term syndrome for describing every possible pathological
reasoning which cause preterm labor (PTL). Although, the activation of these pathways in
physiological manners results in full term labor while the preterm labor occurs when there is an
alteration in these common pathways due to the presence of many pathological reasons which
induces pathological conditions from multiple causes and risk factor (Romero et al., 2006).
Preterm labor is sub-divided into two types;
1. Indicated (Usually associated with maternal and fetal conditions)
2. Spontaneous
One of the four primary pathogenic pathways is the prime responsible factor for the preterm
birth. They may include over-distension of the uterine, cervical disease, infection, endocrine
disorders (Romero et al., 2006) and ischemia, decidual hemorrhage and activation of maternal
and fetal hypothalamic-pituitary axis (Behrman and Bulter, 2007). Intra-Uterine infection and
inflammation is to be considered as the main contributor to preterm birth (Norman et al., 2007).
These are the mechanisms that meet on a mutual pathway which involve in an increase in the
uterotonin and protease expression. In a given woman more than one process can take place at a
time. In some women the combination of inflammation and genetics response are the area of
research that could explain the process of preterm labor with some common risk factors (Challis
and smith, 2001 and ACOG, 2010). Labor is a process which encourages change in chorio-
amniotic membranes that are reliable to acute inflammatory response, regardless the absence of
the inflammatory response (Haddad et al., 2006). NF-Kappa B is activated by the reactive
oxygen species that is responsible for the stimulation of COX-2 expression and it also promotes
inflammation with succeeding parturition.
There is a decrease in the Gx protein in both the preterm and full term labor women and
this was reported by (Khan et a l.., 2010), as compare to non-labor groups. If these data taken
together, they suggest that state of labor whether preterm or full term requires the actions of GPx
for limiting lipid oxidation and is also associated with the reduction in the ROS of antioxidant
defenses (Khan et al., 2010). Mustafa et al., (2010) discovered remarkably increased stages of 8-
OHdG and MDA and suggestively decreased stages of glutathione (GSH) in the blood of
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mothers having preterm labor as compared to those mothers having full term labor. This
conclusion proposed that those women who were in preterm labor have reduced antioxidant
capabilities to secure against the damage cause by the induced Oxygen specie (OS).
Furthermore, abridged events of FRAP, it is an assay which is present in the body to measure the
body ability to defend against the oxidative damage and, also GST, has also been seen in the
women with preterm labor (Hong et al., 2002; Frosali et al., 2004; Mustafa et al., 2010 and
Pathak et al., 2010). Furthermore, the results support that the increase amount of OS in the
maternal environment and low levels of antioxidants extracts both the mother and fetus more
vulnerable to ROS-induced damage. Inflammation persuades the uptake of ROS and can be
reason of apparent OS, which results in tissue injury and ensuing preterm labor (Chadha et al.,
2007).
As a protective response the concentration of Mn-SOD increases to inflammation and OS,
and decrease down the levels of MAPK pathways, NF-kappa B and activator protein-1 (Oberley,
2005). In women having preterm labor were observed with high levels of Mn-SOD in the
membranes of their fetus as compared to those women who were in spontaneous labor at term,
that may recommend a larger degree of OS and inflammatory process in the former. Chorio-
amnionitis is suggested to be linked with preterm labor and histological infection was found to
be related with raised expression of Mn-SOD mRNA in the membranes of fetus of those women
having preterm labor (Than et al., 2009). The increased amount of the Mn-SOD mRNA
expressions may be a compensatory response to the existence of high levels of OS and
inflammation in preterm labor. Suggestively increased amounts of the pro-inflammatory
cytokines IL-1 beta, IL-6, and IL-8, have been detected in the amnion and chorio-decidua of
those women having preterm labor as compared to those women who were having spontaneous
labor. There is an activation of membrane inflammatory response in women having preterm
labor (Keelan et al., 1999). Levels of TAS are slightly lower in women as compared to those
women with simple pregnancies having similar gestational period, and this may indicate the
presence of OS in women with preterm labor (Cinkaya et al., 2010). In comparison to those
women having simple pregnancies those women having preterm labor were observed to have
low levels of PON 1 activity in them (Bakar et al., 2010). This discovery proposed that there are
increase levels of lipid peroxidation and a decrease in the levels of antioxidant activity of PON-
1that may collectively generate a pro-oxidant setting and enhance the hazards for developing
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preterm birth. Moreover, there is a remarkable decrease level of GSH in patients having preterm
labor (Lee and Davis, 2011).
Preterm birth in early gestational age has been associated with the low levels of the
maternal serum selenium (Rayman et al., 2011). GST polymorphism was found to be
expressively greater in those patients who were in preterm labor, demonstrating that these
patients are more susceptible to oxidative damage (Mustafa et al., 2010). Maternal infection
caused by inflammatory setting is concomitant with preterm birth and a state of OS is produced
and the resulting in the decrease of antioxidant defenses and likely to elevate the chances for
preterm birth. The offered suggestion involves the inflammation and repressed the levels of
antioxidant defenses in the pathophysiology of preterm labor. It seems reasonable that in
preventing preterm labor antioxidants supplementation may be associated and also inflammation
associated with birth. Temma-Asano et al 2011 conducted a study and established that NAC was
operative in minimizing OS induced by chorio-amnionitis, and hence protecting preterm labor
(Temma-Asano et al., 2011). Nevertheless, those women with nulliparous complication having
vitamin C and E supplementation in early gestational ages did not reduce the chances of
developing preterm labor. As the results of these studies were contradictive, it is still unclear that
supplementation of antioxidants by the mother is helpful in reducing the event of preterm labor
(Rumbold et al., 2006 and Hauth et al., 2010). Those substances which decrease the variety of
OS by producing free radicals or by reducing the damage which is responsible for creating free
radical chain reactions.
They work in a sense by slowing down or by preventing the damage to the cells from
infection by enhancing the immune system. There are three major categories in which
antioxidants are classified:
(1) Low molecular antioxidants [glutathione (GSH), vitamins C and E, bilirubin, and urate]
(2) Enzymes that neutralize free radicals [superoxide-dismutase, catalase, glutathione peroxidase
(GPx), DTdiaphorase, and peroxiredoxin], and
(3) Non-enzymatic proteins (thioredoxin, glutaredoxin, and metllothionines).
The classification of antioxidants can be in primary and secondary manner. Endogenous
enzymes are classified as primary antioxidants [e.g. Catalase, GPx and superoxide dismutase
(SOD)] and the site of their action is the site where free radicals are produced. Those
antioxidants which are obtained through foods are known as exogenous antioxidants and are
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classified as secondary antioxidants (e.g. Carotenoids and Vitamins A, C, E).Antioxidant
enzymes contribute in a complex communication in oxidizing and reducing those molecules
which defines the cell setting that is essential for keeping cellular, placental, fetal, and postnatal
growth (Dennery, 2004). Such enzymes have low activity in preterm infants and cannot balance
excessive ROS production. The most important antioxidant enzymes are copper-zinc superoxide
dismutases (SOD), which are found in cytoplasm as well as peroxisomes, and manganese SOD
from mitochondria. SOD catalyzes the dismutation of superoxide anion to H2O2. Glutathione
peroxidase (GPx) in mitochondria and catalase (CAT) in peroxisomes catalyze the reaction of
H2O2 to molecular oxygen and water. Together with Vitamin E these enzymes plays an
important part in per-oxidation of those fatty acids which are still poly-unsaturated in the cell
membranes (Thibeault, 2000).
Non- Enzymatic pathways are the other pathways which produces resistance in the
process of oxidative stress. Some of the nutrients like zinc, copper and selenium may have
antioxidants functions as a vital component of antioxidant enzymes. Ceruloplasmin, glutathione
(GSH), transferrin, bilirubin and Vitamins A, E, and C are to be known to have properties of
antioxidants enzymes. ROS induced injury to the full term infants may took place due to the
premature infants because of the deficiency of utero-placental movement of nutrients which are
significantly vital for antioxidant defenses (Shah MD & Shah SR, 2009).Many seleno-enzymes
may include selenium in them having in them GPx as most important part. There is an
association between the serum selenium and Glutathione per-oxidase (GPx) activity and the
weight of the newborns (Trindade, 2007). Preterm diseases may have the involvement of
selenium deficiency in them but this data is not more than enough to hypothesize this theory
(Darlow & Austin, 2003). Vitamin A part is likely intermediated by the action of this vitamin on
retinol-binding protein and also the receptors of retinoic acid, apart from showing its direct
effects as anti-oxidants. In preterm infants the levels of serum vitamin A are consistently to be
shown as low (Darlow& Graham, 2007). With an increase in the gestational age there is an
increase in the concentrations of vitamin C also known as ascorbic acid. Prevention of tera-
togenic effects of maternal diabetes with the supplementation of vitamin C and vitamin E has
been suggested by number of studies but these studies do not show more evidence about other
measurable benefits (Dheen et al., 2009).
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Transferrin, ferroxidase and Ceruloplasmin contribute in the process of iron metabolism
which is a potent oxidizing agent, when bio-availability OP the functions are diminished then an
increase in the susceptibility of oxidative stress. Free iron existence is the sole indicator of the
production of ROS (Brion et al., 2003). There is a decrease in the concentrations of these non-
enzymatic antioxidants that influences those infants which are born in preterm conditions to
difficulties with increased concentrations of ROS. The reduced levels of ceruloplasmin and
transferrin have been detected in those infants which are preterm births with conditions like
asphyxia prior to brain hemorrhage (Lindeman et al., 2000). Glutathione reduction or reduced
synthesis has been anticipated to elaborate the risk of oxidative stress to the newborn infants. A
low level in the bronchi-alveolar fluid foresees that in intubated premature infants have later
developments of BPD in them (Grigg et al., 1993). In vitro, bilirubin has been demonstrated as a
powerful antioxidant vulture of per-oxy radicals. Bilirubin degenerates to its predecessor
biliverdin in the oxidative process as a non-toxic product. In full term as well as preterm births
there is a direct relationship between the plasma concentrations of serum bilirubin and total anti-
oxidants status (Wiedemann et al., 2003).
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
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MATERIALS AND METHODS
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
32
3.0 MATERIALS AND METHODS
For the current study one hundred and seventy one (n=171) preterm infants and about one
hundred (n=100) age and sex matched controls were enrolled. All of the patients were reviewed
after a signed consent form and experimental work was carried out and approved by the
Research Ethics committee of institute of molecular biology and biotechnology (IMBB), The
University of Lahore-Pakistan. Five ml (5ml) blood was drawn from the cuboidal vein and stored
in the vial at -75 degree centigrade for the future analysis.
TABLE 01: DEMOGRAPHIC DISTRIBUTION OF DATA
SEX (n=) MALE 84
FEMALE 87 INFANTS RESUSCITATED (n=)
Yes 57 No 114
SMOKERS (n=) YES 49 NO 122
SEPSIS (n=) YES 56 NO 115
DELIVERY (n=) NORMAL 41
C-SECTION 130 BLOOD TRANSFUSION (n=)
YES 39 NO 132
IRON THERAPY (n=) YES 28 NO 143
DIABETIC (n=) YES 35 NO 136
3.1 INCLUSION CRITERIA
Preterm infants with anemic profile were added in the following study
3.2 EXCLUSION CRITERIA
Infants to mothers with any congenital diseases or diabetes were excluded out of the current study
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
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3.3 CHEMICALS
Reagents and chemicals of high analytical grade were acquired from Sigma Chemical
Co.(St. Louis, Mo, USA).
3.4 BIOCHEMICAL ASSAYS
3.4.1 ESTIMATION OF GLUTATHIONE (GSH)
Glutathione (GSH) was evaluated in the blood of patients according to the method of
Moron et al., 1979. Glutathione is first reacted with the reagent in term to produce a
chromophore TNB taking its absorbance at 412 nm. Amount of Glutathione (GSH) is
calculated according to the formula ([GSH]t = [GSH] + 2 x [GSSG]). The rate of absorbance
change (ΔA 412nm/min) is repeated after fixed interval. Amount of sample is determined by
generating a standardized equation from several standards of glutathione (Tietze, 1969).
3.4.2 ESTIMATION OF CATALASE (CAT)
Catalase was determined according to the generalized procedure of Aebi, 1974.
Evaluation was made with the help of spectrophotometer by reducing the absorbance to 230nm.
Sample was then homogenized by the help of phosphate buffer (pH 7.0) at 1-40C and sample
was then centrifuged at 5000 rpm. The mixture contained phosphate buffer, H2O2 and the
enzyme extract. The specific activity of Catalase will be expressed in terms of units/gram.
Procedure was repeated with the standardized dilutions of Catalase and absorbance was
measured to generate the standard curve.
3.4.3 ESTIMATION OF THIOBARBITURIC ACID REACTIVE SUBSTANCES
(TBARS)
Malondialdehyde (MDA) is the end producduct of oxidative stress that is secreat in the
activation of lipid peroxidation by the binding of free radicals with unsaturated fatty acid s and
the levels of MDA were estimated in subject blood through Thiobarbituric acid reactive
substances (TBARS) method. This method was introduce by Ohkawa et al., 1979. For this
method the take 200µl to 0.2ml of sample in test tube, then add the 0.2ml of 8.1% Sodium
dodecyl sulfate (SDS), then further add the 20% acetic acid by 1.5 ml, finally add the 1.5 ml of
0.8% TBA was purpose about 200µl to 0.2ml of sample 0.2ml of 8.1% Sodium dodecyl sulfate
(SDS) and it stand at centrifugation for 10 min at 3000rpm. After the centrifugation the two
layers were formed in test tube. 2 ml of upper solution ware taken from each wells to take
absorbance at 532nm.
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
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3.4.4 ESTIMATION OF SUPER OXIDE DISMUTASE (SOD)
Activity of Superoxide dismutase (SOD) was estimated by the method of Kakkar, 1972.
It explained the how appropriate amount of samples and buffer were mixed to maintain the
appropriate levels of pH and 0.052M concentration. About 100µl of phenazine methosulfate
was then added followed by the 0.3ml of nitro blue tetrazolium (300μ m) and 0.2 ml of NADH
(750 μmol). As the NADH was added it initiated the reaction instantly afterwards it was
incubated at 300oC for 90 seconds. Finally 0.1ml of glacial was allowed to stand for few
minutes to stop the reaction and it was then centrifuged and n-butanol layer was separated. The
color intensity of the chromogen in butanol layer was then measured at 560 nm. Absorbance
values were then compared with a standard curve generated from known SOD.
3.4.5 ESTIMATION OF GLUTATHIONE REDUCTASE (GR)
Glutathione reductase was determined by the method of David and Richard (1983). It is
employed in the conversion of oxidized glutathione into reduced form Glutathione reductase
catalysis the conversion of oxidized glutathione to reduced glutathione employing NADPH, so
the amount of NADPH is a direct measure for the following enzyme activity. Reagents such as
PBS, EDTA and sodium azide are used in following ratios (0.12 M, 15 milli mole, and 0.1ml),
0.1 ml oxidized glutathione and 0.1 ml sample were added into the cuvette and 600 micro liter
distilled water was added to make the volume up to 2ml, it was then incubated for 3 minutes
and 0.1 ml NADPH. Absorbance was measured at 340nm with help of spectrophotometer after
every 15 seconds interval for 2-3 minutes. Controls contained water instead of oxidized
glutathione. The activity of GR represented as micro moles of NADPH oxidized/minute/g of
sample.
3.4.6 ESTIMATION OF VITAMIN C
The estimation of vitamin C was follow the protocol of Roe and Keuther (1943). It
involvs the conversation that ascorbate is converted into dehydroasorbate whn it is treated with
activated charcoal and later reacted with 2,4-dinitrophenylhydrazine and incubating it for three
hours at 37°C that results in the formation of osazone crystal. The crystals were dissolved in
2.5ml of 85% sulphuric acid and then tubes were cooled in ice and again absorbance was
measured at 540nm with the help of spectrophotometer. A standard graph was made and the
concentration of ascorbate in the samples were observed and expressed in terms of mg/g o
f sample.
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
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3.4.7 DETERMINATION OF ADVANCED OXIDATIVE PROTEIN PRODUCTS
(AOPPs)
AOPPs were estimated in the plasma of patients using semi-automated method
discussed briefly, AOPPs were measured by the help of spectrophotometer (model MR 5000,
Dynatech, Paris, France) and were then calibrated with help of chloramine-T (Sigma, St. Louis,
MO). For the purpose about 200 ml of plasma which was diluted with 1/5 PBS was placed on
microtiter plate (Becton Dickinson Lab ware, Lincoln Park, NJ), and it was then added with
20ml of acetic acid. The absorbance of the reaction mixture is immediately read at 340 nm on
the microplate reader against a blank containing 200ml of PBS, 10 ml of potassium iodide, and
20ml of acetic acid.
3.4.8 PREPARATION AND DETERMINATION OF (AGEs)
The AGE-HSA were determined in vitro by incubating HSA (type V; Sigma; 50 mg/ml)
with 500 mM glucose in PBS for about 65 days at 37°C under sterile conditions. The AGE-
pentosidine which is considered as a marker of non-enzymatic glycation of proteins employing
a modified method by Odetti et al. Plasma proteins or AGE-HAS was allowed to precipitate on
TCA. The pellets were then hydrolyzed in 2 ml of 6 N HCl and were then dissolved in 250ml
0.01 M heptafluorobutyric acid (Sigma). 4 mg of plasma protein was then injected into an
HPLC system (Waters Division of Millipore, Marlborough, MA). A 25-30.46-cm C18 Vydac
type 218TP (10mm) column was used (Separations Group, Hesperia, CA). HPLC was
programmed with a linear gradient of 10 to 17% acetonitrile from 0 to 35 min. Pentosidine was
eluted out almost after thirty minutes by fluorescence excitation at 335 nm and emission at 385
nm.
3.4.9 DETERMINATION OF GAMMA GLUTAMYL TRANSFERASE (GGT)
In the determination of GGT a simple principle is involved method identified by the
Persijin and Van der Silk (1978) that measures the activity of GGT in the given sample for the
reason about 1.0ml of the reagent was incubated at room temperature for about one minute
followed by the addition of the sample in cuvette. Initially the contents were mixed and
absorbance was measured at 405nm by the help of spectrophotometer. The applied step was
repeated after every one, two and three minutes and enzyme activity was calculated and
expressed in IU/L.
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
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3.4.10 DETERMINATION OF NITRIC OXIDE
Nitric Oxide was determined in the group of arthritic patients. NO which is produced in the
response of cells such as picomolar and nanomolar range and they are believed to have a very
shorter half-life (t1/2 <5s) in biological fluids; therefore, their direct assessment is difficult
while they are estimated in the mean of NO2¯ and NO3¯ they are performed to estimate NO
level in biological fluids and cell culture medium (Moncada et al., 1991). Nitrite concentration
is typically measured by a well-known method known as colorimetric Griess assay (Moshage et
al., 1995).
Sulfanilic acid is quantitatively converted to a diazonium salt under the reaction carried out by
reacting nitrite in acid solution. The diazonium salt is then coupled to N-(1-naphthyl)
ethylenediamine, which form a dye which is then measured at 548nm with help of
spectrophotometer and then quantified.
3.4.11 DETERMINATION OF TUMOR NECROSIS FACTOR BY ABCAM’s TNF
ALPHA HUMAN ELISA KIT
TNF alpha is estimated with the help of commercially available ELISA kit by Abcam. The
following kit is designed for the quantitative measurement of TNF alpha in supernatants,
serum, plasma samples and buffered solutions. For the purpose monoclonal antibody specific
TNF alpha has been coated onto the wells of the microtiter strips provided. Samples are added
and the reaction was initiated by adding the appropriate amounts of following reagents in these
wells. When they were incubated for the first time monoclonal antibodies were incubated along
with them. Afterwards they were washed and enzyme Streptavidin-HRP was added which
binds and illuminates it was again incubated followed by washing. A substrate solution was
added which acts on the bound enzyme to induce a colored reaction product. The intensity of
this colored product is directly proportional to the concentration of TNF alpha present in the
samples. And finally absorbance was measured with the help of ELISA reader.
3.4.12 ESTIMATION OF VITAMIN-E
The estimation of vitamin E assessed in blood sample through following method that was
well-established like Emmerie-Engel reaction as reported by Rosenberg in 1992. The method is
followed the reduction method and cause the red colour after the convertion of ferrous from
ferric. The absorbtion of vitamin E was measured at 460nm. About 1.5ml of test sample, abour
standard 1.5ml and 1.5ml of water were pipetted out separately. Abter this step 1.5 ml of ethano
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
37
and xylene were added in all the tubes and centrifuged after the well mixing. Than take the 1.5
ml solution from each well one by one to estimate the levels of vitamin E at 640nm. Although
Ferric chloride solution was added to each well in same quantity, after the addition of ferric
chloride the color of solution is change into red color due to reduction of ferric into ferrous than
after 15 min the take absorbance of each well solution at 520nm with the help of
spectrophotometry method.
3.4.13 DETERMINATION OF VITAMIN A
Rutkowski et al., (2006) explained a unique method for the determination of vitamin A.
1ml of liquid was taken in test tube I with tight stopper and 1ml of KOH solution and it was
then shaken vigorously for 1 minute and heated in tube in a water bath (60oC, 20 minutes),
then it was cooled under cold water and 1 ml of xylene was added, tube was plugged and
shaken vigorously again and absorbance was measured at 335nm against xylene-irradiate the
extract in the test tube II to the UV light for 30 minutes.
3.4.14 DETERMINATION OF INTERLEUKIN-6 “IL-6” LEVELS
Levels of interleukins i.e., IL-6 in serum was evaluated by the help of commercially
available ELISA kits. 0.03 pg/ml is the minimum measureable levels respective method to
callcolate IL6 that is universal cytokine and released due to the inflamtion by any type of
endogenous and exogenous infactions. For the calculation of ILs levels we will face the very
attention for the preparation of specific ranging of standard concention like 0 to 200 pg/mL.
With the help of pipeting each dilution of Aliquots (100 μl) were added in prepared assay
plate. Afterword these prepared solutions were stand in incubation for 2 hours at room
temperature. After passing the incubation time it will be washed with eight times with the
help of washing buffer by specific type of washer called BioTek's ELx50™ Strip. After each
step of washing it will be inverted at specific absorbing paper to eliminate the extra fluids
from kit for taking significant reading through ELISA reader. All the wells are consisting100
μl of solution. After washing process HRP conjugate and antibody solution were added and
incubated at roome temperature for 60 minutes. After the incubation process the ELISA kits
were washed by previous method for eight type. After incubation 150 μL of Sword
substrate/peroxidase mixture were added in the wells of ELISA palate and than it stand at
room temperature at dark light for 15 mintues. 150 μL of development media was finally
added in the wells of ELISA palate and than it stand at room temperature at dark light for half
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
38
hour. 100 μL of TMB substrate were added in wells and were incubated for 30 minutes. After
the incubation the 50 μL of stop solution were added to stop the reaction for the taking of
accurate result through ELISA kit reader with specific principle of ELISA reader.
3.4.15 ESTIMATION OF C-REACTIVE PROTEIN
For the determination of CRP, reagent is used determination employs turbid metric
method by using Synchron® ELIZA Kit. In the reaction, C-reactive protein are allowed to
combine with the specific antibody to form insoluble antigen-antibody complexes. The
employed immune complexes causes increased scattering of light which is determined
proportional to the concentration of CRP in serum. The scattering of light is measured by
taking its absorbance at 570nm. The CRP concentration is determined from a calibration
curve developed from CRP standards of known concentration.
3.4.16 DETERMINATION OF GPx
GPx can be estimated by the help of spectrophotometer. Sodium azide solution was
taken. GPx solution 0.1 along with glutathione was added into the NADPH vial. It was then
mixed by inversion and its pH was maintained upto 7.0 at 25oC with 1M HCl. Finally vial
was added with the 48mM sodium phosphate, 0.38mM ethylene diamine-tetra acetic acid,
0.95mM sodium azide, 0.12mM NADPH, 3.2 units of GPx and 0.075 to 0.15 units of GSH.
After 5 minutes absorbance was measured at 340nm (Aebi and Bergmeyer, 1983).
3.4.17 DETERMINATION OF TOTAL CHOLESTEROL (TCh)
For the estimation of total cholesterol in the serum sample following protocol was
followed reagent and samples were taken in respective amounts reagent at 1 ml and sample
10µl similarly standards were added separately. The mentioned contents were then mixed
together and incubated for ten minutes at the temperature of 37 degree centigrade and finally
absorbance was measured at 505nm and results were expressed in units (mg/dl).
3.4.18 DETERMINATION OF TRIGLYCERIDES (TG)
For the evaluation of triglycerides in the serum sample in the very first step TG gets
hydrolyzed by an enzyme named lipase which converts TG into glycerol and fatty acids. For
the purpose three tubes were taken and were named as blank, calibrator and assay tube. They
were then added with buffer and sample at the rate of 300 and 3µl contents within these tube
were allowed to mix and then stand for ten minutes at 37 degree centigrade and then their
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
39
absorbance was measured at 546nm by the help of spectrophotometer against the blank and
results were expressed in units (mg/dl).
3.4.19 ESTIMATION OF LOW DENSITY LIPOPROTEIN (LDL)
Low density lipoproteins (LDL) were estimated in the serum samples of preterm infants
according to the principle LDL gets hydrolyzed with to fatty acids and free cholesterol and
then oxidized to (CHOD) cholesterol oxidase. Hydrogen peroxide gets reacted with the PAP
and ultimately gives rise to the purple colored compound. Intensity of the color directly
signifies the concentration of LDL in the sample. And finally with the formula of Friedwald’s
levels of LDL were determined.
3.4.20 DETERMINATION OF HIGH DENSITY LIPOPROTEINS (HDL)
High density lipoproteins were determined with the help of special assay known as
HDL-cholesterol assay. First mixture of polyanions and polymers were formed and they were
allowed to attach with the surface of vLDL, chylomicrons and lipodystrophy (LD). These
complexes are may be considered as lipoprotein in nature. pH of the reaction was maintained
with the help of detergent (Reagent II) but HDL remains unstable and separated.
3.4.21 ESTIMATION OF TOTAL BILIRUBIN
Total bilirubin was determined in the serum sample by the commercially available kit.
For the estimation diazotized sulfanilic acid was reacted with bilirubin ti produce a compound
named azobilirubin and then its absorbance was measured at 550nm. The measured
absorption remained directly related with the concentration of total bilirubin in the sample
which was measured in the units (mg/dl). Bilirubin reagent and nitrite reagent were added and
mixed with samples in the well. Titration was done with the help of pipetting up and down for
four to five times. It was then incubated at room temperature for five minutes and at last
absorbance was measured at 550nm.
3.4.22 DETERMINATION OF MYELOPEROXIDASE (MPO)
Standard and samples were prepared by diluting in standard diluents. 100 µl of assay
diluent RD1-27 was added to each well. 50 µl of sample and standard were added in each well
and covered with adhesive strips provided incubated for 2 hours at room temperature. Each
well was aspirated and washed for 4 times with wash buffer. 200 µl of human MPO
Conjugate was added to each well and covered with new adhesive strip and incubated for 2
hours at room temperature. 50 µl of stop solution was added to each well (color change from
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
40
blue to yellow). Wavelength was recorded at 450nm (Klebanoff, 2005). 50 µl of dilutions of
samples and standard were added into appropriate wells. 50 µl of detection reagent A was
added immediately in each well. Plate was shaken using microplate shaker and covered with
plate sealer and incubated at 37Oc for 1 hour. Wash with 350 µl of 1X wash solution and let it
sit for 1-2 minutes. 100 µl of detection reagent B working solution was added to each well.
Incubate it for 30 minutes at 37°c after covering then washed it for 5 times. Then 90 µl of
substrate solution was added to each well and covered with a plate sealer. Incubated for 10-20
minutes at 37Oc. 50 µl of stop solution was added and solution turned yellow. Absorbance
was recorded at 450nm.
3.4.23 ESTIMATION OF SODIUM AND POTASSIUM
For the estimation of sodium and potassium mixture of MgCl2, NaCl, KCl and Tris-HCl
was assayed together in respective amounts 5, 80, 20 and 40mM respectively whereas, pH was
maintained upto 7.4 total volume was made upto 200µl. The reaction was later initiated by
addition of ATP and concentration remained 3.0mM. Similarly controls were processed. At last
activity was estimated by the difference in both assays.
3.4.24 DETERMINATION OF NEUTROPHILS
For the determination of neutrophils whole blood from the patients were obtained and
stored in EDTA vial and were analyzed on the automatic hematological analyzer (Sysmex 3000)
3.4.25 DETERMINATION OF MAGNESIUM AND CALCIUM
The estimation of Mg and Ca was done by their respective protocols explained by Lin
and Way, (1984). Medium of assay was set to about 1ml which contain 50mM of imidazole-HCl
buffer (pH 7.5) after all the additions mixture was incubated for fifteen minutes at the
temperature of 37 degrees n then stop solution was added into it which is ice cold ten percent
TCA. In following the activity of enzymes Mg+2 ATPase and Ca+2 ATPase were determined to
estimate levels of calcium and magnesium.
3.4.26 DETERMINATION OF COMPLETE BLOOD COUNT (CBC) IN THE SERUM
Complete Blood Count includes the determination/estimation of red blood cells,
hemoglobin, values of hematocrit, determination of white blood cells in term to define the
infectious status of patients and platelet count. Estimation of (CBC) was done with the help of
hematology analyzer by the help of automated Coulter (LH780) by Beckman Coulter, Inc.
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
41
3.4.27 ESTIMATION OF SERUM FOLATE (VITAMIN B9) AND VITAMIN B12
Serum folate (folic acid) and vitamin B12 was determined in the serum preterm infants
by their respective protocols that employs the use of HPLC (high performance liquid
chromatography) for the purpose standard and working solutions were prepared and the required
pH and volume was maintained and after the HPLC they were measured for their respective
absorptions for vitamin B9 wavelength was set to (200nm-290nm) while for vitamin B12 was
(210nm-260nm).
3.4.28 DETERMINATION OF TOTAL IRON BINDING CAPACITY
Total iron binding capacity was measured with the help of calorimeter method that
involves two reagents sequentially. First one is acidic reagent that contains iron-binding dye and
excess iron. While the second involved reagent is responsible for increasing pH and restoring the
transferrin affinity for iron. Transferrin is responsible for the extraction of iron from dye-iron
complex and it becomes 100% saturated while the remainder iron stays intact with the dye.
Hence in the particular way decrease in the absorbance of that color dye is then measured with
the help of spectrophotometer by measuring its absorbance at 660nm and this is proportional to
the total iron binding capacity of the serum sample.
3.4.29 DETERMINATION OF RENAL FUNCTION TESTS (RFTs)
Renal function test includes the determination of creatinine, Uric acid, and levels of
electrolytes with the serum sample of preterm infants for the sake of analysis of kidney and its
function. Urinalysis was done which comprise on the investigation of color, blood and turbidity
or any other visual appearance of the urine. Estimation of glomerular filtration rate (GFR), level
of urea and creatinine from serum, for the purpose three to four samples of each subject were
selected and considered for the analysis. Creatinine is the waste material that is given out under
the normal action of muscles in the body these samples are then thoroughly examined under the
microscope for the physical appearances as mentioned up and the levels are then revealed in the
following results.
3.4.30 ESTIMATION OF VITAMIN D (Vit D) IN THE SERUM OF PRETERM
INFANTS
Vitamin D was determined in the serum of preterm infants with the help of commercially
available ELISA kits by (Cayman chemicals USA).
CHAPTER FOUR RESULTS MISBAH, 2017
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RESULTS
CHAPTER FOUR RESULTS MISBAH, 2017
43
4.0 RESULTS
4.1 LEVEL OF HEMOLYTIC CELLS IN PRETERM INFANTS (PTI)
The infants were categorized in different niches such as resuscitation, smoking, gender,
sepsis, mode of delivery, blood transfusion etc Table 02, shows that preterm infants with
significantly decreased levels of RBCs when were compared with control group. Mean of red
blood cells (RBCs) in preterm infants was recorded as (4.35±0.27 s/L) where as in the healthy
group the value remained (4.65±0.15 s/L, p-value=0.013). The levels of white blood cells in
preterm infants ware significantly increased as compare to controle group such as (10.04±1.78)
and (7.57±0.41, p-value=0.042) respectivity shown in table 02. The mean value of platelets in
preterm infants was recorded as (16.34±1.72) but in control group the value remained (307.
±5.27) and it show very signifent results where the p value was (0.035). Table 02 shows that
preterm infants executed significantly decreased levels of Hct when were compared with control
group. Mean of hematocrit (Hct) in preterm infants remained as (35.34±1.67) significantly
decreased as compared to controls (41.29±1.59, p-value=0.012). Neutrophils were significantly
decreased in the preterm infants recorded as (3.35±0.43) as compared to (4.65±0.58, p-
value=0.025) shown in table 02.
Data in Table 02 and regarding hematological biochemical markers reticulocytes, MCH,
MCHC, rHuEPO, serum folate, hemoglobin, sferritin, serum MPO and SFe show highly
prominent results as compare to negative control group because the P<0.05. The consistent
increasing trend in reticulocytes levels (8.35±1.55) were recorded in preterm infants as compared
to control (4.64±0.93) where the p value (0.042). The consistent decreasing trends in case of
MCH, MCHC and increased the value of s-ferritin from groups of preterm infants were recoded
(3.24±0.06), (18.41±3.48) and (6.95±1.79) as compared to healthy group such as (114.87±4.20),
(31.99±2.93) and (1.95±1.05) respectively. The level of rHuEPO and serum folate ware also
shows the consistent decreasing trend (5.45±0.79) and (23.34±2.57) in preterm infants as
campare to normal group such as (7.62±0.83) and (34.63±2.17) respectively. Where the p value
also p<0.05. Table 02 shows that preterm infants executed significantly increased levels of serum
MPO when were compared with control group. Mean of serum MPO in preterm infants was
recorded as (30.84±2.35) where as in the healthy group the value remained (4.81±2.03) where p
value was (0.042). The levels of hemoglobin and sFe in preterm infants ware significantly
decreased (30.84±2.35), (20.56±1.35) as compare to healthy group likewise (14.11±0.87) and
CHAPTER FOUR RESULTS MISBAH, 2017
44
(85.28±3.26) respectivity. Where the p value was (0.039 and 0.040) that is show in table 02.
Levels of TIBC is significantly (p=0.012) decreased in subjects (32.82±4.03) that is the symptom
of hemolysis/anemia as compared to the control (42.23±5.63). There was a significant decrease
(p=0.018) in the level of STFR has been observed in infected (8.77±1.26) as compared to control
(13.21±1.66). ZPP is the significant biomarker of hematolgic profile, our study resulted in the
significant increased (p=0.032) in the level of ZPP in subjects (2.37±1.24) as compared to
normal controls (0.00024±0.000094).
4.2 LEVEL OF RFT and LFT IN PRETERM INFANTS (PTI)
Table 02 shows that preterm infants executed significantly increased levels of uric acid
when were compared with control group. Mean of uric acid in preterm infants was recorded as
(5.37±1.18) where as in the healthy group the value remained (3.85±0.85) where p value was
(0.037). The value of creatinine in preterm infants was recorded as (1.60±0.27) but in control
group the value remained (0.72±0.04) and it show very significant decreased results in pateint
group as compare to healthy group, where the p value was (0.026). The levels of blood urea
nitrogen (BUN) in preterm infants ware significantly increased as compare to controle group
such as (25.59±5.32) and (14.67±2.17) respectivity. Where the p value was (0.035) that is show
in table 02. Furthermore, the levels of other RFT and LFT were increased in the preterm infants
in which including TBILI (3.31±2.64), total protein (6.45±0.81), GGT (53.74±7.98) and hsCRP
(1.40±0.32) as compare to normal group like (0.89±0.36), (6.43±0.29), (43.12±6.82) and
(1.04±0.02) respectively. Where the p value were <0.05.
4.3 LEVEL OF LIPID PROFILE IN PRETERM INFANTS (PTI)
The results that were depicted in Table 02, the levels of lipid profile including
triglycerides (Tg), cholesterol (TCh), high density lipoprotein (HDL) and low density lipoprotein
(LDL) were significantly differ with negative control groups (P=0.038, 0.012, 0.046, .017
respectively). the level of HDL and LDL have the negative correlation with to each other
because if the levels of LDL were increased in this response the levels of HDL automatically
decreased so LDL and triglycerides are also called the bad cholesterol but the higher levels of
HDL were most beneficial effects for human health so it also called good cholesterol. An
increasing trend of TCh (5.32±0.72), Tg (2.53±0.40), LDL (2.96±0.76) was observed in preterm
infants as compared to health group like (4.39±0.32), (1.30±0.13) and (2.29±1.18) respectivitly.
Show in table 02 the mean value of HDL in preterm infants was recorded as (1.37±0.16) but in
CHAPTER FOUR RESULTS MISBAH, 2017
45
control group the value remained (1.69±0.16) and it show very significant increased results in
pateint group as compare to healthy group, where the p value was (0.042).
4.4 LEVEL OF STRESS BIOCHEMICAL MARKERS IN PRETERM INFANTS (PTI)
According to the result of present study (table 02) the levels of oxidative biomarkers were
altered due to the oxidative stress. The major prominent biomarkers including SOD (Superoxide
dismutase), GSHpx (glutathione peroxidase), MDA (Malondialdehyde), GSH (Glutathione),
CAT (Catalase), GSHpr (glutathione reductase) and NO (nitrogen oxide) shows highly
significant outcomes after comparing with negative control group. The consistent increasing
trend in MDA levels (4.07±1.08) were recorded in preterm infants as compared to control
(1.43±0.36) where the p value (0.025). The consistent decreasing trends in case of Glutathione,
GSHpx and increased the value of GSHpr from groups of preterm infants were recoded
(3.35±1.37), (6.64±0.59) and (3.49±1.58) as compared to healthy group such as ( 9.79±1.22),
(8.22±0.69) and (1.47±0.22) respectively. The level of Catalase and SOD ware also shows the
consistent decreasing trend (1.75±1.14) and (0.101±0.004) in preterm infants as campare to
normal group such as (3.9±0.81) and (0.49±0.14) respectively. Where the p value also p<0.05.
The lowest value (0.06 ng/ml) of SOD was recorded in group B (diabetic patients). Table 02
shows that preterm infants executed significantly increased levels of NO when were compared
with control group. Mean of NO in preterm infants was recorded as (58.05±10.385) where as in
the healthy group the value remained (19.82±1.42) where p value was (0.020).
4.5 LEVEL OF DIFFERERENT TYPES OF VITAMINS (B12, A, C, E AND D) IN
PRETERM INFANTS (PTI)
Data in Table 02 and regarding different types of vitamins such as vit B12, vit A,
vit C, vit E and vit D has significant altered their normal pattern after combine with negative
control group. The consistent trend in vit A levels (82.42±4.58) as compared to health group
such as (44.52±6.15). The levels of vitamin A were shows the decreased trends, where it p value
was (0.031). Table 02 shows that preterm infants executed significantly decreased levels of vit
B12 and vitamin C when were compared with control group. Mean of vit B12 and vitamin C in
preterm infants was recorded as (48.49±2.56) and (0.36±0.11) where as in the healthy group the
value remained (58.31±6.65) and (0.56±0.08) respectively. Where the p value ware (0.034 and
0.041 respectivily). Show in table 02 the mean value of vitamin E and D in preterm infants was
recorded as (0.23±0.07) and (9.72±1.25) but in control group the value remained (0.29±0.05) and
CHAPTER FOUR RESULTS MISBAH, 2017
46
(13.22±0.81) respectively. It show very significant decreased results in pateint group as compare
to healthy group, where the p value was (0.011 and 0.029 respectivily).
4.6 EFFECT OF PHYSICAL MARKERS ON PRETERM INFANTS (PTI)
The data presented in table 02 shows that variables of physical marker such as body
weight and age differed significantly (≤0.05). The higher levels of body weight (1.47±0.27) and
age (28.07±3.36) were recorded in preterm infants as compared to normal healthy controls group
such as (2.92±0.33) and (37.65±1.70) respectively.
4.7 LEVEL OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) IN PRETERM
INFANTS (PTI)
Table 02 shows that preterm infants executed significantly decreased the levels of G6PD
when were compared with control group. Mean of phosphate in preterm infants was recorded as
(3.05±2.89) where as in the healthy group the value remained (4.13±4.06) where p value was
(0.0k33).
4.8 LEVEL OF ELECTROLYTES (HCO3, Ca, Na, K, Mg, CL-, Zn, Cu AND P) IN
PRETERM INFANTS (PTI)
Data in Table 02 and regarding stress biochemical markers bicarbonate (HCO3-), sodium
(Na), zinc (Zn), magnesium (Mg), calcium (Ca), chaloride (Cl), cupper (Cu), phosphate (P) and
potassium (K) shows highly significant results as compare to standard group. The levels of
biocarbonate (21.94±3.80) were consistent reduced in preterm infants as compared to control
(28.55±1.63) where the p value (0.015). Table 02 shows that preterm infants executed
significantly decreased levels of Ca nd Cu when were compared with control group. Mean of Ca
and Cu in preterm infants was recorded as (9.03±0.71) and (0.92±0.37) where as in the healthy
group the value remained (9.69±0.32) and (1.60±0.08) respectively. Where p value ware (0.021
and 0.027 respectivily). The levels of Mg in preterm infants ware significantly decreased
(1.53±0.32) as compare to controle group such as (1.71±0.17). Where the p value was (0.029)
that is show in table 02. The consistent increasing trends in case of Na, K and Cl from groups of
preterm infants were recoded (5.28±0.36), (5.92±1.03) and (90.59±6.18) as compared to healthy
group such as (4.37±0.34), (4.04±0.48) and (1.03±1.31) respectively. Where the p value were
(p<0.05). The level of Zn was also shows the consistent increasing trend (0.28±0.08) in preterm
CHAPTER FOUR RESULTS MISBAH, 2017
47
infants as campare to normal group such as (0.016±0.06). The p value also recorded (0.012).
Table 02 shows that preterm infants executed significantly increased the levels of P when were
compared with control group. Mean of phosphate in preterm infants was recorded as (5.75±0.72)
where as in the healthy group the value remained (3.92±0.58) where p value was (0.047).
4.9 LEVEL OF INFLAMMATORY BIOMARKERS IN PRETERM INFANTS (PTI)
The levels of inflammatory biomarkers are also disturbed in the preterm infants due to the
oxidative stress and some other complication. The levels of inflammatory biomarkers ware
increased in preterm infants including IL6 (6.80±0.78) and TNFα (31.63±4.42) versus the
normal healthy group like (5.64±0.51) and (29.90±1.14) respectively. Where the p value were
(0.043 and 0.038 respectively). Table 02 depicts that there was an increased level of AOPP in
subjects (1.36±0.40, p=0.040) that is significantly higher than controls (0.84±0.04). Present study
also resulted in the higher level AGE in infected subjects (2.73±0.20) as compared to normal
controls (2.56±0.10). Where the p value was (0.036).
TABLE 02: VARIABLES OF MEDICAL IMPORTANCE IN THE PRETERM INFANTS (PTI) SUFFERING FROM ANEMIA VARIABLES CONTROL (n=100) PATIENTS (n=171) P˂(.05) RBCs/L 4.65±0.15 4.35±0.27 0.013 WBCs/L 7.57±0.41 10.04±1.78 0.042 PLTs/L 307.83±5.27 16.34±1.72 0.035 Hct% 41.29±1.59 35.34±1.67 0.012 Neutrophil Counts/L 4.65±0.58 3.35±0.43 0.025 ReticulocytesK/mm3 4.64±0.93 8.35±1.55 0.042 MCHpg 114.87±4.2 3.24±0.06 0.022 MCHCg/L 31.99±2.93 18.41±3.48 0.021 rHuEPO IU/L 7.62±0.83 5.45±0.79 0.041 Serum Folate 34.63±2.17 23.34±2.57 0.029 Hbg/L 14.11±0.87 9.90±1.82 0.039 SFerritin ng/ml 1.95±1.05 66.95±1.79 0.028 Serum MPO 4.81±2.03 30.84±2.35 0.042 SFe ng/ml 85.28±3.26 20.56±1.35 0.040 UricA mg/dL 3.85±0.85 5.37±1.18 0.037 Creatinine mg/dL 0.72±0.04 1.60±0.27 0.026 BUN mg/dL 14.67±2.17 25.59±5.35 0.035 TBILI mg/dL 0.89±0.36 3.31±2.64 0.021 TotalP g/L 6.43±0.29 6.45±0.81 0.019 GGT U/L 43.12±6.82 53.74±7.98 0.016 hs-CRP mg/L 1.04±0.02 1.40±0.32 0.042 TCH mg/dl 4.39±0.32 5.32±0.72 0.038 Tg mg/dl 1.30±0.13 2.53±0.40 0.012 LDL mg/dl 2.29±0.18 2.96±0.76 0.046 HDL mg/dl 1.69±0.14 1.37±0.16 0.017 MDA nmol/ml 1.43±0.36 4.07±1.08 0.025
CHAPTER FOUR RESULTS MISBAH, 2017
48
SOD µg/ml 0.49±0.14 0.101±0.004 0.001 GSH µg/ml 9.79±1.22 3.35±1.37 0.008 CAT nmol/mol 3.95±0.81 1.75±1.14 0.017 IL-6 pg/ml 5.64±0.51 6.80±0.78 0.043 TNF-α pg/ml 29.9±1.14 31.63±4.42 0.038 AOPPs 0.84±0.04 1.36±0.30 0.040 AGEs 2.56±0.10 2.73±0.20 0.036 TIBC mcg/dl 42.23±5.63 32.82±4.03 0.012 STfR 13.21±1.66 8.77±1.26 0.018 ZPP µmol/mol 0.00024±0.000094 2.37±1.24 0.032 GSHPx µg/ml 8.22±0.69 6.64±0.59 0.027 GSHPr µg/ml 1.47±0.22 3.49±1.58 0.033 NO µmol/L 19.42±1.42 58.05±10.38 0.020 BWeight kg 2.92±0.33 1.47±0.27 0.028 Age year 37.65±1.70 28.07±3.36 0.042 VitB12 mg/g 58.31±6.65 48.49±2.56 0.034 VitA mg/g 44.52±6.15 82.42±4.58 0.031 VitC mg/g 0.56±0.08 0.36±0.11 0.041 VitE mg/g 0.29±0.05 0.23±0.07 0.011 VitD mg/g 13.22±0.81 9.72±1.25 0.029 HCO3 mmol/L 28.55±1.63 21.94±3.80 0.015 Ca mmol/L 9.67±0.32 9.03±0.71 0.021 Na mmol/L 4.37±0.34 5.28±0.36 0.037 K mmol/L 4.04±0.48 5.92±1.03 0.039 Mg mmol/L 1.71±0.17 1.53±0.32 0.044 Cl mmol/L 1.03±1.31 90.59±6.18 0.041 Zn mmol/L 0.16±0.06 0.28±0.08 0.012 Cu mmol/L 1.60±0.08 0.92±0.37 0.027 G6PD IU/L 4.13±4.06 3.05±2.89 0.033 P mmol/L 3.97±0.58 5.75±0.72 0.047
CHAPTER FIVE DISCUSSION MISBAH, 2017
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DISCUSSION
CHAPTER FIVE DISCUSSION MISBAH, 2017
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5.0 DISCUSSION
Status of oxidative stress was reviewed in preterm infants suffering from anemia.
Different antioxidative enzymes namely, glutathione peroxidase (GSH-Px), superoxide
dismutase (SOD), reduced glutathione (GSH), malondialdehyde-hemoglobin (MDA-Hb),
catalase (CAT), and biomarkers viz., IL-8, IL-6 and TNF-alpha including many other blood
factors are responsible for the preterm labor and anemia. Pregnancy is normally a time frame of
felicity and anticipation, but due to many reasons it can contribute in the preterm delivery. About
12% of total pregnancies are thought to have increased risk for the premature birth but with
proper knowledge risks of preterm can be decreased. In normal cases pregnancy usually last for
about 40 weeks but in the cases of pretermies labor may be commenced before the completion of
37 weeks due to physical and biochemical reasons. Uterine contraction opens the cervix that
increases the chanes for preterm delivery. Other risk factors for the development of preterm birth
includes high blood pressure, excessive use of alcohol, are reason for certain cervical or uterine
abnormalities, high blood pressure, smoking, use of alcohol, drugs etc. Anemia is characterized
as reduced numbers of red blood cells (RBCs), hematocrit and/or hemoglobin concentration.
Reasons of anemia may include increase loss of erythrocyte or inadequate production of red
blood cells. At gestational period of 2 weeks, erythropoiesis may occur in the yolk sac that
generates cell expresses embryonic hemoglobin.
At gestational period of 6 weeks, red blood cells production initiates in liver that later
expresses fetal hemoglobin. At 6 months of gestation, bone marrow becomes the site of
hematopoiesis. During fetal life, size of erythrocytes decreases and number increases, 30-40% of
hematocrit increases on onset of second trimester. In later gestation RBCs shift from fetal to
adult hemoglobin production under the influence of increased oxygen concentration. Red blood
cell counts continue to decrease unless delivery of oxygen is inadequate and production of
erythropoietin is induced again. Preterm babies, after birth also go through reduction in
concentration of hemoglobin. After birth, level of hemoglobin in preterm infants declines
gradually and progressively in the two to three months of age. Anemia of prematurity is due to
lower level of hemoglobin at birth, reduced lifespan of red blood cells and a suboptimal response
of erythropoietin. This anemia of prematurity possibly exaggerated through factors that include
blood sampling frequently for laboratory tests, or through substantial clinical signs and
symptoms. Oxidative stress production is generally in two ways. First way is when reactive
CHAPTER FIVE DISCUSSION MISBAH, 2017
51
molecules are created by endogenous metabolites and second way is when environmental
exogenous reactive contents get into biological system. Cell physiology changes because of these
two ways. The instability between generation and excretion of reactive oxygen species is known
as “oxidative stress”. Reactive oxygen species are unstable type of molecules having oxygen that
can react easily. Reactive oxygen species can damage RNA, DNA and lipid proteins, and even
cell death can occur. The reactive oxygen species (ROS) synthesized during metabolic pathways
commonly are superoxide anion, hydroxyl radicals, hydrogen peroxide and singlet oxygen Many
studies revealed that reactive oxygen species are generated from mitochondrial oxidative
phosphorylation (Trachootham et al., 2009).
Throughout oxidative phosphorylation, O2 is utilized and reduced into water and
approximately 1% oxygen changes to superoxide anion (O2 −). Cellular organs are damaged
through reactive oxygen species (ROS) molecules. Reactive oxygen species affects lipids,
proteins and DNA molecules. Reactive oxygen species effects mainly mitochondrial DNA as it
dont have the ability to repair like nuclear DNA. Thus, function of mitochondrial DNA is lost
due to repeated hurts. Defense system of antioxidant is generated by living cell which also
protects cellular membranes, nucleic acids and proteins. These may include compounds viz
lipoic acid, coenzyme, glutathione and enzymes etc. Enzymes viz catalase, glutathione
peroxidase, glutathione reductase and superoxide dismutase protects against oxidative stress.
Lipid peroxides are released through unsaturated lipid membrane components that undergo
oxidative damage. Studies revealed that isoprostanes are produced when arachidonic acid is
injured by oxidative stress. DNA guanosine is oxidized to 8-hydroxydeoxyguanosine (8OHdG),
that is closely related to neuronal oxidative stress. (Pandey and Rizvi, 2010 b).
Oxidative stress damages the erythrocyte membrane and also causes hemoglobin
oxidation. During aerobic respiration different oxidants come through endogenous and
exogenous means, which altogether known as called Reactive Oxygen Species (ROS). The
endogenous source of ROS includes mitochondrial respiratory chain, resistance reactions,
enzymes like nitric oxide synthase, transition metal interceded oxidation and xanthine oxidase.
(Lykkesfeldt, 2007). Erythrocytes have such enzymes which limits ROS effect. The main cause
of oxidative stress is the superoxide of ROS present in the cells. Red blood cells are mainly
responsible for transport of oxygen and carbon dioxide between tissues and lungs. Antioxidant
protection is provided to erythrocytes through anti-oxidative system and red blood cells also
CHAPTER FIVE DISCUSSION MISBAH, 2017
52
move freely which facilitate other organs and cells of the body. (Siems et al., 2000). As red
blood cells have high O2 and hemoglobin concentration, hence oxidative stress chances increases
(Bryszewska et al., 1995). As RBCs lack mitochondria, so in mature red blood cells formation of
adenosine triphosphate takes place through glycolysis. Various studies proved that normally 90%
glucose produces ATP through glycolysis it was proved by different studies that in normal
conditions about 90% of glucose produced ATP by glycolysis as remaining 10% get into hexose
monophosphate shunt. In tissues glutathione is reduced via HMP shunt by using NADPH
pathway (Sivilotti, 2004). In vivo various studies explained that high level of oxidative stress
negatively affects red blood cells parameters which includes deactivation of enzymes and
membrane bound receptors (Halliwell and Gutteridge, 2007), high glutathione (GSH), lipids and
proteins oxidation (Pandey and Rizvi, 2009; Pandey and Rizvi, 2010a).
Red blood cells membrane has increase concentration of polyunsaturated fatty acids that
can oxidize easily. The cell membrane of red blood cells has two fields viz: lipid bilayer and
cytoskeleton. In the bilayer, phospholipids are spread asymmetrically while in entire lipid
domain cholesterol is dispersed (Bryszewska et al., 1995). Lipids are considered vital for shape
protection of red blood cells. For normal and regular functioning, fluidity is very essential for
plasma membrane of erthrocytes. Reactive oxygen species assail membrane and via lipid
peroxidation various unnecessary products are formed. (Pandey and Rizvi, 2010b).
Malondialdehyde (MDA) is the universal oxidative stress marker and it secreated in the blood by
the process of lipid peroxidation. Pryor and Stanley (1975) introduced that MDA is produced as
product of decomposition through lipids oxidation and the mechanism is believed to involve
prostaglandins formation, just like from PUFA, endo-peroxides are formed with two or more
double bonds. Alkaline phosphatase (ALP) enzyme is present in each cell of the body. The major
concentration of ALP is present in bone, liver and bile ducts. Alkaline phosphatase test is done
for the diagnosis of liver or bone problem. As liver damages, cells of liver releases ALP in
concentration more than normal in the blood. Alkaline phosphatase test is done to diagnose any
bile duct blockage. With the increase in bone tissues activities, ALP level also increases.
Aspartate aminotransferase (AST) enzyme is present in heart, muscle cells and liver. Aspartate
aminotransferase was formally known as glutamic oxaloacetic transaminase. Aspartate
aminotransferase level indicates stage of liver or other organ damage. Through the level of AST,
it can be diagnosed that jaundice is due to the disorder of liver or blood (Nyblom et al., 2006).
CHAPTER FIVE DISCUSSION MISBAH, 2017
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Bilirubin test is done to measure the bilirubin level in the blood. Bilirubin, a yellow
pigment, is present in bile which is formed through liver. Premature babies have to face number
of complications. Hyperbilirubinemia is one of the common problems of infants.
Hyperbilirubinemia condition is such in which babies have elevated bilirubin level, which is
produced by normal red blood cells breakdown. Elevated level of bilirubin contributes to
jaundice. Extremely raised bilirubin level causes damage to brain, thus preterm infants are
rapidly treated for jaundice before bilirubin attains life-threatening level (Liu et al., 2008).
Albumin formation is through liver cells and it’s continues circulation in the blood gives balance
to the fluid of the body. Albumin is one of the blood plasma protein and also play role as a
carrier in the body. Albumin level can be altered due to any liver abnormality. Serum albumin
test is helpful in diagnosis of many problems like hepatic disorders, deficiency of nutrition,
chronic pancreatitis and renal disease (Suzuki et al., 2006). Catalase is an enzyme, which act as
catalyst. Catalase enzyme through catalyzes reaction decomposes hydrogen peroxide into oxygen
and water. It is present in those organisms which can survive in presence of oxygen. Catalase
plays its role to prevent from accumulation and cellular tissues and organelles are protected from
peroxide damage that is produced continuously through various metabolic reactions. Glutathione
reductase is an enzyme which acts as catalyst. Glutathione reductase also regulates the reduction
of glutathione (GSSG) that exists in the oxidized form and reduced to glutathione (GSH).
Glutathione reductase is substantive for redox cycle of glutathione through which reduced
cellular glutathione (GSH) level is maintained.
Glutathione (GSH) have the ability to reduce the levels of oxidative stress by reacting
with organic peroxides and free radicals, it can also act as a substrate for glutathione S-
transferases and glutathione peroxidases in organic peroxides detoxification and xenobiotics
metabolism (Deponte, 2013). Glutathione transferase (GST) or glutathione S-transferases
enzymes are mainly present in the cytosol. Glutathione transferase has different activities and is
involve in various reactions. Glutathione transferase plays an important role in transformation of
compounds like cancerous products, therapeutic drugs and also produces compounds against
oxidative stress (Tew et al., 2011). Adenosine triphosphatase (ATP-ase) enzyme acts as catalyst
and decomposes ATP into ADP and releases free phosphate ion. Energy releases during
dephosphorylation reaction. Calcium use to control metabolic activities and enzymes of various
CHAPTER FIVE DISCUSSION MISBAH, 2017
54
cells. Calcium ions regulate soluble enzymes activities in the liver. Calcium ATPase enzyme is
P-ATPase form which after contraction of muscles transfer the calcium (Jensen et al., 2004). In
the fetus, the main origin of Epo is liver macrophage (Falke et al., 1987). There comes a point in
between the gestational age of third trimester and first month, where Epo produces from
peritubular cells inside the kidney, instead of liver macrophage and reason of this alteration in
still unknown. Either Epo reduces through kidney and/or liver, there is high demand of
hypoxemia for Epo to be produce. Epo production is regulated by physiological or non-
physiological stimulants through increase gene expression of Epo (Bondurant and Koury, 1986
and Schuster and badiavas, 1989). Rich (Rich, 1986), found out that Epo could be produced
through macrophages of bone marrow. Researchers influenced that macrophages were reactive to
alterations inside oxygen tension. They also contended that macrophages beneath firm condition
can play its role to regulate erythropoiesis.
Preterm infants fail to produce Epo significantly on anemia contempt an evident demand
towards oxygenation of tissue (Bifano et al., 1992). Still, it is not known whether premature
babies rely on production of Epo through liver / kidney and/or both. This study resulted that
production of Epo mRNA and proteins seem to be integral in macrophages isolated of premature
infants cord blood. Production of Epo from macrophages of preterm infants is as like term
infants, it brings doubt that defective production of Epo through macrophages is responsible for
anemia of prematurity. To understand the molecular level of premature anemia, studies proposed
to determine regulation from gene expression of Epo in the development of peritubular renal
cells. Erythropoietin (Epo) is the cytokine that play its role in stimulation of erythropoiesis, and
is available since 1985 for clinical usage and studies. Strauss et al, (1995) established the criteria
that describe the hematocrits in infants suffering with respiratory problem, it indicated the need
of blood transfusion is necessary in such infants also receiving Epo. They concluded, it would be
wrong to say that tried medicines reduces blood transfusions rather critical health condition of
the patients make it more frequent to be blood transfuse. In 1996 Bechensteen et al,(1996)
reported that patients whether receive Epo or not, it does not affect the length, weight and head
perimeter of the infants, while the reason of this observation in unknown. It is clear that through
increase erythropoiesis stimulation, need of protein and vitamin also increases, as protein and
vitamin are not supplied through diet by preterm infants. In 1990 Shannon et al, described that
treatment with Epo caused weight gain more faster in patients while most researches did not
CHAPTER FIVE DISCUSSION MISBAH, 2017
55
shown any significant difference in weight gain in between the treated and non-treated groups.
Vera et al drew in 2001 that treatment with EPO increased the level of hematocrits significantly.
They also proposed that count of reticulocytes increases with the treatment, which proves that
preterm babies have the ability to react with exogenic EPO through increase erythropoiesis,
Although interruption may come from endogenic EPO production, which may results from either
raise hemoglobin or absence of hemoglobin reduction which is require for stimulation of Epo.
Similar report was given by Bechesteen et al, (1993) that treatment affects the reticulocyte count.
At any stage of life, erythropoiesis depends mostly on enough nutrition, particularly
intake of iron and protein, deficiencies of iron and protein are significant determining factors
towards erythropoiesis (Brown and Howard, 1996). For newborns nutritional adequacy is
important due to their fast growth (Doyle, 1997). It is necessary to adjust intake of protein in
accordance with neonatal growth, however enough quantity of protein for erythropoiesis
stimulation during treatment of Epo is not yet defined (Brown and Howard, 1996). Authors
described that erythropoiesis enhances as intake of protein increases, as Epo dosage decreases.
Preterm babies induce lower iron stores because of reduce gestational age and decrease intake of
iron. Epo usage increases deficiency of iron in this means so supplementation of iron becomes
important during treatment of Epo. Presently, it has been confirmed that necessary amount of
iron supplemented for effective treatment of Epo should be no more less than 6mg/kg/day
(Donato et al., 1996 and Soubasi et al., 1995). Studies have been conducted on administration of
oxygen at resuscitation through variation in titration schemes of oxygen within the first ten
minutes after delivery. (Escrig et al., 2008). Kumar et al, 2014 modified this to fix the oxygen
concentration at resuscitation. The study was designed to fine the unbalance of oxidative stress
markers and how exposure to oxygen affects it at the time of birth. In 2005 according to NRP
(Neonatal Resuscitation Programme) preterm infants were resuscitated in between 21% to 100%
of O2 and objective saturation was not defined for the initial ten minutes. All preterm infants
with 100% of O2 received positive-pressure ventilation (PPV) which shows that ventilation or
hypoxia was responsible for intubation instead of sustained hyperoxia or apnea. Results depicted
that saturated oxygen had higher values as compared with the neonatal resuscitation program
upper limit, thus 100% of O2 concentration is not right for resuscitation of preterm infants
(guidelines of 2010) at the time of birth. Saturated oxygen does not bring any significant change
in between 21% and /or 40% of O2.
CHAPTER FIVE DISCUSSION MISBAH, 2017
56
Preterm infants are suggested to wean at 21% of O2 through six minutes of lifetime.
Gestational age could be the significant factor which influenced resuscitation of O2 at the time of
birth, as extremely preterm infants less than 26 weeks of gestation need approximately oxygen at
the initial stage. Gestation of 28 weeks might get away oxygen for SpO2 (oxygen saturations)
maintenance within the defined range. The issue; exposure of oxygen following through titration
frequently to maintain range of SpO2 at resuscitation have been addressed by many studies. In
case of 100% O2 initial titration concentration was observed more effective than still
concentration in premature infants in maintaining targeted SpO2. (85-92). However, treatment
fails with 21% of O2 at resuscitation. (Rabi et al., 2011). (Kumar et al., 2014) In a study it was
indicated that 100% of O2 at resuscitation had higher oxidative glutathione (GSSG) than reduced
glutathione (GSH), at one month of age. In preterm infants having asphyxia from modest to
control had high total hydroperoxide while redox potential was lowered in resuscitation of 100%
O2 which indicated that is inspired concentration of oxygen is lowered it can help to reduce
oxidative stress (Ezaki et al., 2009). Preterm infants at 30% O2 resuscitation can have reduction
in oxidative stress (Vento et al., 2009). Plasma nitrotyrosine (NT) significantly increases in 40%
and 100% O2 resuscitation. In the plasma, nitrated proteins indicate inflammation and/or form
peroxynitrite from superoxide and nitric oxide (Ischiropoulos, 1998). In an analysis, preterm
infants had high plasma nitrotyrosine (NT) in case of bronchopulmonary dysplasia (BPD) while
NT was found lower infants without BPD (Banks et al., 1998).
At birth, polycythemia of preterm infants is well distinguished along the rapidly
decreasing values of hemogram in the first 8 months of age. After birth, total count of heme
declines (Sisson et al., 1959) and iron gained is maintained in different tissues by baby (Langley,
1951) As the formation of blood rate exceeds the destruction rate, mass of hemoglobin raises and
iron tissue storage re-utilizes (Seip and Halvorsen, 1956). Schulman et al, (1954) show that
preterm infants from different weights retrieve the mass of hemoglobin present at 3 to 4 months
of initial life and at this state preterm infants had relatively excess iron. Preterm infants reveal
minimum quantity iron of non-hemoglobin in the bone marrow at the time of birth, number
increases from 1st to 2nd month and amount disappear by the 3rd month. At birth, if tissue iron
stores reutilized for the formation of novel hemoglobin, it would not anticipate that preterm
infants response to administrated iron until and unless mass of hemoglobin in the circulation gets
equivalent. However, different researches proposed that early anemia ameliorated in preterm
CHAPTER FIVE DISCUSSION MISBAH, 2017
57
infants give oral iron preparations from 1st week of age and few studies show even more speedy
response. (Tuttle and Etteldorf, 1952; Sisson and Whalen, 1958 ). Gaisford and Jennison (1955)
intramuscularly fed iron to ten preterm infants on the third week of life. This group, near 16
weeks of age showed high hemoglobin concentration as compared with the control group.
Preterm infants when parenterally fed iron on the first four weeks of age, concentration of
hemoglobin, counts of hematocrits and erythrocytes rapidly decreases. Same results were find
out from the group without iron supplementation, rather the group supplemented with iron
rapidly recovered from early anemia and also hemoglobin concentration increased on the 3rd
month of age. This proves that at the time preterm infants had raised tissue iron stores, even then
iron administered exogenically accelerate the range of formation of hemoglobin.
Schulman (1958) depicted that erythropoietic resumption time is not linked with
maturity, preterm infant’s birth weight but is associated with initial hemoglobin concentration. In
this study, preterm infants with corresponding birth weight along decreases concentration of
hemoglobin took up erythropoiesis before. The alteration in concentration of hemoglobin was
about 7 or 8mg/100ml at the time of birth and remained for more than four weeks of life.
Gardner et al, (1955) established relationship among activity of erythroid in the bone marrow
and hemoglobin concentration of preterm infants. This study described that occurrence of
erythropoiesis takes place after the level reduces beneath 11gm/100 ml without depending on the
concentration of hemoglobin or birth weight. Seip and Halvorsen (1956), observed that preterm
infants with below 1500gm birth weight had occurred earlier reticulocytosis comparable with the
preterm infants of birth weight 1500gm to 2000gm. This research aimed to associate the
erythropoietic intensity and time response towards the overall preterm infants rate of
development. Denman and Arlene (1960), studied the hemoglobin concentration and found no
significant difference in the first 8 weeks of life. Only the initial deviation in the mass of
hemoglobin was 6gm in preterm infants who had 2000gm birth weight. The range and mean of
birth weight and growth rate was quite same. Both groups resumed erythropoeisis at the same
time of age, the time when concentration of hemoglobin and mass of hemoglobin were nearly
identical. Thus, preterm infant’s hematological behavior differences are not explicated through
any evident underlying deviations among the studied group.
CHAPTER FIVE DISCUSSION MISBAH, 2017
58
The evident consequence of iron supplemented during early anemia of preterm infants is
not explained well. Just a suggestion not establishment that erythropoietic stimulation might be
due to exogenic irons (Josephs, 1953). It could be possible that raised iron readily accelerate
and/or stimulate formation of hemoglobin through action of mass. Shoden and Sturgeon (1959)
point out that formulations of iron-dextran parenterally fed incline to deposit at sites of storage as
ferritin instead of hemosiderin. Heilmeyer and Granick (1958, 1954) proposed that physiological
form of iron is ferritin and is available promptly for body rather than iron of hemosiderin. In this
study the observed effects could be due to these deviations. In preterm infants, iron deficiency
anemia might be the result of various influences. Iron storage of preterm infants might be
influenced by frank parental anemia and /or impaired parental iron. (Sisson and Lund, 1958).
Sturgeon (1959) suggested that no detectable change produce during the period of non-anemic
pregnant mothers by parental supplementation of iron in hemotoligical profile of preterm infants
during the initial 1½ year of life. In the study by Denman and Arlene 1960, no possible change
was seen in between the groups because of parental iron deficiency anemia. Results showed that
one mother had concentration of hemoglobin beneath 9.7gm/100ml while four had under
11gm/100ml. None of the mother effected of erythrocytes microcytosis or hypochromic anemia.
Parental iron storage differences potentially evaluated by measuring iron binding capacity, iron
binding protein saturation, serum iron and free protoporphyrin of erythrocyte. Such findings
indicated an improved total nutritional iron in termed women (Holy, 1953) as compared with the
women with oral supplementation of iron during the time of pregnancy (Sturgein, 1959). Both
groups had good nutritional iron and so no variation was observed that may influence the iron
storage of preterm infants. Preterm infants in this research were properly nourished. To observe
the mothers nutrition, they were also registered. Questionary emphasized on the diet and weight
of preterm infants. Though uner observation preterm infants had no medicative iron after 2-3
months of age they started taking iron riched cereals. The recorded diet indicated that preterm
infants of both groups were identical while feeding. At birth, preterm infants had minimum iron
storage and iron in the mass of circulatory hemoglobin estimates the total available iron for the
formation of hemoglobin. (Langley, 1951). At birth, the most significant determinant is the size
of the mass of hemoglobin for the need of exogenic iron inorder to compete the development
requirement in late infancy. (Sisson et al., 1959).
CHAPTER FIVE DISCUSSION MISBAH, 2017
59
Differences actually in gestational age, weight at birth, weight gain and hemoglobin
concentration were little and so are not likely substantial. if these factors had influence then that
would in the form of nutritional iron towards the control group. Contemporary, experimental
subjects had increased hemoglobin concentration below 12 weeks of age and high level
continues to maintain onwards from the 3rd month of life. In the experimental preterm infants
circulating mass of hemoglobin exceeds than that from control group by 10 weeks of age, and all
through the whole study duration. The reason could be the administration of iron to the
experimental preterm infants. Parenterally iron was administered to assure the consistent and
measurable amount. Research was not dependent on the iron form utilized and/ or administration
route of iron. This study showed how preterm infants absorb compounds of iron easily when
administered orally. (Oettinger et al., 1954). As hemoglobin concentration and absorption of iron
are interlinked instead of iron storage (Hahn et al., 1943), it is expected that preterm infants
would easily absorb oral administration of iron during the period of early anemia. Generally,
course of hematology in the control group which was fed with diet rich of iron underlined that
not necessarily preterm infants become anemic if super nutrition are given. This data reveals that
preterm infants by the age of six weeks take up hematopoiesis and as soon as possible
supplementation of exogenic iron provides benefit to them. On should not wait for iron storage to
be utilized.
Henny et al, 2016 conducted a study on iron profile based on the level of hemoglobin,
transferrin saturation and serum ferritin. Two markers of iron in combination i.e. transferrin
saturation and zinc protoporphyrin ware used for iron deficiency diagnosis, as these markers
have the ability to detect dysregulation of supplemental iron in erythropoiesis before the
occurrence of anemia (Rao and Georgieff, 2009). Serum ferritin is the protein of acute phase
which during any inflammation or infection increases. This study excluded infected patients and
diagnosis of infection was examined clinically and through laboratory tests. C-reactive protein,
the marker of acute infection was also not assesst. Indonesians have recommended
supplementation of iron as standard for preterm infants. Negative iron balance is found in
preterm infants as their need is increases with the rapid development. This usually occurs at 8
weeks of life. At this time period, erythrocytes production along mass of muscles and also
enzymes need more iron for consumption. (Hammond and Murphy, 1960 and Ziegler et al.,
2011). A Cochrane analysis described that preterm infants who were supplemented with iron had
CHAPTER FIVE DISCUSSION MISBAH, 2017
60
higher level of hemoglobin than the preterm infants without supplementation of iron at 6-9
months of age. (Mills and Davies, 2012).
Iron level is lower in breast milk as compared with the formula milk which had high
amount of iron. However, the gut of preterm infants can easily assimilate the iron of mother feed.
A case study reported that preterm infants who were breastfed solely and partially showed a
significant difference of 4.1g/dL in the level of hemoglobin among subjects at 6-8 weeks of age.
(Mills and Davies, 2012). In preterm infants of gestational age less than 36 weeks iron deficiency
is linked with low weight at birth, abrupt duration of fort1ified formula fed and weight gain
initially at 1½ month of life. (Rao et al., 2002). However, it is difficult to figure out how much
iron is needed to prevent preterm infants from iron deficiency. Few studies report that high
amount of iron is needed from formula milk as compared with the mother feed to reduce iron
deficiency but still it’s doubtful that this will prevent preterm infants from iron deficiency.
Preterm infants fed with human milk might have protein mal nutrition.
Preterm infants had deficiency of vitamin E due to hemolytic anemia (Oski and Barness,
1967). This study reports that preterm infants would not have this deficiency if vitamin E is
supplemented with soluble water and the whole study showed normal vitamin E concentration of
plasma. Likewise, in preterm infants no deficiency of folic acid, vitamin C and/or vitamin B12
was found. Hemolytic anemia might be due to low superoxide dismutase along glutathione
peroxidase activity within the erythrocytes. It is indicated that selenium and copper could be
responsible for activity decline in such enzymes. Rudolf et al (1981) reported that hemoglobin
concentration had no correlation with values of selenium RBCs or activity of glutathione
peroxidase in the preterm infants with very low birth weight. Ronnholm and Simes, (1985)
design the study which resulted that selenium intake and RBC selenium concentration
subsequently depend on intake of protein. Therefore, correlation found among the intakes of both
with concentration of hemoglobin is not storming. The level of ceruloplasmin and albumin of
plasma concentration decreases in preterm infants with very low birthweight rather than the full
term normal infants (Raiha et al., 1976). In copper transport chain both ceruloplasmin and
albumin are seem to be active. Albumin transports ingested copper towards liver while
ceruloplasmin is involved in donating copper too extrahepatic tissues (Evans, 1973). Haga et al,
CHAPTER FIVE DISCUSSION MISBAH, 2017
61
(1981) suggested neither activity of superoxide dismutase nor ceruloplasmin play any role in
anemia of prematurity.
CHAPTER FIVE
FIGURE 01consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the mother and fetoplacental unit. of 20-37 weeinclude reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emoof events are involved such as activation of fetal HPA axis (
CHAPTER FIVE
FIGURE 01: Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the mother and fetoplacental unit.
37 weeks which cause the complication approximately 7include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emoof events are involved such as activation of fetal HPA axis (
Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the mother and fetoplacental unit. Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration
ks which cause the complication approximately 7include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emoof events are involved such as activation of fetal HPA axis (
Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the
Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration ks which cause the complication approximately 7
include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic predisposition, environmental and emotional factors. For the initiation of labor, there are various endocrine cascade of events are involved such as activation of fetal HPA axis (
62
Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the
Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration ks which cause the complication approximately 7
include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic tional factors. For the initiation of labor, there are various endocrine cascade
of events are involved such as activation of fetal HPA axis (
Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the
Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration ks which cause the complication approximately 7-10% of all delieveries.
include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic tional factors. For the initiation of labor, there are various endocrine cascade
of events are involved such as activation of fetal HPA axis (hypothalamic
Labour is the normal mechanism in which the fetus is ejected form the uterus.consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the
Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration 10% of all delieveries. Major factors of preterm birth
include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic tional factors. For the initiation of labor, there are various endocrine cascade
hypothalamic-pituitary-adrenal
DISCUSSION MISBAH, 2017
Labour is the normal mechanism in which the fetus is ejected form the uterus. This process requires the consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother athe end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the
Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration Major factors of preterm birth
include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic tional factors. For the initiation of labor, there are various endocrine cascade
adrenal axis). This cascade of
DISCUSSION MISBAH, 2017
This process requires the consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the process of labor, there are several changes occur in the concentrations of circulating hormone of mother and fetus at the end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the
Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration Major factors of preterm birth
include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic tional factors. For the initiation of labor, there are various endocrine cascade
). This cascade of
DISCUSSION MISBAH, 2017
This process requires the consistently painful uterine contractions to enhance the duration, intensity, frequency in the cervical dilatation. In the
nd fetus at the end of pregnancy. However, the human parturition is due to the fluctuation in biochemical events between the
Preterm birth (PTB) is decricbed as the delievey of mother wintin the the time duration Major factors of preterm birth
include reproductive tract infection, behavioral factor, obstetrical complications, high and low body mass, genetic tional factors. For the initiation of labor, there are various endocrine cascade
). This cascade of
CHAPTER FIVE DISCUSSION MISBAH, 2017
63
event is responsible to enhance the levels of estrogen and diminish the concentrations of progesterone. The steroid hormones have important role in the stimulation and activation of myometrial cell by increasing the synthesis of prostaglandin and Oxytocin hormone. Stimulatory event of prostaglandin raise the expression of gap junction protein as well as prostaglandin F receptor and Oxytocin receptor. The expression of contraction associated protein is tightly regulated by estrogen positively, whereas negatively regulated by progesterone hormone. The contraction associated proteins (CAPs) are also expressed on the uterus to regulate the concentrations of Cyclooxygenase, Lipoxygenase and Oxytocin endopeptidase. CRH (Corticotropin releasing hormone) which is the peptide hormone secreted from the hypothalamus that is present on the chorionic and placental membrane as well as decidual and amnionic cells. Corticotropin releasing hormone has the capability to stimulate the secretion of ACTH and cortisol synthesis. In the negative feedback loop, the mother pituitary secrete cortisol hormone which hampers the activity of hypothalamic Corticotropin releasing hormone (CRH). While in positive feedback loop, CRH further involved in the activation of maternal and fetal HPA axis. After the activation of fetal HPA axis, it further triggers to increase the secretion of ACTH (Adrenocorticotropin hormone) which leads toward the release of DHEAS (Dehydroepiandrostenedionsulfate) which is actually the precursor of steroid hormone. DHEAS is basically expressed on the zone of fetal adrenal gland of fetus and mother as well. Dehydroepiandrostenedionsulfate is than further metabolize into Estrone, estradiol and estriol. CRH has also the capability to enhance the production of prostaglandin in decidual, chorionic and amnionic cells. This increase level of prostaglandin has crucial role in parturition. Corticotropin releasing hormone (CRH) has some other actions such as in the contraction of smooth muscle cells and uterine vessels dilation. Moreover the deficiency of 15-OH-PGDH enzyme triggers to enhance the degradation of prostaglandin which is responsible to enhance the levels of PGE2 and cause myometrial contractions. Moreover, genital infections have also important role in the synthesis of inflammatory cytokines. During infections, macrophages and granulocytes are synthesized in the myometrial cell which further release proteases such as matrix metalloproteinases, Lipoxygenase and Cyclooxygenase as inflammatory cytokines including IL-6, IL-8 and TNF-α. Cytokines have the capability to increase the production of prostaglandin that causes contraction of myometrial membrane, preterm prelabour rupture of membrane and cervical ripening. On the other hand, genital infections trigger to reduce the enzyme levels of 15-OH-PDGH, results in preterm labour. Due to oxidative stress and poor nutritional diet in pregnant mother, elevated levels of free radicals and inflammatory cytokines are produced. Furthermore, the disruption in mitochondrial electron transport chain of fatty acid and prostaglandin synthesis as well as ischemic and hypoxic condition have major role in the formation of reactive oxygen species (ROS). Stimulation of macrophages, monocytes and neutrophils are also contributed in the synthesis of free radicals. On the other hand, reactive nitrogen species (RNS) are implicated in the damaging of cellular DNA by disrupting nitric signaling (NO) that cause vasoconstriction. The decrease blood flow in the uterus and placental wall triggers to cause pre-clampsia, preterm labour and PPROM (Preterm prelabour rupture of membrane). Poor nutritional diet of mother such as lack of vitamins, iron and folic acid is also one of the major factors for the progression of reactive oxygen species and reactive nitrogen species. Due to infection or inflammation, macrophages, neutrophils and granulocytes secrete various inflammatory cytokines that are responsible to stimulate NADPH oxidase enzyme which is basically ROS generating enzyme. This enzyme is the main contributor to decline the formation of anti-oxidants and increase the synthesis of free radicals that cause lipid peroxidation by MDA and Isoprostane. MDA forms an adduct in the nucleus and trigger the DNA damaging, stands breakage and chromosomal abnormalities. Oxytocin is a non-peptide hormone which has peripheral and central actions during pathological and physiological conditions including lactation, parturition, ejaculation, erectile dysfunction and material behavior. It has secreted from paraventricular nuclei of hypothalamus and also synthesized via peripheral tissues such as lining of uterus (uterine epithelium), amnion, placenta and corpus luteum. The Oxytocin hormone which is formed by maternal and fetal stress has major effect on myometrium. The Oxytocin binds with its specific receptor OTR (Oxytocin receptor), it belongs to the family of G-protein coupled receptor family. In the uterus, the alpha subunit of Oxytocin receptor Gα/1i couple with PLC-β (Phospholipase C-β) and triggers the hydrolysis of PIP2 (Phosphoinositide-bis-phosphate) into DAG (Diacylglycerol) and IP3 (Inositol-tris-phosphate). IP3 is implicated in the mobilization of Ca2+ in SR (Sarcoplasmic reticulum) whereas DAG has involved in the regulation of PKC (Protein kinase C). The Ca2+ is released from Sarcoplasmic reticulum and form Ca2+ dependent Calmodulin complex that further stimulate MLCK (Myosin light chain kinase). The phosphorylation of myosin chain by myosin light chain kinase enzyme triggers the contraction of myometrium wall. VOCCs (Voltage-operated Ca2+ channels) which are also known as L-type Ca2+ channels that trigger the entry of Ca2+ ions in the myometrial cell and important for impulsive activity. These channels are open during the mechanism of action potentials and consequently open the channels for the Ca2+ entry. The contraction in smooth muscles of uterus builds upon the balance between Myosin light chain phosphates (MLCP) and myosin light chain kinase (MLCK). The phosphorylation of myosin chain has the capability to cause contraction in the smooth muscle cells in myometrium. Whenever Oxytocin binds to its specific receptor, it activates Rho proteins and triggers
CHAPTER FIVE DISCUSSION MISBAH, 2017
64
the interaction between actin and myosin and cause the Ca2+ sensitization. In myometrial cell, the Rho protein is stimulated by the activation of alpha subunit of Oxytocin receptor that further activates ROCK. It is basically Rho kinases which contribute in the inhibition of myosin light chain phosphatase (MLCP) and consequently cause myometrium contraction and lead to preterm labour. Moreover, Rho kinase and protein kinase C enzymes are stimulated by DAG (Diacylglycerol) that activate CP1-17 (C-kinase-activated protein phosphatase-1 inhibitor) and influence MLCP-P. Since, the Ca2+ sensitization by the Oxytocin receptor metabolism to cause smooth muscle cells contraction of myometrium is still debatable. In the body system, iron level is regulated by hepcidin which is bascially peptide hormone and formed in the liver than released into the blood circulation. The levels of hepcidin enhanced due to the response of inflammation/infection and iron concentrations. While its levels declined due to the result of erythropoiesis. The basic purpose of Hepcidin is to regulate iron homeostasis via interacting with exporter protein (Ferroportin). This transmembrane exporter protein is located on the membrane of duodenal Enterocytes as well as on the surface of RE (Reticuloendothelial) macrophages of liver, bone marrow and spleen. In mother, low iron intake in diet and excessive bleeding during delivery are responsible to cause infection and inflammation which trigger macrophages activation and secretes interleukins and tumor necrosis factor. These inflammatory cytokines are the causative factor to enhance the levels of Hepcidin in the liver. The Hepcidin released into the blood circulation and hampers iron absorption in the small intestine of fetus. It has also the capability to bind with Ferroportin receptor and cause its degradation. The increase level of Hepcidin is involved to decrease iron absorption in liver, whereas inflammation trigger to reduce red blood cells survival and cause bone marrow suppression. On the other hand, the preterm babies have immature red blood cells due to preterm delivery. These babies have increased breakdown of red blood cells and decrease formation of blood cells which further lead towards low erythropoietin response and resultantly anemia is occur.
CHAPTER SIX SUMMARY MISBAH, 2017
65
SUMMARY
CHAPTER SIX SUMMARY MISBAH, 2017
66
6.0 SUMMARY
Almost 12% of babies born are known as premature babies as they were born before the
thirty seventh week of pregnancy such babies are known to have underdeveloped organs and are
subjected to multiple problems that comprise of issues related to their digestion or respiration,
additionally, they are found to have problems in their brain development and growth. Such
babies do not have fully developed lungs which is not able to produce sufficient ‘surfactant’ this
complication is termed as respiratory distress syndrome. Especially preterm babies who may
suffer any infection at the time of birth usually have low blood pressure and hence they may be
supplemented by the appropriate medication. In digestive system they may not have fully
developed intestines so they are provided with very low quantity of feed which might be as low
as one teaspoon. Breast milk is best for the new born babies especially the premature babies,
such babied fed on breast milk are on less chance to develop any infection or serious intestinal
complications and such babies have encountered to have improved growth. Coming on to the
vessels blood vessels of premature babies are more fragile and are subjected to rupture easily.
Bleeding problem in such babies may be from minor to severe. Premature babies are kept under
the oxygen tanks because of their uncontrolled oxygen pressure and they are also aided with IV
nutrition for the deficiencies. Causes beyond the preterm birth are common out of which one
common maternal cause remains preeclampsia, certain kind of infections, abuse of drug,
abnormality in structure of uterus, preterm birth history, placental abruption and premature
rupture of membrane of uterus. Such babies even if they survive they remain prone to infections
and may suffer through medical conditions throughout their life span.
Moreover, preterm infants are at higher risk of diabetes and hypertension. Rate of
premature births are increasing especially in the developed countries under the reason which
includes modern lifestyle. Hence if such preterm baby is born in high socioeconomic status attain
higher rate to lead healthy life as compared to those who born in relative lower socioeconomic
status. During pregnancy iron deficiency counts for a common cause in developing anemia and
increasing chance to have a preterm birth. Therefore sufficient iron is supplemented in common
clinical practice to preterm infants. Umbilical cord is the importents part of placenta that connect
the mother with baby for transpotation of specific nutrients. Typically, umbilical cord are
attached with two clamps and the cord is cut at this clamp. It has been reported that the levels of
CHAPTER SIX SUMMARY MISBAH, 2017
67
Hb secnificantly increased at the time of birth by the two clamps assignment within the 35 to 175
seconds. It has been observed that cord clamping is not associated with the specifically infants or
preterm infants and they have significantly high levels of Hb and due to delayed cord clamping
there is reduce in RBC transfusion. It has been defined by the author that reduce levels of RBC
transfusion leads to induce delayed cord clamping in preterm infants, so the levels of RBC
reduced by the removal of blood that leads to use for various blood tests performed by the blood
analyzer. Clinical trial is obligatory to perform to monitor the device by using 93 preterm infants
having weight less than 1Kg.
Preterm infants fail to produce Epo significantly on anemia contempt an evident demand
towards oxygenation of tissue. Still, it is not known whether premature babies rely on production
of Epo through liver / kidney and/or both. This study resulted that production of Epo mRNA and
proteins seem to be integral in macrophages isolated of premature infants cord blood. Production
of Epo from macrophages of preterm infants is as like term infants, it brings doubt that defective
production of Epo through macrophages is responsible for anemia of prematurity. To understand
the molecular level of premature anemia, studies proposed to determine regulation from gene
expression of Epo in the development of peritubular renal cells. Erythropoietin (Epo) is the
cytokine that play its role in stimulation of erythropoiesis, and is available since 1985 for clinical
usage and studies. Established the criteria that describes the hematocrits in infants suffering with
respiratory problem, it indicated the need of blood transfusion is necessary in such infants also
receiving Epo.
Studies concluded, it would be wrong to say that tried medicines reduces blood
transfusions rather critical health condition of the patients make it more frequent to be blood
transfuse. In 1996 it was reported that patients whether receive Epo or not, it does not affect the
length, weight and head perimeter of the infants, while the reason of this observation in
unknown. It is clear that through increase erythropoiesis stimulation, need of protein and vitamin
also increases, as protein and vitamin are not supplied through diet by preterm infants. In 1990
scientists described that treatment with Epo caused weight gain more faster in patients while
most researches did not shown any significant difference in weight gain in between the treated
and non-treated groups. Treatment with EPO increased the level of hematocrits significantly.
They also proposed that count of reticulocytes increases with the treatment, which proves that
CHAPTER SIX SUMMARY MISBAH, 2017
68
preterm babies have the ability to react with exogenic EPO through increase erythropoiesis.
Although interruption may come from endogenic EPO production, which may results from either
raise haemoglobin or absence of haemoglobin reduction which is require for stimulation of EPO.
Similar report support that treatment affects the reticulocyte count. Preterm infants are suggested
to wean at 21% of O2 through six minutes of lifetime. Gestational age could be the significant
factor which influenced resuscitation of O2 at the time of birth, as extremely preterm infants less
than 26 weeks of gestation need approximately oxygen at the initial stage. Gestation of 28 weeks
might get away oxygen for SpO2 (oxygen saturations) maintenance within the defined range.
At birth, polycythemia of preterm infants is well distinguished along the rapidly
decreasing values of hemogram in the first 8 months of age. After birth, total count of heme
declines and iron gained is maintained in different tissues by baby. As the formation of blood
rate exceeds the destruction rate, mass of hemoglobin raises and iron tissue storage re-utilizes.
Studies reveal that preterm infants from different weights retrieve the mass of hemoglobin
present at 3 to 4 months of initial life and at this state preterm infants had relatively excess iron.
Preterm infants reveal minimum quantity iron of non-hemoglobin in the bone marrow at the time
of birth. Therefore, correlation found among the intakes of both with concentration of
haemoglobin is not storming. The level of ceruloplasmin and albumin of plasma concentration
decreases in preterm infants with very low birth weight rather than the full term normal infants.
In copper transport chain both ceruloplasmin and albumin are seem to be active. Albumin
transports ingested copper towards liver while ceruloplasmin is involved in donating copper too
extra-hepatic tissues.
CHAPTER SEVEN CONCLUSION MISBAH, 2017
69
CONCLUSION
CHAPTER SEVEN CONCLUSION MISBAH, 2017
70
7.0 CONCLUSION
The current study concludes that imbalance between oxidant and antioxidants in the body
leads to the overproduction of reactive oxygen species and inflammatory cytokines that are
involved in the preterm delivery of babies. These inflammatory cytokines in the number of ways
are involved in the uptake of several proteins and hampers the iron absorption in liver. Moreover,
preterm babies have immature red blood cells due to preterm delivery. So they may execute
increased breakdown of red blood cells and decrease formation of blood cells that further leads
to low erythropoietin response and resultantly anemia may occur.
PART II MISBAH, 2017
71
PART-II
EXPRESSION OF UPSTREAM MATERNAL VARIABLES REGULATING PRETERM
BIRTH
CHAPTER ONE INTRODUCTION MISBAH, 2017
72
INTRODUCTION
CHAPTER ONE INTRODUCTION MISBAH, 2017
73
9.0 INTRODUCTION
Preterm birth is defined as birth of a baby earlier than 37th week of gestational period and
is one of the leading and major causes of mortality and long-term morbidity in perinatal
medicine (Lawn et al., 2006 and Goldberg et al., 2008). Premature babies are classified as per
their gestational age (GA) and they are; Extremely preterm with Gestational Age < 28 weeks,
second is the Severely preterm with Gestational Age between 28-31 weeks, and the third one is
the Moderately preterm births with Gestational Age between 32-33 weeks and the last one is the
Near term with Gestational Age between 34-36 weeks. The incidence of preterm birth is 5-9 %
of all the deliveries in Europe out of which 20% are those infants who are born before the 28th
week of gestational period (Goldberg et al., 2008 and Muglia and Katz, 2010). Over the last few
decades the number of incidence of preterm births has been rising, and the reason of this is the
increase numbers of preterm deliveries for example the earlier intervention in preeclampsia and
increased number of assisted pregnancies. A very major issue is that premature infancy accounts
for 75% of perinatal mortality and about more than half of the long-term morbidity (Lawnet et
al., 2006 and Muglia and Katz, 2010). In 1970s before the widespread use of assisted ventilation,
there were only a few numbers of survivors among the infants which were born extremely
premature and even more infants who were full mature and they died due to respiratory distress
which is cause by absence of pulmonary surfactant therapy (Saigal and Doyle, 2008).
Usage of antenatal corticosteroids may have improved many methods of assisted
ventilation and also it’s introduces the surfactant administration, as survival rates for preterm
born have improved remarkably. Improvement in the survival rate has not been yet escorted by a
low levels in major neonatal diseases occurs. The National Institute of Child Health & Human
Development (NICHD) Neonatal Research Network reported a decreased numbers in the
mortality of the babies who are born at preterm but also reported an increased numbers in some
major morbidity among those infants who are born with a birth weight of 501-750 grams (Stoll et
al., 2010). The deliveries at preterm commonly known to us as Preterm Births (PTD) are defined
as birth occurring at or before 37 weeks of gestational age and are the major reason of perinatal
diseases and often deaths in the United States (Demissie et al., 2001). Despite a jointly arranged
effort to decrease down the incidence level of Preterm Births, there has been a non-stoppable
increased incidence level of PTB in last two decades. In 2005, specifically the incidences of
preterm births were 12.7% as compared to 9.1% which was in 1981 (Goldberg et al., 2008). The
CHAPTER ONE INTRODUCTION MISBAH, 2017
74
increasing level in the incidence of Preterm Birth and the effect of Preterm deliveries on the
infant survival and health, General Surgeon has made the level of preterm births to a low level of
7.6% and it is a very major goal of the initiative Healthy People 2010. Majority of the women’s
who had a preterm delivery doesn’t have any known risk factor of the specific disease.
The best determinants before pregnancy are history prior to a Preterm delivery. Risk
factors other than these during Pre-Pregnancy may include American-African race, more than
one gestation, short sized cervix, and a BMI (Body Mass Index). When a woman gets pregnant,
the presence of fetal fibronectin and intra uterine infection are also the determinants of preterm
delivery. The success rates of intervention in preventing the rates of preterm births are at limited
rates. In a single study, administration of 17α-hydroxyl progesterone caproate in women with a
history of Preterm Birth results in a low level of repeated Preterm delivery ranging from 55% in
the placebo group up to 36% in women which were receiving α-hydroxly progesterone caproate
(Meis et al., 2003). A recent editorial reported that “our limited ability to stop preterm labor once
it has started is even more disheartening than our frustrated efforts to predict it.”(Lockwood,
2002) As soon as the preterm contraction starts, to decrease down the preterm delivery no
effective intervention exists. Even, if the preterm labor has started no specific anti biotic and
tocolytic therapy has been introduced to reduce the effect of preterm birth (Alex Stagnaro-Green,
2009). At the level of hypothalamus and pituitary the concentration of the circulating thyroid
hormones is regulated by a negative feedback system. Thyroid stimulating hormone (TSH) is
responsible for the production of thyroid hormone by the thyroid gland produced by the anterior
pituitary, and it is itself control by the thyroid-releasing hormone (TRH) in the hypothalamus.
For the production of thyroid hormone Iodide is the main element which is required. Thyroid
peroxidase (TPO) is an enzyme which is expressed by the thyroid stimulating hormone (TSH) in
the thyroid gland, and iodine is oxidized by iodide by this enzyme. Subsequently iodine is
arranged in a glycoprotein known as thyroglobulin (Tg) which in turn is responsible for the
formation of thyroid hormones, formation is in a sense that major biologically inactive form
thyroxine (T4) and lower amounts of the biologically active tri-iodothyronine (T3).
In the circulation T3 and T4 are bind to the circulation due to a protein called Thyroxine-
Binding Protein (TBP). The entrances of free T3 and T4 into the target cells can only be happen
by the mean of the thyroid hormone transporters (MCT8, Oatp1c1and MCT10). Thyroid
hormones can be activated (T4 to T3) and can be inactivated (T3 to T4 or T2) in the target cells,
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called as de-iodinates (D1, D2 and D3).Transcription can be modulated by the binding of T3
with the nuclear thyroid receptors (TR-alpha and TR-beta) (Miot et al., 2012). For the
development of neonatal and fetal brain thyroid hormone is very essential, it also helps in other
stages of pregnancy such as development of placental and fetal growth. Various physiological
functions occur to ensure the release of optimal amount of thyroid hormones to maintain a
normal pregnancy and fetal development and this all happens as soon as the pregnancy is
initiated. Human chorionic gonadotropin (hCG) stimulates the thyroid glands in the first
trimester due to its structural resemblance to thyroid stimulating hormone (TSH). This may
results in a temporarily higher concentration of free T4 levels and lower concentrations of TSH
in circulation in the first trimester of pregnancy as compared to outside the pregnancy
(Guillaume et al., 1985 and Glinoer et al., 1993). After this period there comes a phase where
free T4 levels began to drop to a lower concentration levels and serum TSH levels rise to a
normal level in circulation. In early gestational age, estrogen levels also increases, which in turn
stimulates the release of thyroxine-binding globulin (TBG) from the liver. During gestation,
there is an increase in the levels of TBG concentrations; there is a peak in the mid gestational age
and maintained afterwards (Glinoer, 1991).
This causes a rise in the concentrations of T3 and T4 levels in the circulation. Renal
blood flow and glomerular filtration rate also increases and it leads to more iodide removal from
plasma and more loss of iodine (Dworkin et al., 1966). This causes an increase in demand of
release of thyroid hormones from the thyroid glands during pregnancy. In the first trimester, all
the fetuses are totally dependent on mother’s thyroid hormone production and their release. After
the 18th to 20th week of gestational age most of the hormones synthesis starts to happen but in the
case of T4 is detected in serum approximately as soon in 10th week to 12th week of gestation.
Maternal thyroid hormone can move across the placental barrier. The conversion of T4 to rT3 is
carried out by de-iodinate which is present in placenta (Burrow et al., 1994). In reproductive age
disorders of thyroid hormones are very common. The estimation of incidence of thyroid hormone
disorder is to be equal to 2% to 3% (Krassas et al., 2010). Hyperthyroidism and Hypothyroidism
are the two most basic thyroid hormone disorder clinically. Hyperthyroidism is to be detected in
about 0.1% to 0.4% of total pregnancy and it has been connected with the risk factors in
pregnancy which may include pre-eclampsia, miscarriages, intrauterine growth restriction,
placental abruption and preterm birth (Casey et al., 2007; Earl et al., 2010 and Reid et al., 2010).
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The incidence of hypothyroidism is estimated to be 0.3% to 0.5% of total pregnancy (Krassas et
al., 2010). The most common disease which is cause by the hypothyroidism is the Hashimoto’s
disease and it is closely related to the presence of TPO-Ab. Miscarriages, preeclampsia, impaired
psychomotor development and preterm births are some of the major diseases which are closely
associated with hypothyroidism (Haddow et al., 1999 and Reid et al., 2010). The presence of
active thyroid hormones both in fetus and mothers is required for a normal fetal development.
Any process which affects the bio-availability of the free thyroid hormones and intra cellular
interactions of thyroid hormones may cause an abnormal fetal development and its growth
(Kester et al. 2001). In 2006, (Ghafoor et al 2006) assessed the outcomes of pregnancy and
status of TPO antibody in approximately 1500 having normal working thyroid hormone
Pakistani women. For the entire cohort, the prevalence of positive Thyroid Per-Oxidase was
11.2%. Thyroid peroxidase (TPO) positive-antibody women had a remarkable amount of
increase in the levels of Preterm deliveries as compared with the negative-antibody women
(26.8 vs. 8.0%, P < 0.01). In the study, thyroglobulin (TGB) assessment was not performed.
The prevalence of low birth weight in Pakistan is defined as a birth weighing less than
2.5 kg, is 25%. The resemblance in the prevalence of low birth weight and preterm birth was
26.8% and 25%, respectively which encourages the authors to conjure a theory without firm
evidence that Auto-Immune Thyroid Disorder AITD brings about a high incidence of low birth
weight present in Pakistan (Ghafoor et al 2006). There have been remarkable understandings in
the deleterious impacts of thyroid disorders in the last two decades. Specifically, clinical
hypothyroidism has been related with miscarriages in both the first and second trimester (Allan
et al., 2000 and Abalovich et al., 2002). The presence of thyroid antibodies in normal working
thyroid gland women has been linked with increase in numbers of miscarriage in both unselected
women and also in women with a previous history of recurrent (Stagnaro-Green et al., 1990 and
Stagnaro-Green et al., 2004). Although, a low IQ levels of the new born are closely related to the
minor thyroid diseases in mothers (Pop et al., 1999 and Haddow et al., 1999). Finally, in general
population approximately 5% to 10% postpartum thyroidal dysfunction has been reported and
about 25% is reported in those women who were suffering from type I DM (Diabetes Mellitus)
(Alvarez-Marfany et al., 1994 and Stagnaro-Green, 2004). Given the effect of clinical
hypothyroidism and autoimmune thyroid disease (AITD) on multiple features regarding maternal
and fetal health, and the large amounts of Preterm Births across United States, an evaluation of
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relationship between the thyroid hormones and Preterm Birth is indicated. In 1969 an article
made by Jones and Manin, about 13 different studies have shown analysis between the
relationship of different thyroidal disorders and either preterm Delivery or low birth weight.
Together taken, these different studies have reported fascinated results. Now, to express the
effect of hypothyroidism and Auto-Immune Thyroidal Disease on Preterm birth, the current
documents just show a summary of the present literature. A large-scale prospective study of
approximately 25,765 pregnant women describes that clinical hyperthyroidism does not show to
have any type of effect on the occurrence of Preterm Births (Casey et al., 2006).
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REVIEW OF LITERATURE
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10.0 LITERATURE REVIEW
Before the 24th week of gestation, the loss of pregnancy is known as Miscarriage which
has an effect in every one woman out of five who receive pregnancy due to which it has become
the common complication of pregnancy (NHS, 2010). The delivery of babies between the 24th
week to 37th week of gestation is known as Preterm Birth and it has a incidence ratio of about 6-
10% of all the pregnancies (Creasy, 1991).More than 10% off all the pregnancies are the preterm
babies. The most important cause of neonatal mortality is the preterm birth and it is also the
second most major reason of mortality before the age of 5 (after pneumonia) globally. The main
reason for neonatal short term and long morbidity worldwide is the preterm birth. The incidence
range of preterm birth is approximately 5% to 18% with lowest in Northern European countries
and highest in sub-Saharan African countries (Blencowe et al., 2012).Over the last decades, the
ratio of preterm births have raised in the developed as well as the developing countries
worldwide. The possible debate to this phenomena may include broader use of ultrasound-based
estimation of gestational age, improved registration, infertility treatment, increased maternal
age, multiple pregnancies, maternal health conditions (particularly with increasing age), and
changes in obstetric practice with preterm deliveries induced because of fetal or maternal
indication. The highest rate of preterm births has been recorded in moderate (between 32-33
weeks’ gestation) and also in late preterm (between 34-36 weeks’ gestation) pregnancies
(Shapiro-Mendoza et al., 2012).
The preterm birth rates in Norway decreased down to 7.2% in year from 2000-2002 and
there was another decrease in the rates of preterm to 6.5% in year 2009-2011. There have been
large impacts on preterm infants over the last few decades due to the advance care of neonatal
care. Before labor for maternal and fetal indication about only one third of total preterm birth are
medically induced (Zeitlin et al., 2013). Maternal indications include Eclampsia, Pre-Eclampsia,
HELLP syndrome, uterine rupture, maternal disease and a history of previous caesarean
(Gyamfi-Bannerman et al., 2011). Some of the common fetal indications may include IUGR
(Intra-Uterine Growth Retardation), intrauterine infection, oligohydramnion, a poor flow of
blood to umbilical cord, placenta previa, abnormal fetal heart rate and placental abruption
(Gyamfi-Bannerman et al., 2012). Off all the preterm births about two third are the spontaneous
births, and the causes are largely unknown. It is said that the biological pathways are dependent
upon multiple factors including genetic and environmental which includes the fetal and maternal
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genetic as well as environmental factors such as IUF (intrauterine infection), inflammation and
other immunological processes, placental ischemia or hemorrhage and also uterine distension.
Those mothers who are herself preterm deliver their child too early (Wilcox et al., 2008).
There is a high risk for preterm birth for the fetuses who are male (Zeitlin et al.., 2002)
there is a high risk of preterm birth in those child who had a birth defect during their birth
(Rasmussen et al., 2001).Those mothers who are in the Asian, African and Caribbean region
gives birth to child early as compared to those mothers who are in European region (Patel et al.,
2004) and it suggests a wide range of variation in pregnancy length with social status. There are
several social and other characters which are associated with preterm birth. Those mothers who
have a low social status and are less educated and those with young and advanced age are at risk
of delivering early births (Thompson et al., 2006 and Sukhov et al., 2011) Increased risk of
preterm birth may also include psychological and physical factors for example low BMI before
pregnancy and stressful environment, and also may include depression (Hendler et al., 2005;
Grigoriadis et al., 2013; Witt et al., 2014). From the conception to the weeks around the
expected date of delivery the pregnancy continuum is known as FETAL MATURATION. The
disruption of this process is known as preterm birth, and before the organs of the baby are fully
developed they are exposed to the external environment. Those children who are born in early
stages before its normal duration undergoes neonatal complication due to anatomical and
functional immaturity and above all organs such as brains, digestive organs, lungs and heart are
more vulnerable.
As compare to full term birth the babies with preterm birth are more affected to apnea,
ARDS (Acute Respiratory Distress Syndrome), necrotizing enterocolitis, uncontrolled body
temperature, hypoglycemia, intra-cerebral hemorrhage, difficulty in feeding and bacterial
infections (McIntire et al., 2008; Hibbard et al., 2010). With the decrease in gestational age there
is an increase in the neonatal complication such as morbidity and fetal death (Markestad et al.,
2005). Those children who are born preterm are at risk of developing various numbers of
complications in later life, and with the decrease in gestational age there is an increase in long-
term disabilities (Moster et al., 2008; Saigal et al., 2008) There is a high risk in case of preterm
child for cerebral palsy (CP), asthma, vision and hearing deficit, epilepsy, impaired mental
function, behavioral and psychological disorders, and learning disabilities. (Moster et al., 2008;
Crump et al., 2011; Mackay et al., 2013) There is an extremely increased rate of risk of
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developing early chronic obstructive pulmonary disease in those adults who are born in preterm
births (Vollsaeter et al., 2013). Those preterm babies who are born preterm are different to those
babies which are born at full term in functional manner. Those adults who are born preterm
undergo a lower educational status, lower economic status and have fewer children, and are less
often married. However, it is important to note that despite the increased risk of untoward long-
term outcomes, most individuals born preterm are healthy and have a normal level of function
(Moster et al., 2008).
10.1 SELENIUM ROLE IN PRETERM LABOR
Preterm labor which is less than 37 weeks of gestation is one of the primary cause of
mortality and morbidity of mother and as well as to fetal life which occur in approximately 6%
to 7% of all the total pregnancies in developing world and about 25% in the under-developed
countries (Steer,2005). Number of studies has detected the correlation between the selenium and
the preterm births. Few Cross-sectional researches in Holland, India, Iran and Germany reported
that those women delivering preterm births have tend to have lower levels of selenium in their
serum as compared to those women who are delivering full term births (Sievers et al., 2001;
Gathwala et al., 2003; Iranpour et al., 2009 and Rayman et al., 2011). A study done in Poland
also reported about low levels of selenium concentrations in maternal serum and also low levels
of GPx activity in those women who deliver preterm births as compared to those mothers who
delivered at full term. The low GPx activity and reduced concentrations of selenium in the blood
of preterm infants may subsidize them to a higher risk of developing sepsis and other premature
diseases (Dobrzynski et al., 1998). There was a study conducted in United States which include
13 infants which were born preterm and 15 those infants who were born at full term and
postulated that there was a low concentration of selenium in the blood of those infants who were
born preterm as compared to those who were born at full term, however the concentration of
selenium was not significant in the mothers blood (Mask and Lane, 1993).
As predicted, intake of selenium in the diet was about 2 to 3 times higher (96-134 g) in
those target subjects which were reported in the Polish population. The intake of selenium
supplementation shows some variations in different studies. Preterm is the premature rupture of
the membranes before the onset of labor (PPROM) is a primary factor in the initial stages of
preterm births and affects approximately 10% to 12% of all the total pregnancies. At a
worldwide scale, PPROM is strongly related to the maternal and fetal morbidity and mortality
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(Parry and Strauss, 1998 and ACOG, 2007). In the pathophysiology of PPROM there is a vital
role of antioxidants activity and different generations of ROS that has been correlated with
augmentation of collagen degradation and succeeding damage to fetal membrane veracity. With
selenium there has been an underlined association between the active potential through a
prospective, placebo-controlled, double blind, a small RCT in Iran, which randomized 166 primi-
gravid pregnant women in their first trimester to receive a100_g/d selenium until their labor
(Wood et al., 2001 and Wall et al., 2002). There was a significant increase in the concentration of
the serum selenium concentration in the women with dietary supplementations of selenium and a
reduce levels in the occurrence of PPROM (Tara et al., 2010).
10.2 VITAMIN D AND SPONTANEOUS PRETERM BIRTH
Before the completion of 37 week of gestation spontaneous preterm birth (SPB) occurs.
There are number of reasons according to pathophysiological point for SPB for developing the
intra-uterine infection and inflammation. Bacterial vaginosis is one of the major factors in
developing the infection in SPB and vaginosis is the disturbance between the normal vaginal
floral balance with a rapid increased growth of anaerobic bacteria that is the main factor that
secretes the inflammatory cytokines, phospholipase A2 and prostaglandins (Hillier, 1993;
Lamont, 2000 and Allsworth and Peipert, 2007). A study was conducted by Bodnar et al., (2009)
and his study reported that in early pregnancy there is an inverse proportion between the
maternal vitamin D status and the prevalence of bacterial vaginosis. Hensel et al., (2011) uses an
intensive data set of about 523 women and proved that there is a strong correlation between the
25 (OH) D3 insufficiencies (< 75 nmol/l) with bacterial vaginosis between the pregnant women.
A cross-sectional study of American-African adolescents shows a strong association between the
vitamin D deficiency (25(OH) D3< 50 nmol/l) with bacterial vaginosis (Davis et al., 2010). As
vitamin D shows anti-inflammatory and immune-dulatory effects like the production and
regulating the functions of cytokines and de-granulation of neutrophils products which are vital
and appropriate in the prevention of invasion of micro-organisms that may induce the risk of
developing the risk of SP birth (Nizet et al., 2001; Liu et al., 2006 and Chesney, 2010). Many
cells in the immune systems are responsible for the expression of the VDRs and they are
controlled by the vitamin D (Muller et al., 1991). In response to auto-immunity vitamin D tends
to diminish the activation of acquired immune system, and innate immune system is enhanced by
this key hormone.
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It reduces the production of inflammatory cytokines such as IL 1, 6 and TNF that are
involve in the SPB by involving in the cell mediated immunity (Helming et al., 2005; Bouillon et
al., 2005; Liu et al., 2006 and Diaz et al., 2009). Active 1, 25(OH) 2D3 is readily synthesized by
the human decidual cells. Hence, number of studies shows that vitamin D is associated with
innate and as well as acquired immunity responses at maternal-fetal edge crossways gestation
(Diaz et al., 2009 and Liu et al., 2009). By helping the myometrium dormancy vitamin D helps
to reduce the risk of SPB. Myometrium contractility is reliable on calcium secretion in the
muscle and this is controlled by vitamin D (Delorme et al., 1983 and Tribe, 2001). Regarding the
experimental data there is a limited observation available that shows a relationship between
vitamin D and spontaneous preterm birth (SPB). In Canada, a prospective cohort study was
conducted including 221 women and this study does not show any relationship of 25(OH) D3
deficit (< 50 nmol/l) or inadequacy (< 75 nmol/l) with preterm births (Shand et al., 2010) while
there was another observational study which reported that maternal 25(OH) D3 levels of(< 28
nmol/l) at 28th week to 32nd week of gestational period were correlated to 0.7 weeks lesser
gestation in white skin women sample (Morley et al., 2006). Another cohort study with 4,225
women with SPB at ≤34 gestational period shows that the incidence of vitamin D insufficiency
(25 (OH) D3< 50 nmol/l) among those women who deliver preterm births were comparable to
those women who deliver at full term (Baker et al., 2010).
Tanzanian pregnant women with vitamin deficiency (25(OH) D3 levels < 80 nmol/l) were
not associated with an increased level of developing preterm births (Mehta et al., 2010). Bodnar
et al. (2008) conducted a cohort study of 82,213 pregnant women with single live births and
found out that seasonal sunlight exposure and vitamin D are significant to preterm births. The
incidence of spontaneous preterm births among those women who conceive in summer season
and fall season were comparatively lower as compared to those women who were conceiving in
winters and spring season where the chances of developing the spontaneous preterm births were
higher (Bodnar et al., 2007). In the near future, there may be a study to reported the strongest
evidence regarding that vitamin D sufficiency may protect from developing preterm births and
this study will be reported by Hollis and his colleagues on their work on approximately 600
white, black, and Hispanic pregnant women and by giving external supplementation of 400 IU,
2000 IU, and 4000 IU/d of vitamin D3 respectively during early gestational age (Hollis and
Wagner, 2009).
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10.3 ROLE OF THYROID HORMONE IN PRETERM DELIVERY: ANATOMY AND
PHYSIOLOGY OF THYROID GLAND
Thyroid gland, a bi-lobed gland shaped as a “Butterfly”, is to be considered as one of the
main and largest glands of the endocrine glands whose weight is about 15-20 grams in a normal
adult that depend upon body weight, iodine, age and gestational status (Sari et al., 2003).
Thyroid gland is situated anteriorly to trachea just below the sternal notch. Right and left both
the lobes are approximately 4 cm in length from top to bottom and are wrapped around trachea,
they are attach superiorly to trachea and inferiorly to the larynx. The blood to the thyroid gland is
mostly supplied by the superior thyroid artery which is the branch of external carotid and also
from the inferior thyroid artery which is a branch of thyroid-cervical trunk of the sub-clavian
artery bilaterally and usually there is a fifth artery known as the thyroid eaima from which the
arch of the aorta enters the midline. From the blood ejected by the heart during cardiac output
thyroid gland blood supply covers up a total of 2% of total blood ejected none the less thyroid
gland accounts only 0.4% of total body weight. Inferiorly the blood drains into the
brachiocephalic vein and the internal jugular vein. The growth of thyroid gland is potentially
larger in amount and the term used for an enlarged gland is known as “GOITER”. The main
constituent of the thyroid glands are the follicular and the para- follicular cells which are also
known as the C-Cells of the thyroid glands. The functional unit for thyroid synthesis and storage
of the thyroid gland are the thyroid follicles (Conn et al., 2005). The follicular cells of the
thyroid glands produces certain follicles which vary in sizes, then thyroid hormones is secreted
from these follicles and then they are stored in the lumen.
The lumens have many homogenous colloids present in them known as the
THYROGLOBULIN (TG) and many other proteins also such as the ALBUMIN. C-cells are
present in between the follicles that secrete calcitonin in return to an increase level of calcium.
Calcitonin act directly on bone by preventing its reabsorption and hence lowering the level of
calcium in blood circulation. The first ever gland to be discovered in the 24th week of gestation is
the thyroid hormone (Trueba et al., 2005). Its formation is from the mesodermal layer of the
epithelial cells over median surface of the pharyngeal floor which is developing and it arises in
the first pharyngeal arch. From the neural crest cells the formation of C-cells occurs and from
here they are migrated towards the ultimo-brachial body which later on fuses with the thyroid
gland. Congenital hypothyroidism occurs when the above mentioned process fails to occur and
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thyroid hormone does not produced. The effects of congenital hypothyroidism is approximately
1 in 4000 newly born babies, thyroid dysgenesis is one of the cause (85%) while the rest 15% is
due to other causes including thyroid hormone synthesis (Trueba et al., 2005). Thyroid
dysgenesis occurs at irregular intervals having only 2% chances of family history (Castanet et
al., 2001).
The site for thyroid hormones synthesis is the thyroid gland. In the thyroid gland the
iodide is actively taken into from the diet by the help of sodium-iodide pump and is readily
converted to iodine. Pre-Thyroglobulin is formed by the coupling effect of tyrosine. In response
to binding with the thyroid stimulating hormone, thyroid hormones are secreted in the
circulation. Thyroid stimulating hormone (TSH) is a trans-membrane receptor that is linked to a
second messenger system G-protein (Hennemann and Visser.1997). As compared to the active
T3 more in-active T4 is secreted into the blood stream. In-active T4, by the help of peripheral
tissues is converted into the active T3 in the circulation. More than 99% of all the total T3 and
T4 in plasma are bounded to hormone binding protein and for cellular uptake only the unbound
form thyroid hormone is available (Friesema. 2001). Thyroid stimulating hormone (TSH) is the
key that regulates the above mentioned process and it is secreted by the pituitary gland. The
pituitary gland is stimulated by Thyroid Releasing Hormone which is situated in the
Hypothalamus. Thyroid Stimulating Hormone (TSH) and the Thyroid Releasing Hormone
(TRH) both are regulated by a negative feedback loop by the circulating T3 and T4 levels in the
circulation (Brent and Springer, 2010). For the synthesis of thyroid hormones several
components are required. The most necessary components for the synthesis of thyroid hormones
include Thyroid Peroxidase TPO, Hydrogen Peroxidase and Iodine.
Out of the total iodine in the body 70-80% is utilized by the thyroid, which is equal to a
roughly estimate of 15-20 mg. For an adequate thyroid hormone production thyroid gland must
extract approximately about 60 µg of iodine OR iodide from the circulation (Md Consult et al.,
2011). Sodium-Iodine symporter (NIS) transports inorganic iodine to the thyroid gland; Where
TPO-HPO system oxidizes it, and then uses it in the thyroglobulin to iodinate tyrosyl. By the
coupling effect of iodinated tyrosyl in the thyroglobulin T3 and T4 are produced. Then these
hormones are hydrolyzed and then from here are secreted into the circulation (Conn et al., 2005).
These above mentioned process are so linked to each other that if any disorder in the steps occurs
at a single point it may cause impairment in the production of thyroid hormones.
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10.4 THYROID FUNCTION DURING PREGNANCY
The net effect of pregnancy is an increase in the demand of thyroid hormones by the
thyroid gland (Glinoer et al., 1997). Pregnancy is a normal physiologic function that induces the
body normal volume and function hence affecting the rate of production of thyroid hormones.
During pregnancy a normal intake of 250μg iodine is recommended to compensate the
remarkable change in the iodine metabolism during pregnancy. This increased level of iodine is
not always achieved, so in order to prevent fetal damage (because of low iodine levels) iodine
supplementation may be required in areas of iodine depletion (Lazarus, 2011). In pregnancy, the
synthesis of thyroid hormones is increased up to 50% and this increase in the synthesis of thyroid
hormone is due to the increased concentration of estrogen induced in the thyroglobulin (Glinoer
et al., 1997; Kennedy et al., 2010).According to the assays used for thyroid hormones
measurements, the concentration of thyroid hormones in the circulation is to be decreased, or
increase or it can be unchanged (Brent and Springer, 2010). T4 assays measuring shows an
increase amount of T4 levels of 1.5 times greater as compared to non-gestational period. There is
a general agreement that there is a frequent rise in the release of free T4 due to the increase level
of circulating human chorionic gonadotropin hormone (hCG) during the first trimester and there
is a downfall in the levels of free T4 during the second and the third trimester, but it stays in their
normal ranges (Glinoer et al., 1997).
Changes in free tri-iodothyronine (fT3) concentration are also seen in which it parallels
the concentration of fT4, again within the normal range. The placenta secretes human chorionic
gonadotropin (hCG) in the first week after conception and the level is the highest at week 10
before it begins to decrease and reach a plateau at week 20 (Brent and Springer, 2010; Lazarus,
2011). Human Chorionic-Graphic hormone (hCG) acts as a agonist for the thyroid stimulating
hormone (TSH) to contribute the occurrence of transient hyperthyroidism due to its elevated
levels in circulations that accounts for almost 2% to 3% of all the total pregnancies (Lazarus,
2011). There is a similarity between the molecules of TSH and hCG, as in the case of both the
TSH and hCG receptors. Consequently, Thyroid gland stimulation is mainly controlled by the
weekly peak of the hCG hormone and the amplitude and the duration of hCG peak greatly
control the production of TSH. The first indicator regarding the thyroidal dysfunction in non-
gestational female is usually the TSH levels in the serum. It is due to the direct relation between
the TSH level and the free T4 levels (fT4) that a very minor change in T4 levels will greatly
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affect in the levels of freeT4 levels in the serum. In early gestational age, there is a change in the
levels of TSH that it is suppressed to about 50-70% due to the over secretion of hCG in serum
and this level does not provide any information in controlling or treating this thyroidal
dysfunction (Lazarus, 2011). Hence, it is important to rely over T3 and T4 rather in bound or
unbound state as an indicator of thyroidal function in early gestational age, but in case of overt
hyperthyroidism there is a definite rise in the level of TSH. When gestational age reach to about
16 weeks TSH become the only indicator for thyroidal status (Brent and Springer, 2010).
10.5 MATERNAL THYROID FUNCTION AND FETAL BRAIN DEVELOPMENT
Before 12 to 14 weeks of gestational age the fetus is incapable of producing its own
thyroid hormone and hence its total relies on the thyroid hormone supplied by the mother. An
adequate amount of T3 and T4 are mandatory for the fetal brain for its normal growth and
nourishment. Until the third trimester the fetus is unable to produce thyroid hormone adequately
(Connors et al., 2010). It is not until full term that the normal levels of thyroid hormones are
achieved.
10.6 IMPACT OF THYROID AUTOANTIBODIES
Thyroidal antibodies measurement (i.e., thyroid peroxidase antibodies) do not give any
information regarding thyroidal status, their presence does have a remarkable inference over
pregnancy. Thyroid Per-Oxidase (TPO) antibodies are indicator for an increased risk of
miscarriage, preterm delivery and infertility (Lazarus, 2005). Thyroid Per-Oxidase (TPO)
antibodies are present in pregnant women in a ratio of 10% and are interlinked with the thyroidal
reserved dysfunction (Brent and Springer, 2010). In addition the presences of Thyroid Per-
Oxidase (TPO) antibodies have a 50% risk of producing postpartum thyroid dysfunction
(Browne-Martin et al., 1997; Stagnaro-Green, 2004). Most of the thyroidal disorders are
autoimmune disease and occurs due to the presence of auto-antibody. These antibodies attribute
to the pathogenesis of many thyroidal disorders as dis turn the thyroidal function by attacking the
thyroid gland (Larsen et al., 2003).
Pop et al. (1995) states that in a report about a strong relationship between the presence
of anti-Thyroid Oxidase and altered developmental stages in the offspring’s. Another study by
Kvetny and Hedvig in 2006 reported that those mothers having raised levels of anti-Thyroid
Oxidase during pregnancy will have neonates with congenital hypothyroidism. They postulates
that the neonates thyroid function was greatly affected by these auto-bodies in the form of
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cytotoxic, and brain development and other developmental factors goes beyond neonatal period,
Infant and neonatal development can be impaired by this transient hypothyroidism. In the same
case when these antibodies crosses the maternal placental barrier and attacks the fetal thyroid
gland, the transfer of these anti-bodies from the breast of the mother in milk to the neonates is
also a similar fashion. Tornhage and Grankvist (2006) conducted a research and reported that
mothers having positive Thyroid Stimulating Hormone (TSH) receptor antibody TRAb have
chances of developing thyroidal disorders in their infants and this may be worsen and can be
prolonged during the lactation phase through the breast milk. These reports shows that the
disorder caused in the fetal and neonatal development are caused by the transfer of auto
antibodies from the mother to the fetus through uterus or through the breast milk (Kvetny and
Hedvig, 2006; Tornhage and Grankvist, 2006).
10.7 PRETERM PREMATURE RUPTURE OF MEMBRANES (PPROM)
The spontaneous rupture of fetal membranes before the onset of labor contraction is
known as preterm premature rupture of membranes (PPROM).This occurs before the completed
37th week of gestational age (Simhan and Canavan, 2005). Approximately 2% to 3% off all the
pregnancies it PPROM complicates the normal pregnancies and about 25% to 30% off all the
preterm births (Mercer, 2003 and Goldenberg et al., 2008). Within 24 to 48 hours about 50% of
women goes into PPROM and those who go within seven days are about 70% to 90%and some
are also which deliver after few weeks and months (ACOG Committee, 2007 and Caughey et al.,
2008). Latency period is the time duration which starts from PPROM to the onset of labor
contractions. Gestational age is inversely proportional to the latency phase (Test et al., 2011).
When duration of PPROM exceeds above 72 hours is known as prolonged PPROM (Richardson
et al., 1974). In most of the cases of PPROM the mechanism of membrane rupture is still
unknown. In general the risk factors of PPROM are the same as listed in the case of preterm
births that include e.g. tobacco use, previous preterm delivery, vaginal bleeding and the most
important one is the infection (Minkoff et al. 1984; Harger et al., 1990; Ekwo et al., 1993;
Berkowitz et al., 1998; Silverman and Wojtowycz 1998 and Mercer et al. 2000). Against the
ascending infection fetal membranes form a barrier, there are chances of high risk of developing
intra-uterine infection after PPROM (Caughey et al., 2008). In prolonged PPROM chances of
developing infection increases (Test et al., 2011).
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10.8 COMMON BIOLOGICAL PATHWAYS TO PRETERM BIRTH
Spontaneous preterm births are sub-divided into mechanistic pathways such as intra-
uterine inflammation and infection, fetal stress, fetal anomalies, estrogen metabolism pathways
and extracellular matrix degradation.
10.9 INTRAUTERINE INFLAMMATION AND INFECTION PATHWAY
The alteration in maternal normal vaginal flora, which causes the normal flora
lactobacilli to be replaced by anaerobic bacteria (Hillier et al., 1993), is known bacterial
vaginosis (BV) and it is diagnosed by Gram-stain by the Amsel criteria. In asymptomatic
women, positive results of bacterial vaginosis are the markers of intra-uterine inflammation and
infection (Goldenberg et al., 2000) and there is an increase in two folds times of developing
spontaneous preterm birth when the presence of the bacterial vaginosis in the women serum is
detected (Meis et al., 1995 and Balu et al., 2003). The mechanism which is important to leading
preterm birth is the intra-uterine infection (Goldenberg et al., 2000). Numbers of cytokines are
produces by the activation of chorio-decidua due to the bacterial invasion and these markers are
tumor necrosis factor- [TNF-], C-reactive protein [CRP]) and Interleukins [IL] 1, 2, 6, and 8
have been estimated as biomarkers in ensuing preterm birth. Interleukin-6 (IL-6) is a pro-
inflammatory cytokine and is a major facilitator to response according to infection and
inflammation. It can be analyzed by the mother serum samples and the cervical fluid, one of the
most well studied bio-marker of preterm births is the IL-6 (Menon et al., 2011). In a case-control
study matched for parity, study center and race for asymptomatic women, the measurement of
IL-6 at 24th week of gestation were raised in those women who delivered at less than 32nd week
of gestation and at less than 35th week of gestational agein comparison to those women who
delivered at full term (Goepfert et al., 2001).
In a study of meta-analysis, at the 37th week of gestational age of asymptomatic women
shows that the high risk of preterm births were closely related to the raised levels of cervical IL-6
(Wei et al., 2010) . Paternoster et al. (2002) conducted a study and in that study he sustained that
among the pregnant women those who were asymptomatic cervical IL-6 was to be considered
the most powerful indicator for preterm births. Kramer et al.,(2010) found out that around the
24th week of gestation there was no association between the cervical IL-6 and preterm births,
while another case-control study conducted by Goldenberg et al., (2001) perceived that at 24th
week of gestation, there is a weak effect of cervical IL-6 over SPTB, and at less than 32nd week
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and at less than 35th week of gestational period of asymptomatic women (Wei et al, 2010). In a
evaluated literature C-reactive protein (CRP) is considered as a potential marker in case of
preterm birth and is a protein which is involve in the maternal systemic inflammatory response.
Hvilsom et al., (2002) conducted a case-control study and observed that in early second trimester
(14 to 18 weeks) C-reactive protein (CRP) shows association to SPTB in asymptomatic pregnant
women. Ranges of C-reactive protein concentrations ranges from 5.6 g/mL to 16.4 g/mL, and
there is high risk of developing preterm births in those women who have high concentrations of
C-reactive protein in their serumin comparison to those women who have lower levels of C-
reactive proteins.
Catov et al., (2007) described an increasing menace of early to moderate spontaneous
preterm births at 34th weeks of gestational age (OR = 2.8, 95% CI: 1.1, 7.5) and later
spontaneous preterm births which is at 34th week to 37th week of gestational age (OR = 2.6, 95%
CI: 1.3, 5.5) in those women who are asymptomatic. Riboni and his colleagues observed that in
mid-second trimester there is an increase in the risk of spontaneous preterm births (OR = 3.1,
95% CI: 1.4, 6.8) in those women who have elevated concentrations of serum C-reactive protein
(8.4 g/mL) (Riboni et al., 2012). Another study shows no association to C-reactive protein in
envisaging preterm births in asymptomatic pregnant women; hence not validating the previous
studies results (Kramer et al., 2010). The assessment of C-reactive protein and IL-6 in
calculating succeeding preterm birth, many studies also studied the association of other cytokines
(IL-1, IL-2, IL-8, and TNF) but no evidence suggested the raised cytokine standards were related
to a high risk of spontaneous preterm births (Goldenberg et al., 2001; Paternoster et al., 2002;
Kramer et al., 2010 and Riboni et al., (2012).
10.10 EXTRACELLULAR MATRIX DEGRADATION PATHWAY
So far, the presence of fibrinoectin is the most effective marker for predicting preterm
birth (Goepfert et al., 2000 and Goldberg et al., 2001). Fetal fibronectin is a glycoprotein which
is formed by the fetal membranes and it follows the fetal membranes as well as placenta to the
uterine lining (lockwood et al., 1991 and Goldberg et al., 2005) and plays a vital part after
delivery in separating the placenta from the uterus (Conde-Agudelo et al., 2011). Between 16th
week to 22nd week, there is absence of fetal fibrinoectin at the level of 50 ng/mL, whereas
previous studies had proved that from 22nd week of gestation there is presence of fetal
fibrinoectin at the level of 50 g/mL in the cervix and vagina and also observed that it is a
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powerful predictor of spontaneous preterm birth (Morrison et al., 1996; Goldberg et al., 1996;
Goldberg et al., 2000; Goldberg et al., 2001 and Roman et al., 2005). In an another study a test
of was conducted in which they show that 29% women were positive result with cervical and
vaginal fibrinoectin if the last fetal fibronectin test done was positive, and other 42% women
were those who had a ensuing positive result if the last two tests done were positive, only 3%
women were those who have fibronectin tests positive if the last fetal fibronectin test performed
was negative (Goldberg et al.1997).
Data from this study provide the evidence that greater numbers of positive fetal
fibronectin result are associated with an increased risk of sPTB at 35th week to 37th week of
gestational age. Morrison and his coworkers also verified the efficacy of cervical fetal
fibronectin for assessing different tools in expecting spontaneous preterm birth in between who
were high risk asymptomatic women (Morrison et al., 1996). Regardless of very small sample
size, the data from a cohort prospective study shows that those women with positive fetal
fibrinoectin results (50 ng/mL) were at higher risk of developing spontaneous preterm births
earlier to 34th week of gestational period in comparison to those women having fetal fibronectin
negative results, spontaneous preterm risks were additionally exaggerated when positive uterine
contraction were coupled with positive fetal fibrinoectin. In the same way, at 24th week of
gestational period positive cervical fetal fibronectin (50ng/mL) was observed to have association
with spontaneous preterm birth at 32nd week to 35th week of gestation as compared to those
women having results negative (Paternoster et al., 2002). Roman et al. (2005) conducted a study
on asymptomatic women and found out high negative prognostic values and specification for
vaginal fetal fibronectin in predicting spontaneous preterm births in between 14 to 21 days of
assessment and also for spontaneous preterm births at the 34th week of gestation. Honest et al.
(2002) conducted a meta-analysis and found out the association between the asymptomatic
women and the presence of fetal fibronectin.
10.11 FETAL STRESS PATHWAY
Corticotrophin-releasing hormone (CRH) is a hormone which is uttered by the human
placenta and also by the fetal membranes, and during the third trimester the highest peak of this
hormone secretion is seen (Yeast and Lu, 2005). Berkowitz et al. (1996) conducted a
retrospective study and examined the plasma Corticotrophin-releasing hormone (CRH) levels at
four gestational age intervals and found out nil differences in complete mean Corticotrophin-
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Releasing hormone (CRH) between the asymptomatic women who delivered at preterm and the
mean of Corticotrophin-releasing hormones (CRH) amongst those women who delivered at full
term. Gestational age related corticotrophin-releasing hormone (CRH) values were not
associated with envisaging succeeding spontaneous preterm births. Analysis done through a
case-control study of 254 asymptomatic women shows that raised levels of maternal plasma
Corticotrophin-Releasing Hormone (CRH) at the 28th week of gestation shows association with
spontaneous preterm births at the 35th week but higher concentrations of Corticotrophin-
Releasing Hormone (CRH) at 24th to 28th week of gestation do not foresee spontaneous preterm
births at the 32nd week of gestational age or less than 37th week of gestation (Moawad et al.,
2002). Goldenberg et al. (2001) performed a study and additionally established similarities in
finding association among Corticotrophin-Releasing Hormone (CRH) levels with successive
preterm birth.
10.12 FETAL ANOMALIES PATHWAY
Several studies have recognized raised concentrations of human-chorionic gonadotropin
(hCG) and fetoprotein association with increased risk for developing spontaneous preterm births
in cervical and vaginal fluids between pregnant women who were asymptomatic (Goldenberg et
al., 2002; Sanchez-Ramos et al., 2003 and Goldberg et al., 2005 ). Early and late preterm births
were associated with raised concentrations of fetoprotein. Moawad et al. (2002) performed a
case-control study which includes 127 asymptomatic women and found out that there is an
increase in the risk of developing spontaneous preterm birth (SBP) at less than 32nd week of
gestation and for spontaneous preterm birth at 35th week. At 28th week of gestation levels of
fetoprotein measured shows association with following spontaneous preterm birth.
10.13 ESTROGEN METABOLISM PATHWAY
The major form of circulating estrogen is the estriol (Goodwin, 1999). Some earlier
studies shows that raised salivary estriol (2.1ng/mL) was a well indicator for late spontaneous
preterm birth in those pregnant women who were asymptomatic and use of estriol from saliva
testing has multiple benefits, the use of saliva as a bio marker has one limitation (McGregor et
al., 1995 and Heine et al., 2000) and that is confusing by issues such as, posture, food
consumption, diurnal variations and patient activity in estriol levels (Ramsey and Andrews, 2003
and Yeast and Lu, 2005). Though medical advances have enhanced the endurance of preterm
infants and also treatment of short and long terms morbidities, a very little success has been
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achieved in accepting and stopping preterm births. Great efforts have been done in explaining the
usage of biologic fluids from different sources in expecting spontaneous preterm births. Not only
the identification of biochemical indicators which shows association with spontaneous preterm
births allows the researchers to understand much better about the fundamental mechanisms or
pathways which lead to preterm birth but also helps in guiding the most active targeted
intervention approaches aimed for women who were at risk for preterm birth. There are many
studies which evaluate the association between the different biochemical indicators and
spontaneous preterm births between the asymptomatic women. Understanding spontaneous
preterm births is a challenge for the researchers because of its multiple causes and
pathophysiologic different characters. Moreover, maternal and fetal risks factors are also of great
worry. Fetal fibronectin (50ng/mL at 24th-26th week of gestation), so far, is the most effective
indicator in predicting spontaneous preterm births in asymptomatic women.
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MATERIALS AND METHODS
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11.0 MATERIALS AND METHODS 11.1 SOURCE OF DATA
The present study was designed to investigate the key processes involved in thyroid
hormone and their related auto-antibodies, oxidative stress markers and antioxidant capacity in
the development of acute myocardial infarction. All the selected patients were screened at Gulab
Devi Chest Hospital, Lahore. Informed consent was obtained before being included in this study.
Twenty clinically apparently healthy individuals were included as controls. The experimental
protocol was approved by the Research Ethical Committee of The Institute of molecular biology
and biotechnology, The University of Lahore. Five ml of venous blood sample were taken from
the anti cubital vein of each participant. The sample bottles were centrifuged within one hour of
collection, after which the serum were separated and stored at -70°C until assayed.
11.2 INCLUSION CRITERIA
The AMI disease patients included were based on the following criteria: 1) patients with Age
20-70years were included in study 2) All patients of AMI were included in this study.
11.3 EXCLUSION CRITERIA
The subjects with the history of taking drugs (including alcohol and cigarette), pre-
diagnosis medications (e.g. antiparkinsonian/antipsychotic), were excluded from this study.
None of the controls were on any medication, history of chronic infections, malnutrition
syndrome, depression, psychosis or metabolic dysfunction (Such as diabetes mellitus, liver
diseases, cancer) that could interfere with their oxidative metabolites and thyroid hormone
status.
11.4 CHEMICALS
All chemical reagents of analytical grades were purchased from sigma/Invitrogen
Chemical Co. (St. Louis, Mo, USA).
11.5 FOLLOWING PARAMETERS WILL BE ESTIMATED
I- THYROID BIOMARKERS
II- STRESS BIOMARKERS
III- ANTI-OXIDANT
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11.5.1 ESTIMATION OF THYROID PROFILE IN SERUM SAMPLES
11.5.2 ESTIMATION OF T3 (Tri-iodothyronine) ELISA
Were estimated by using human ELIZA kit (BioVendor)
11.5.3 ESTIMATION OF T4 (Thyroxine) ELISA
Were estimated by using human ELIZA kit (BioVendor)
11.5.4 ESTIMATION OF TSH (Thyroid stimulating hormone) ELISA
Were estimated by using human ELIZA kit (BioVendor)
11.5.5 ESTIMATION OF TPO (Thyroid peroxidase) IgG ELISA
Were estimated by using human ELIZA kit (BioVendor)
11.5.6 ESTIMATION OF Thyroglobulin IgG ELISA
Were estimated by using human ELIZA kit (BioVendor)
11.5.7 ESTIMATION OF STRESS BIOMARKERS IN SERUM SAMPLES
11.5.8 ESTIMATION OF THIOBARBITURIC ACID REACTIVE SUBSTANCES
(TBARS)
Lipid peroxidation in blood samples was estimated calorimetrically by measuring
Thiobarbituric acid reactive substances (TBARS) by the method of Ohkawa et al., 1979. To
0.2ml of sample, 0.2ml of 8.1% Sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid and
1.5 ml of 0.8% TBA was added. After centrifugation at 3000 rpm for 10 min the upper organic
layer was taken and its OD was read at 532 nm against an appropriate blank without the sample.
The levels of lipid peroxides were expressed as milimoles of Thiobarbituric acid reactive
substances (TBARS)/g of liver tissue using standard curve.
11.5.9 ESTIMATION OF SUPER OXIDE DISMUTASE (SOD)
Superoxide dismutase (SOD) activity was determined by the method of Kakkar, 1972.
The assay mixture contained 0.1ml of sample, 1.2ml of sodium pyrophosphate buffer (pH 8.3,
0.052 M), 0.1 ml of Phenazine methosulphate (186μm), 0.3 ml of nitro blue tetrazolium (300μ
m), 0.2 ml of NADH (750 μmol). Reaction was started by addition of NADH. After incubation at
300 C for 90 sec, the reaction will be stopped by the addition of 0.1 ml of glacial was allowed to
stand for 10 min; centrifuged and n-butanol layer will be separated. The color intensity of the
chromogen in butanol layer will be measured at 560 nm against n-butanol and concentration of
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SOD will be expressed as units/g of liver tissue. Absorbance values were compared with a
standard curve generated from known SOD.
11.5.10 ESTIMATION OF GLUTATHIONE (GSH)
Blood GSH was estimated according to the method of Moron et al., 1979.GSH reacts
with Ellman’s reagent (5, 5-dithio bis (Nitrobenzoic acid) or DTNB) to produce a chromophore
TNB with maximal absorbance at 412 nm and oxidized glutathione GSSG. The amount of
glutathione measured represents the sum of reduced and oxidized glutathione in the sample
([GSH]t = [GSH] + 2 x [GSSG]). The rate of absorbance change (ΔA 412nm/min) is made to be
linear for the convenience and consistence of measurement, and is linearly proportional to the
total concentration of GSH. The concentration of an unknown (sample) is determined by
calculating from the linear equation generated from several standards of glutathione (Tietze,
1969).
11.5.11 ESTIMATION OF CATALASE (CAT)
Catalase was assayed according to the method of Aebi, 1974. The estimation was done
spectra photo metrically following the decrease in absorbance at 230 nm. The blood sample
was homogenized in M/150 phosphate buffer (pH 7.0) at 1-40C and centrifuged at 5000 rpm.
The reaction mixture contained 0.01 M phosphate buffer (pH 7.0) 2mM H2O2 and the enzyme
extract. The specific activity of Catalase was expressed in terms of units/gram. Absorbance
values were compared with a standard curve generated from known Catalase.
11.5.12 DETERMINATION OF NITRIC OXIDE
NO is produced by various cell types in picomolar to nanomolar range and has a very
short half-life (t1/2 <5s) in biological fluids; therefore, a direct measurement of its production is
difficult and the analysis of NO2- and NO3
-, the stable products of NO oxidation, is often
performed to estimate NO level in biological fluids and cell culture medium (Moncada et al.,
1991). Nitrite concentration is typically measured by a well-known method such as colorimetric
Griess assay (Moshage et al., 1995).
11.5.12.1 PRINCIPLE
Sulfanilic acid is quantitatively converted to a diazonium salt by reaction with nitrite in
acid solution. Thediazonium salt is then coupled to N-(1-naphthyl) ethylenediamine, forming an
azo dye that can be spectrophotometrically quantitated based on its absorbance at 548 nm.
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11.5.12.2 CHEMICALS REQUIRED
N-(1-naphthyl) ethylenediamine dihydrochloride (Component A)
Sulfanilic acid (Component B)
Nitrite standard solution (Component C)
11.5.12.3 PROTOCOL
Mixed together equal volumes of N-(1-naphthyl) ethylenediamine (Component A) and
sulfanilic acid (Component B) to form the Griess Reagent. Mixed the following in a
spectrophotometer cuvette:
100 μL of Griess Reagent
300 μL of the nitrite-containing sample
2.6 mL of deionized water
The mixture was incubated for 30 minutes at room temperature. A photometric reference sample
was prepared by mixing 100 μL of Griess Reagent (above) and 2.9 mL of deionized water.
Measured the absorbance of the nitrite-containing sample at548 nm relative to the reference
sample.Absorbance values were compared with a standard curve generated from known NO.
11.5.13 ESTIMATION OF INTERLEUKIN-6 (IL-6) (BY ELISA KIT)
11.5.13.1 Procedure
All the solutions present in the kit were mixed and reagents were prepared according to
the instructions and allowed to reach at room temperature for at least 30 min.
At the end of the assay, store immediately the reagents at 2-8ºC: avoid long exposure to
room temperature.
Unused coated micro well strips should be released securely in the foil pouch containing
desiccant and stored at 2-8ºC.
To avoid potential microbial and/or chemical contamination, unused reagents should
never be transferred into the original vials.
As it is necessary to perform the determination in duplicate in order to improve accuracy
of the test results, prepare two wells for each point of the calibration curve (C0-C5), two
for each sample, and one for blank.
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Reagent Calibrator Sample/control Blank
Sample 50μL
Calibrator (C0-C5) 50Μl
Diluted conjugate 100Μl 100μL
Incubate 1h at room at room temperature (22-28ºC).
Remove the content from each well; wash the wells 3 times with 300μL of diluted Wash
Solution.
Important note: during each washing step, gently shake the plate for 5 seconds and
remove excess solution by tapping the inverted plate on an absorbent paper towel.
TMB Substrate 100μL 100μL 100μL
Incubate 15 min in the dark at room temperature (22-28ºC).
Stop Solution 100μL 100μL 100μL
Shake the micro plate gently.
Read the absorbance at 450nm against a reference wavelength of 620-630nm or against blank
within 5 min.
11.5.14 DETERMINATION OF TUMOR NECROSIS FACTOR BY ABCAM’s TNF
ALPHA HUMAN ELISA KIT
Sample type: cell culture supernatant, serum, plasma
Assay type: sandwich (quantitative)
Sensitivity: <10pg/ml
Range: 25pg/ml-800pg/ml
Recovery: 107%
Assay time: 3h 40 min
Assay duration: multiple steps standard assay
Species Reactivity: reacts with human
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Abcam’s TNF alpha human in vitro ELISA (enzyme linked immunosorbent assay) kit is
designed for the quantitative measurement of TNF alpha in supernatants, serum, plasma samples
and buffered solutions. A monoclonal antibody specific TNF alpha has been coated onto the
wells of the microtiter strips provided. Samples including standards of known TNF alpha
concentrations, control specimens or unknown samples were pipette into these wells. During the
first incubation, the standards or samples and a biotinylated monoclonal antibody specific for
TNF alpha were simultaneously incubated. After washing, the enzyme Streptavidin-HRP that
binds the bio-tinylated antibody was added, incubated and washed. A TMB substrate solution
was added which acts on the bound enzyme to induce a colored reaction product. The intensity
of this colored product is directly proportional to the concentration of TNF alpha present in the
samples. This kit recognized both endogenous and recombinant Human TNF alpha. The platform
used was microplate.
11.5.15 DETERMINATION OF GLUTATHION PEROXIDASE (GPx)
Glutathione peroxidase was determined by the method of spectrophotometer with the
help of buffer/ enzyme reagent (Aebi Bergmeyer 1983).
BUFFER/ENZYME REAGENT
9.20 sodium azide solution
NADPH
Glutathione
0.1 GPx solution
48mM sodium phosphate
0.38mM ethylene diaminetetra acetic acid
HCL
ASSAY
Wavelength 340nm
Optical path 1cm
Temperature 25°C
Ph 7.0
Measurement against blank
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11.5.15.1 PROCEDURE
The GPx concentration can be measured spectra photo metrically. The 9.02 sodium azide
solution had taken 0.1 GPx solution and glutathione into β-NADPH vial. Mixed by inversion and
pH was adjusted upto 7.0 at 25°C with 1M HCL. In the 3.05 reaction mixture, final
concentration at 48mM sodium phosphate, 0.38mM ethylene diaminoteraacetic acid, 0.12mM β-
NADPH, 0.95mM sodium azide.3.2 units of glutathione peroxidase and 0.075 to 0.15 unit of
glutathione, 0.02mM DL-dithiothreitol , hydrogen peroxide. Absorbance was measured at
340nm after 5 minutes.
11.5.16 ESTIMATION OF GLUTATHIONE REDUCTASE (GR)
Glutathione reductase was evaluated by using method of David and Richard (1983). The
amount of NADPH employed is a direct measure of enzyme activity. Reagents used are PBS
(0.12 M), EDTA -15 milli mole, Sodium azide (0.1ml), Oxidized Glutathione-6.3mili mole and
NADPH. The phosphate buffer (1ml),EDTA (0.1ml), sodium azide (0.1ml),oxidized glutathione
(0.1ml) and sample (0.1ml) were added into the cuvette and distilled water (600 micro litre) was
also added to make the volume upto 2ml,then incubated for 3 minutes and NADPH (0.1ml) was
added. The absorbance was read at 340nm in a spectrophotometer at every 15 seconds interval
for 2-3 minutes. For each series of measurement, controls were set up that contained water
instead of oxidized glutathione. The activity of GR represented as micro moles of NADPH
oxidized/minute/g of sample.
11.5.17 DETERMINATION OF AOPPs
AOPP were determined in the plasma using the semi-automated method previously
devised in our laboratory. Briefly, AOPP were measured by spectrophotometry on a microplate
reader (model MR 5000, Dynatech, Paris, France) and were calibrated with chloramine-T
(Sigma, St. Louis, MO) solutions that in the presence of potassium iodide absorb at 340 nm (27).
In test wells, 200 ml of plasma diluted 1/5 in PBS was placed on a 96-well microtiter plate
(Becton Dickinson Lab ware, Lincoln Park, NJ), and 20ml of acetic acid was added. In standard
wells, 10 ml of 1.16 M potassium iodide (Sigma) was added to 200ml of chloramine-T solution
(0-100mmol/liter) followed by 20ml of acetic acid. The absorbance of the reaction mixture is
immediately read at 340 nm on the microplate reader against a blank containing 200ml of PBS,
10 ml of potassium iodide, and 20ml of acetic acid. The chloramine-T absorbance at 340 nm
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
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being linear within the range of 0 to 100mmol/liter, AOPP concentrations were expressed as
micromoles per liter of chloramine-T equivalents.
11.5.18 DETERMINATION OF VITAMIN A
Measured 1 ml of the analyzed liquid to the test-tube I (centrifugal) with a tight stopper
and added 1 ml of the KOH solution, plug the tube and shake vigorously for 1 minute- heated the
tube in a water bath (60oC, 20 minutes), then cooled it down in cold water-add 1 ml of xylene,
pluged the tube and shaked vigorously again for 1 minute- centrifuge the tube (1500×g, 10
minutes), collected the whole of the separated extract (upper layer) and transferred it to the test
tube II made of “soft” (sodium) glass-measured the absorbance A1 of the obtained extract at 335
nm against xylene-irradiate the extract in the test tube II to the UV light for 30 minutes, then
measured the absorbance A2-calculate the concentration cx of vitamin A (µM) in the analyzed
liquid, using the formula:
cx = (A1 - A2) · 22.23
Where: 22.23 multiplier received on basis of the absorption coefficient of 1% solution of vitamin
A (as the retinol form) in xylene at 335 nm in a measuring cuvette about thickness = 1 cm.
11.5.19 ESTIMATION OF VITAMIN-E IN SAMPLE
Vitamin E was evaluated in samples by the Emmerie-Engel reaction as reported by
Rosenberg (1992).
11.5.19.1 PRINCIPLE
The Emmerie-Engel reaction is based on the reduction of ferric to ferrous ions by
tocopherols, which, with 2, 2’-dipyridyl, forms a red colour. Tocopherols and carotenes were
first extracted with xylene and read at 460nm to measure carotenes. For correction, ferric
chloride solution is added and absorption ia taken at 520nm.
11.5.19.2 REAGENTS
1. Absolute alcohol
2. Xylene
3. 2, 2-dipyridyl (1.2g/L in n-propanol)
4. Ferric chloride (1.2g/L in ethanol)
5. Standard solution
6. H2SO4 (0.1N)
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11.5.19.3 PROCEDURE
1.5ml of liver extract, 1.5ml of the standard and 1.5ml of water were pipetted out
separately. To all the tubes, 1.5ml of ethanol and 1.5ml of xylene were added, mixed well and
centrifuged. Xylene (1.0ml) layer was transferred into another stopper tube. To each tube, 1.0ml
of dipyridyl reagent was added and mixed well. The mixture (1.5ml) was pipetted out into a
cuvette and the extinction was read at 460nm. Ferric chloride solution (0.33ml) was added to all
the tubes and mixed well. The red colour developed was read exactly after 15 min at 520nm in a
spectrophotometer. The concentration of tocopherols in the sample was calculated using the
formula,
Sample A520 - A460
Tocopherols (µg) = --------------------------------- × 0.29 × 0.15
Standard A520
11.5.20 ESTIMATION OF VITAMIN-C IN LIVER SAMPLE
Vitamin- C was estimated by using the method of Roe and Keuther (1943).
11.5.20.1 PRINCIPLE
Ascorbate is converted into dehydroascorbate on treatment with activated charcoal,
which reacts with 2, 4-dinitrophenyl hydrazine to form osazones. These osazones produce an
orange colored solution when dissolved in sulphuric acid, whose absorbance was measured by
spectrophotometer (540nm).
11.5.20.2 REAGENTS
1. TCA (4%)
2. 2,4-dinitrophenyl hydrazine reagent (2%) in 9N H2SO4
3. Thiourea (10%)
4. Sulphuric acid (85%)
5. Standard ascorbic acid solution: 100µg / ml in 4% TCA
11.5.21 EXTRACTION OF VITAMIN-C
Ascorbate was extracted from 1g of the liver sample using 4% TCA and the volume was
made up to 10ml with the same. The supernatant obtained after centrifugation at 2000rpm for 10
min was treated with a pinch of activated charcoal, shaken vigorously using a cyclomixer and
kept for 5min. The charcoal particles were removed by centrifugation and aliquots were used for
the estimation.
CHAPTER THREE MATERIALS AND METHODS MISBAH, 2017
104
11.5.21.1 PROCEDURE
Standard ascorbate ranging between 0.2-1.0ml and 0.5ml and 1.0ml of the supernatant
were taken. The volume was made up to 2.0ml with 4% TCA. DNPH reagent (0.5ml) was added
to all the tubes, followed by 2 drops of 10% Thiourea solution. The contents were mixed and
incubated at 37°C for 3 hours resulting in the formation of osazones crystals. The crystals were
dissolved in 2.5ml of 85% sulphuric acid, in cold. To the blank alone, DNPH reagent and
Thiourea were added after the addition of sulphuric acid. The tubes were cooled in ice and the
absorbance was read at 540nm in a spectrophotometer. A standard graph was constructed using
an electronic calculator set to the linear regression mode. The concentration of ascorbate in the
samples were calculated and expressed in terms of mg/g of sample.
CHAPTER FOUR RESULTS MISBAH, 2017
105
RESULTS
CHAPTER FOUR RESULTS MISBAH, 2017
106
12.0 RESULTS
A total of fifty preterm mothers and twenty control subjects were included in the study.
Thyroid biomarkers, circulating stress biomarkers, inflammatory biomarkers and antioxidants
were assessed in array to set their effective roles in causing preterm delivery and also to
underline the consequence of these markers as valuable PTD prognostic markers.
12.1 THYROID BIOMARKERS PROFILE OF PRETERM MOTHER PATIENTS Vs
CONTROL
The levels thyroid hormone and its associated antibodies portray a high contribution of
this hormone in causing preterm delivery. Statistically Significant (p≤0.036, p≤0.008) higher
level of FT4 (16.35±3.11pmol/L) and TSH (4.98±1.03IU/L) were observed in preterm mothers
in comparison with normal delivering mother’s FT4 (11.25±2.25pmol/L) and TSH
(3.26±1.005IU/L). On the other hand the mean FT3 of controls and patients is 5.26μg/dl and
3.58μg/dl respectively. An overall significant decreasing trend was also observed in GSH levels
between PTD patients and controls (p=0.019). As far as levels of auto antibodies related with
thyroid hormone is concerned, a persistent higher pattern was observed in preterm mothers TgAb
(47.16IU/L),TPOAb (9.28IU/L) as compared to control group’s TgAb (31.25IU/L) and
TPOAb(6.35IU/L) and is statistically highly significant (p=0.032, p=0.004)respectively.
12.2 CIRCULATING STRESS BIOMARKERS PROFILE OF PRETERM MOTHERS VS
CONTROL
The results regarding stress biomarkers MDA, GSH, NO, AOPPs IL-6 and TNF-α shown
in table 1 demonstrate the critical role of stress markers in the consequences of causing preterm
labour. The mean MDA of control subjects is 0.951μmol/L and that of oral preterm mothers is
2.06μmol/L. An overall increasing trend was observed in MDA levels between PT mothers and
controls. On the other hand the mean GSH of controls and patients is 8.06μmol/L and
6.28μmol/L respectively. An overall decreasing trend was also observed in GSH levels between
PTD patients and controls. The nitrosative stress biomarker, NO, in control and preterm delivery
patients showed a mean value of 23.26μmol/L and 40.26μmol/L respectively, scrutinizing an
increasing level of NO in patients. Likewise the mean value of AOPPs in controls was
99.26±4.23μmol/L and in preterm mothers 131.26±1.06μmol/L respectively. An increasing drift
was observed in AOPPs levels between diseased patients and controls. The inflammatory stress
markers (IL-6, TNF-α) depicted that mean value of IL-6 in control and diseased is 3.73 pg/ml
CHAPTER FOUR RESULTS MISBAH, 2017
107
and 6.29 pg/ml respectively and the mean value of TNF-α in healthy and diseased individual is
21.26 pg/ml and 39.26 pg/ml respectively. Hence, pointing towards the existence of increased
trend among preterm mothers. It is apparent from our results that the stress biomarker MDA,
GSH, NO, AOPPs IL-6 and TNF-α had an increasing trend in patients vs. controls while GSH
showed a decreasing trend in patients when compared to healthy subjects. All stress biomarkers
exhibited statistically significant values (p<0.05).
12.3 ANTIOXIDANT BIOMARKERS PROFILE OF PRETERM MOTHER PATIENTS
Vs CONTROL
The enzymatic antioxidant biomarkers SOD, Catalase, GPx and GRx and non-enzymatic
antioxidant biomarkers vitamin E, vitamin A, vitamin C, vitamin B6, vitamin B9 AND vitamin
B12 results fluctuations clearly illustrated their role at the time of preterm labour. As compared
to normal, all foresaid variable showed significant (p=0.025, 0.001, and 0.015 respectively) low
levels of SOD (0.14±0.0011μmol/L vs. 0.625±.0011μmol/L), CAT (3.19±0.15U/L vs.
4.26±0.016U/L), GPx (7.18±1.09 μmol/L vs. 6.26±1.22 μmol/L), GR (2.29±1.28 μmol/L vs.
4.26±0.0015 μmol/L) in preterm mothers group were recorded. While the mean value for GPx in
controls was found to be 6.26±1.22 μmol/L whereas in diseased it was observed to be 7.18±1.09
μmol/L. An overall increasing trend was seen between both PTD patients and healthy subjects.
The mean value of antioxidant vitamin A in controls was 6.01±1.76nmol/L whereas in preterm
delivery females the mean value was 4.29±1.09nmol/L, hence a decreasing trend was seen in
vitamin A levels between diseased and controls. The vitamin C mean value in controls was 0.65
nmol/L while in diseased individuals it was 0.462 nmol/L. The mean value for GPx in controls
was found to be 1.59 U/g Hb whereas in diseased it was observed to be 6.51 U/g Hb. An overall
decreasing trend in vitamin E was seen between both preterm mothers (0.36±0.0019nmol/l) and
normally delivering subjects (0.498±0.001nmol/L). The low levels of vitamin B6
(61.28±4.26nmol/L), vitamin B9 (2.18±0.168nmol/L) and vitamin B12 (206.15±4.26pmol/L)
were observed in preterm delivering mothers as compared to vitamin B6 (77.26±6.25nmol/L),
vitamin B9 (3.26±0.0018nmol/L) and vitamin B12 (226.25±8.26pmol/L) respectively. All
antioxidant showed statistical significance with the condition (p≤0.05).
CHAPTER FOUR RESULTS MISBAH, 2017
108
Table 03: THYROID BIOMARKERS PROFILE OF PRETERM DELIVERY FEMALE
PATIENTS Vs CONTROL
PARAMETERS CONTROL (20) PRETERM MOTHERS(40) P VALUE
T4 (pmol/L) 11.25±2.25 16.35±3.11 0.036
T3 (μg/dl) 5.26±1.25 3.58±0.94 0.019
TSH (IU/L) 3.26±1.005 4.98±1.03 0.008
TgAb (IU/L) 31.25±3.26 47.16±5.26 0.032
TPOAb (IU/L) 6.35±1.05 9.28±1.28 0.004
Table 04: STRESS BIOMARKERS PROFILE OF PRETERM DELIVERY FEMALE
PATIENTS Vs CONTROL
PARAMETERS CONTROL (20) PRETERM MOTHERS(40) P VALUE
MDA (μmol/L) 0.951±0.001 2.06±0.056 0.016
NO (μmol/L) 23.26±4.26 40.26±3.68 0.000
GSH (μmol/L) 8.06±1.26 6.28±1.88 0.012
AOPPs(µmol/L) 99.26±4.23 131.26±1.06 0.016
IL-6 (pg/ml) 3.73±2.76 6.29±1.44 0.041
TNF-α (pg/ml) 21.26±7.26 39.26±3.29 0.011
CHAPTER FOUR RESULTS MISBAH, 2017
109
Table 05: ANTIOXIDANT BIOMARKERS PROFILE OF PRETERM MOTHERS Vs
CONTROL
PARAMETERS CONTROL(20) PRETERM MOTHERS(40) P VALUE
SOD (U/ml) 0.625±.0011 0.14±0.0011 0.025
CAT (U/L) 4.26±0.016 3.19±0.15 0.001
GPx (μmol/L) 6.26±1.22 7.18±1.09 0.032
GRx (μmol/L) 4.26±0.0015 2.29±1.28 0.015
Vit A(nmol/L) 6.01±1.76 4.29±1.09 0.033
Vit C(nmol/L) 0.65±0.0016 0.462±0.0016 0.016
Vit E(nmol/L) 0.498±0.001 0.36±0.0019 0.035
Vitamin-B6 (nmol/L) 77.26±6.25 61.28±4.26 0.011
Vitamin-B9 (nmol/L) 3.26±0.0018 2.18±0.168 0.000
Vitamin-B12 (pmol/L) 226.25±8.26 206.15±4.26 0.045
CHAPTER FIVE DISCUSSION MISBAH, 2017
110
DISCUSSION
CHAPTER FIVE DISCUSSION MISBAH, 2017
111
13.0 DISCUSSION
Because of the high mortality and high morbidity rate preterm labor is one of the most
common and important obstetrical and perinatology disorder which is responsible for the
inhibition of the body organs and system. Hence, in term of those whose early detection has been
diagnosed must be given preventive steps. The prevalence of preterm labor is relatively higher in
developing countries as compared to developed countries. Rate of preterm birth in year 2005 was
calculated to be about 9.6%. In which approximately the incidence of preterm labor in Africa and
Asia continent was 85%. The reason or the causes which are responsible for the preterm labor
are not yet fully known. Still, there are many mechanisms which are responsible for preterm
labor: maternal and fetal H-P-A (Hypothalamic- Pituitary- Adrenal) axis activation,
inflammation and other infections, excess enlarging of uterus and decidua bleeding, which
produces certain clinical signs and also shows a synchronize movements between the
myometrium contractions, rupture of the fetal membranes and opening of the cervix also known
as the cervical ripening. Reactive species (ROS) may also cause the activation of myometrium
muscle contraction. Hence, a study was performed to assess the oxidative markers in the body.
One function is also the evaluation of relation between the thyroid hormones and the anti-bodies
which are responsible for its aggravation. Inflammatory markers have certain characteristics
which are responsible for the pathophysiology of term and preterm labor. Preterm labor is said to
be a state in the female pregnancy that intends to inhibit the uterine physiology that is
responsible for the organ maturity of the fetus. About 50% off all the preterm births, Intra-
amniotic infection (IAI) and inflammation are associated with preterm labor. Cytokines that are
associated with the labors are characterized by the immune penetrations and also by expressing
tumor necrosis factor alpha (TNF-α), IL-8, IL-, interleukin (IL)-1β and monocyte chemo-
attractant protein (MCP-1), amniotic fluid, cervix, placenta and in the fetal membranes.
Expressing these pro-inflammatory mediators results in
(i) There is an increase in the prostaglandins levels which are responsible for increase
uterine contractions;
(ii) Chorio-amnion degradation in the extracellular matrix (ECM); (iii) Cervix ripening
An important pro-inflammatory cytokine and a basic organizer that respond to
inflammation and infection is the Interlueiken-6. In mother serum samples interlueiken-6 can be
CHAPTER FIVE DISCUSSION MISBAH, 2017
112
assessed, IL-6 is one of the most important preterm bio-marker that is studied until now (Menon
et al., 2011). In fetal membranes, placenta and uterine epithelial cells activation of TLRs along
with their ligands tends to induce the release of pro-inflammatory cytokines. These pro-
inflammatory cytokines may be responsible for the link between the PPROM, uterine contraction
and cervical ripening (Schaefer et al., 2004; Noguchi et al., 2010). In a study, there is an
increased in the levels of IL-6 in mothers with preterm labor in comparison to control healthy
group which are simultaneous with another study of Paternoster et al. (2002) in which he shows
that in asymptomatic pregnant women levels of cervical IL-6 was well thought out to be the
most strong parameter regarding preterm births. Wei et al., (2010) conducted a study and
sustained that at the 37th week of pregnancy of women with no symptoms have higher risk of
preterm births association with elevated values of cervical IL-6.
Macrophages, VSMCs, foam cells, fibroblasts, inflammatory cells and monocytes are
various cells that are responsible for the production of the TNF-α and it increases the synthesis of
protein, macrophages phagocytosis and growth at cellular level and discernment by enhancing
response to inflammation. In case of preterm birth there is an up-regulation of TNF-α. In our
conducted study, elevation of TNF- α was observed in women with those who were delivering
preterm births in comparison to those mothers delivering normal births and this was similar to a
study done by Lyon et al. (2010) in which he observed a relationship between preterm birth and
the increased values of IL-6, IL- Beta and TNF- α in the circulation. Those women having both
TNF- α polymorphism and also bacterial vaginosis in them tend to have higher risk of
developing preterm births as compared to those women having only TNF- α or bacterial
vaginosis alone in them (Macones et al., 2004).
Reactive Oxygen Specie aids as important molecules that regulate the normal procedures
but it also have involvement in the pathological procedures that happens in the female
reproductive system by affecting numerous physiological procedures ranging from maturation of
oocyte to fertilization, development of embryo and even pregnancy. It has also a role in the
ignition of labor. Reactive RNS (Reactive Nitrogen Specie) has affect over vasodilation by
showing effects on nitric oxide signaling and also it causes DNA damage. Vasodilation ability in
the body will be damaged and get depressed by the over presence of the free radicals in the
blood; hence, flow of blood will get low due to the activation of vasoconstriction, which
ultimately leads to IUGR (Intra-Uterine Growth Retardation), pre-eclampsia and preterm labor
CHAPTER FIVE DISCUSSION MISBAH, 2017
113
(Stein et al., 2008). Raised levels of NO were observed in our study in preterm births apart from
those mothers who were delivering at full term due to the presence of nitric oxide which get
stimulated by the inflammatory cytokines which may include TNF-α, IL-1 and lipo-
polysaccharides. During the follicular development granulose cells, theca cells and the oocytes
expressed the endothelial NO synthase. Inducible NO in cases of pathological state is sole
responsible for the production of Nitric Oxide NO. In response to immunologic cases inducible
NO synthase is expressed in different organs.
During the oxidative stress proteins are at high risk of being affected by the free radicals.
AOPPs (Advanced Oxidation Protein Products) are those biomarkers that contain di-tyrosine that
are produced due to the stressed state and it initiates inflammatory process (Piwowar et al.,
2010). In our study, raised levels of AOPPs were observed in the serum of women with preterm
labor in comparison to control group in which there is an increase in the production of hypo-
chlorous acid from an enzyme known as the myeloperoxidase under the circumstances of stress
caused by oxidation and carbonyl that react with albumin, in human blood it is the most plentiful
proteins present and which oxidizes it to AOPPs that triggers NADPH oxidase by the RAGE
receptor. Ultimately, there is an increase in inflammatory process and oxidative stress. There are
many studies that show that significant relationship between the decrease levels of antioxidants
and preterm labor. Antioxidants may be endogenous or they may be exogenous that may be
helpful in the prevention of formation of dangerous oxidants and it also in activates the already
present oxidants that tends to blocks the proliferation reactions that are due to these toxic agents.
Enzymatic antioxidants may include catalase; superoxide dismutase (SOD), glutathione
reductase (GR) and glutathione peroxide (GPx) whereas non-enzymatic antioxidants are
Vitamins A, C and E and reduced glutathione (GSH). The human myometrium soft muscles are
gifted with the enzymatic hunting system that tends to lower the possible destruction caused by
ROS (Telfher et al. 1997, Matharoo-Ball & Khan 2003). A large number of studies suggest that a
low level of enzymatic antioxidants is the basic reason for the onset of preterm labors.
The major anti-oxidant defense system against the reactive O2- is the superoxide
dismutase (SOD), and in mammals there are three forms of SOD present: cytoplasmic Cu/Zn
SOD (SOD1), mitochondrial Mn/SOD (SOD2) and extracellular Cu/Zn SOD (SOD3). All of
which catalytic metals (Cu or Mn) are mandatory for their stimulation. Relative studies show that
in each sub-cellular region conversion of O2- and H2O2 is catalyzed by the SOD, which may
CHAPTER FIVE DISCUSSION MISBAH, 2017
114
contribute in the cell signaling process. Furthermore, inhibition of oxidative nitric oxide is
carried out by the SOD; hence formation of per-oxy nitrite is prevented (Fukai and Ushio-Fukai,
2011). In our study levels of SOD were decreased to a low level in preterm births women as
compared to those women who delivered at full term. A protein that uses Fe as a co-factor and is
encoded on chromosome 11 by a gene is the catalase. Low catalase levels may happen due to the
mutation of this gene and relatively high oxidation state known as acatalasemia. Furthermore to
SOD activity, catalase hydrolyses free radicals and peroxidase into oxygen and water. By
breaking the oxidative chain reaction these enzymes helps to inhibit the formation of free
radicals and convert them into more stable products known as the anti-oxidants. The levels of
catalase were reduced in our study as compared to the healthy women groups and it was
simultaneous to another study conducted by Negara et al. 2010 in which he reported a decrease
in the values of catalase in women delivering preterm births.
H2O2 is catalyzed by the catalase and glutathione by sharing same nature of catalyzing.
Regardless GPx has more affinity of against H2O2 when compared with catalase. Difference is
between there mode of kinetics. In accordance to concentration of H2O2 catalase catalyzes it, and
in case of GPx it becomes saturated when concentration of H2O2 falls below 10-5 mol/L. GPx is
more potent in catalyzing H2O2 in comparison to catalase when the concentration falls to a lower
level or normal value Kobe et al., 2012. As compared to healthy groups levels of GPx falls in
diseased groups was stated in the present studies. Reduction in the levels of SOD and CAT
shows that the increased levels of GPx are due to this reduction and this enhancement in the GPx
is to respond against stress state and removal of H2O2. Production of GPx may be also due to the
increased formation of lipo-peroxidase. Conversion reaction to reduced form of GSH from
oxidized glutathione is catalyzed by the glutathione reductase. Present studies shows a fall in the
levels of GR in pregnant women delivering preterm births when compared to normal healthy
women
Pituitary gland secretes a hormone through thyroid stimulating hormone which is the
main regulator of the above mentioned literature. In the hypothalamus, thyroid releasing
hormone is present which is responsible for the stimulation of thyroid hormone. In the
circulation, regulating T3 and T4 are present which regulates the production of thyroid hormone
by the TRH and TSH from a mechanism known as the negative feedback mechanism (Brent and
Springer, 2010). Several components are in demand for the synthesis of thyroid hormone. Iodine,
CHAPTER FIVE DISCUSSION MISBAH, 2017
115
Hydrogen Per-oxidase and Thyroid Per-oxidase are the most important components which are
required for the synthesis of thyroid hormones. In some studies preterm birth is associated with
clinical hypothyroidism and increased levels of TRH (Allan et al., 2000; Casey et al., 2005;
korewaar et al., 2013). In preterm mothers at the time of delivery there is an increase in the
levels of thyroid hormone in comparison to healthy group delivering and this study shows
similarity with a study which was conducted by Johns et al. (2017) which reported that changes
in the levels of thyroid hormones increases the risk of preterm births. There are many
physiological mechanisms which are regulated by tri-iodothyronine (T3) that include
differentiation, proliferation, metabolism, inflammation and cellular growth through binding with
thyroid hormone response elements (TREs) by regulating the target genes. In our study there is a
decrease in the levels of T3 when it was compared with healthy women. This fall in the level of
thyroid hormone triggers the hypothalamic-pituitary-thyroid axis; there is an increase in the
levels of human chorionic gonadotropin (hCG), this hormone share a structural similarity
between the TSH, and it also stimulates and increase in the levels of T3 and T4.
Apart from its various functions Vitamin A which is actually stored in the liver is considered
as a good antioxidant, in immune functions it has a vital role. A form of vitamin A known as the
retinoic acid is formed by the dendritic cells and the macrophages of lymph nodes and mucosa.
When state of inflammation is achieved, conversion of CD4 T lymphocytes into T-helper cells
leads to formation of inflammatory cytokines (Green and Mellanby, 1928 and Raverdeau and
Mills, 2014). In our study levels of vitamin A were decreased in case groups and these results
show correlation with a study conducted by Azaïs-Braesco and Pascal, 2000; Mactier and
Weaver, 2005. Low levels of vitamin A in this group shows that they have more affinity towards
catching infection. Furthermore low levels of vitamin A induce pro-inflammatory cytokines to
secrete that may lead to inflammation and triggering oxidative stress through NADPH.
The damage caused by the hydroxyl radicals are eliminated actively by the non-toxic
vitamin E. Initially this antioxidant is found in the lipoproteins and cellular membranes where its
most important function is to delay the lipid peroxidation process. Inflammation has been
associated with decrease levels of vitamin E. To minimize the wound healing time is one of the
functions of vitamin E and maintaining the stability and normal physiology of membranes.
Vitamin E normal levels in the body prevent it from the onset of disease. In our study we
observed low levels of vitamin E which is responsible for more growth of hydroxyl radicals,
CHAPTER FIVE DISCUSSION MISBAH, 2017
116
resulting in more lipid peroxidation leading in the disturbance of normal cell integrity and
eventually leading to increase chances of preterm births. For the synthesis and degradation of
collagen vitamin C has an important role and is also essential for maintaining chorio-amniotic
fluids. Decrease presence of vitamin C in pregnancy leads to an increase risk of premature
rupture of chorio-amniotic membranes. In a study conducted earlier we showed that PROM can
be a standard protocol for the estimation of vitamin C throughout the pregnancy (Casanueva et
al., 1998). There was a fall in the levels of vitamin C in our study in those women who were
delivering preterm births and this study was in accordance to a study which was conducted by
Casanueva et al., 2005. In our study vitamin C was derived in accordance to the collagen
metabolism and that of collagen in maintaining the integrity of membranes throughout the
pregnancy (Bryant-Greenwood, 1998; Tejero et al., 2003). For collagen post-transcriptional
modification vitamin C was considered as an important co-factor and a down regulator for the
gene 72-kDa type IV collagenase, matrix metalloproteinase (MMP-2), in amnion cells; it also
controls the catabolism of collagen (Pfeffer et al., 1998).
An observation was made in the increase levels of homo-cysteine supports the role of FA
in placenta on which induces a cytotoxic as well as oxidative stress on placental circulation and
function. Apoptosis may increase when homo-cysteine are introduced with trophoblastic cells
(Clarke, 2000; Steegers-Theunissen and steegers, 2003; Parazzini et al., 2011). Recent studies
shows an association between the folate metabolizing genes and incidence of spontaneous
preterm birth (johnson et al., 2005; Zafarghandi et al., 2013) which is similar to our finding.
Present study shows that levels of vitamin B6 and B12 fall to a low level in those females who
were delivering preterm births when compared to normal healthy delivering births and this study
was similar to another study conducted by Van Sande et al., 2013.
CHAPTER FIVE
FIGURE 0several causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes the neutrophilOxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate aimbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
CHAPTER FIVE
FIGURE 02: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes the neutrophils to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate aimbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate aimbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the antithe maternal and fetal HPA axis to activate and secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Cacontraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
117
: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the anti
nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in the oxytocin receptors to activate and causes the Ca++ ions influx activating actin myosin cascade causes the muscles contraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the anti
nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of TTBG causing more uterine contraction, cervical ripening resulting in PROM and preterm labor. Oxytocin activates
ions influx activating actin myosin cascade causes the muscles contraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive Oxygen Specie) +and results in the lipid peroxidation due to decrease in the anti-oxidants. This mechanism causes
nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of T
PROM and preterm labor. Oxytocin activates ions influx activating actin myosin cascade causes the muscles
contraction resulting in preterm labor. Other factors like placental abruption and pregnancies decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
DISCUSSION MISBAH, 2017
: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTHseveral causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive oxidants. This mechanism causes
nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of T
PROM and preterm labor. Oxytocin activates ions influx activating actin myosin cascade causes the muscles
contraction resulting in preterm labor. Other factors like placental abruption and pregnancies anomalies causes decidua and fetal membrane disturbances that lead to cervical ripening results in preterm labor.
DISCUSSION MISBAH, 2017
: MECHANISM OF DIFFERENT PARAMETERS INDUCING PRETERM BIRTH There are
several causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive oxidants. This mechanism causes
nd secretes cortisol and oxytocin. Decreased cortisol levels causes the imbalance between the estrogen and progesterone levels that is responsible for the over production of T3 through
PROM and preterm labor. Oxytocin activates ions influx activating actin myosin cascade causes the muscles
anomalies causes
DISCUSSION MISBAH, 2017
There are
several causative factors which are responsible for preterm birth through series of chain cascade. They constitute of some major mechanisms which act as an aggravating these causatives factors. Now, when infection occurs it causes
s to infiltrate and causes the cytokines to release which causes the production of ROS (Reactive oxidants. This mechanism causes
nd secretes cortisol and oxytocin. Decreased cortisol levels causes the through
PROM and preterm labor. Oxytocin activates ions influx activating actin myosin cascade causes the muscles
anomalies causes
CHAPTER SIX CONCLUSION MISBAH, 2017
118
CONCLUSION
CHAPTER SIX CONCLUSION MISBAH, 2017
119
14.0 CONCLUSION
Current work concluded the facts and figures that may contribute as the reason behind
preterm delivery involves both the endocrinal and immunological responses that may lead to the
activation of inflammatory markers which in term are responsible for increased oxidative stress,
may be involved in the over activity of thyroid gland. Hormones such as oxytocin and estrogen
were increased while progesterone remained decreased which may initiates preterm labor. The
levels thyroid hormone and its associated antibodies portray a high contribution of this hormone
in causing preterm delivery. Statistically Significant higher level of free T4 and TSH were
observed in preterm mothers. On the other hand the level of free T3 falls when compared to
normal healthy pregnant women.
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