CORD BLOOD CALPROTECTIN CORD BLOOD CALPROTECTIN CONCENTRATIONS IN FULL-CONCENTRATIONS IN FULL-TERM PREGNANCIES WITH TERM PREGNANCIES WITH NORMAL AND RESTRICTED NORMAL AND RESTRICTED

FETAL GROWTHFETAL GROWTH

Sofia LiosiSofia Liosi 11 , Despina D. Briana, Despina D. Briana 11

Dimitr ios GourgiotisDimitr ios Gourgiotis 22 , , Maria Boutsikou, , Maria Boutsikou 11 , Stavroula , Stavroula BakaBaka 11 , Antonios Marmarinos , Dimitr ios Hassiakos , Antonios Marmarinos , Dimitr ios Hassiakos 11 , ,

Ariadne Malamitsi-PuchnerAriadne Malamitsi-Puchner 11 . .

1.1.Neonatal Division, 2nd Department of Obstetrics and Gynecology, Athens Neonatal Division, 2nd Department of Obstetrics and Gynecology, Athens University Medical School, Athens, GreeceUniversity Medical School, Athens, Greece

2.Research Laboratories, 2nd Department of Pediatrics, Athens University 2.Research Laboratories, 2nd Department of Pediatrics, Athens University Medical School, Athens, GreeceMedical School, Athens, Greece

CalprotectinCalprotectin

• a 36-kDa calcium and zinc binding a 36-kDa calcium and zinc binding protein protein

• comprises two heavy chains of 14 kDa comprises two heavy chains of 14 kDa and one l ight chain of 8 kDaand one l ight chain of 8 kDa

• constitutes approximately 60% of soluble constitutes approximately 60% of soluble cytosol proteins in human neutrophil cytosol proteins in human neutrophil granulocytesgranulocytes

CalprotectinCalprotectin

• a marker of neutrophil turnover

• immunomodulatory and antimicrobial effects

• antiproliferative effects (inducing apoptosis)

Intrauterine growth Intrauterine growth restriction (IUGR)restriction (IUGR)• Failure of the fetus to achieve his/her

intrinsic growth potential

• Consequence of anatomical and/or functional disorders and diseases in the feto-placental-maternal unit

• Results to increased morbidity and mortali ty in intra- and extra-uterine l i fe

Neutrophil activationNeutrophil activation

is present in preeclampsia and isolated intrauterine growth restriction

Possible mechanisms may be: • hypoxia• the presence of soluble mediators of inflammation• endothelial stimulation

IUGRIUGR

• associated with an increased incidence of apoptosis in IUGR-affected fetal membranes

HypothesisHypothesis

• Umbilical cord blood Umbilical cord blood concentrations of concentrations of calprotectin in IUGR cases may differ calprotectin in IUGR cases may differ from respective concentrations in from respective concentrations in appropriate-for-gestational-age (AGA) appropriate-for-gestational-age (AGA) controls since the former present controls since the former present increased neutrophil activation and increased neutrophil activation and

excessive apoptosisexcessive apoptosis ..

AimAim

• Investigate cord blood calprotectin concentrations in IUGR, and AGA pregnancies at birth

• Correlate determined concentrations with gestational age, gender and mode of delivery ..

SubjectsSubjects

• 160 healthy, singleton full-term 160 healthy, singleton full-term pregnanciespregnancies

- - 111010 AGA AGA (placental weight: (placental weight: 480-621480-621 g)g)- 50- 50 asymmetric IUGR asymmetric IUGR ( placental weight ( placental weight

230230 -400g)-400g)

• Apgar scores:Apgar scores: >>8 in 8 in 11 s ts t and and 55 t ht h minuteminute

Gestation Related Optimal Gestation Related Optimal Weight (GROW)Weight (GROW) computer-computer-

generated programmegenerated programme

www.gestation.net

Custom baseDaysBirthweight (g)Gestation: WeeksBooking weight (kg)Maternal height (cm)SexEthnic groupParity at booking56Customised centile =

Causes of IUGRCauses of IUGR

• Preeclampsia (Preeclampsia ( n=6 )n=6 ) • Pregnancy induced hypertension (n=5Pregnancy induced hypertension (n=5 ))• Various diseases : Various diseases : severe type I DMsevere type I DM

(( n=5),iron deficiency anemia (n=3), n=5),iron deficiency anemia (n=3), hypothyroidismhypothyroidism (n=7)(n=7)

• Maternal smoking ( n=24 )Maternal smoking ( n=24 )

Demographic dataDemographic data

IUGR AGAIUGR AGA• Gestational age (weeks) Gestational age (weeks) 3838 .36.36 ±1±1 .27 * .27 * 338.988.98 ±±1.07 *1.07 *

• BW (g) BW (g) 25250707±±264.5 * 264.5 * 33310310 ±±310.5*310.5*

• BW centile BW centile 33 .0 (0-5) 44.5 (20-99).0 (0-5) 44.5 (20-99)

• Gender (male/female) Gender (male/female) 21/29 66/4421/29 66/44

• Mode of delivery (VD/ECS)** Mode of delivery (VD/ECS)** 22/28 77/3322/28 77/33

• Parity (1Parity (1stst /other) /other) 3131 // 19 76/3419 76/34

* * values are mean ± SD**VD: vaginal delivery/ ECS: elective cesarian section **VD: vaginal delivery/ ECS: elective cesarian section

MethodsMethods

Blood collected from:Blood collected from:• Doubly-clamped umbilical cords (mDoubly-clamped umbilical cords (m ixed ixed

arteriovenous bloodarteriovenous blood ) – reflecting fetal ) – reflecting fetal statestate

• Determination of plasma Determination of plasma calprotectin calprotectin concentrations by enzyme immunoassayconcentrations by enzyme immunoassay ((Human Calprotectin ELISA, HBT Hycult Human Calprotectin ELISA, HBT Hycult Biotechnology b.v , Uden, The Biotechnology b.v , Uden, The Netherlands)Netherlands)

• Statistical analysis (parametric tests)Statistical analysis (parametric tests)

Figure 1. Error bars Figure 1. Error bars of the concentrations of of the concentrations of Calprotectin in IUGR cases and AGA controls.Calprotectin in IUGR cases and AGA controls.

IUGR GROUP (N=50)

AGA GROUP (N=110)

C

AL

PR

OT

EC

TIN

LE

VE

LS

(n

g/m

l)

320

300

280

260

240

220

0

Results (1)Results (1)

• NoNo statistically signif icant differences in statistically signif icant differences in cord blood calprotectin concentrations cord blood calprotectin concentrations between IUGR and AGA groupsbetween IUGR and AGA groups ..

Results (2)Results (2)

• In a combined groupIn a combined group

• Cord blood calprotectin concentrations Cord blood calprotectin concentrations were signif icantly decreased in cases of were signif icantly decreased in cases of caesarean sectioncaesarean section ..

• The effect of birthweight, customized The effect of birthweight, customized centi le, gestational age, gender and centi le, gestational age, gender and parity on calprotectin concentrations was parity on calprotectin concentrations was not signif icantnot signif icant ..

Results (3)

• In the IUGR groupIn the IUGR group

• Calprotectin was significantly elevated Calprotectin was significantly elevated for every week increase in gestational for every week increase in gestational age.age.

• Calprotectin was posit ively correlated Calprotectin was posit ively correlated

with birthweight.with birthweight.

Conclusions (1)Conclusions (1)

• Cord blood calprotectin concentrations at Cord blood calprotectin concentrations at term are independent of intrauterine term are independent of intrauterine growth and probably do not reflect the growth and probably do not reflect the increased neutrophil activation and increased neutrophil activation and excessive apoptosis expected in IUGRexcessive apoptosis expected in IUGR

• PParity, gender and maternal age do not arity, gender and maternal age do not seem to have any impact on umbil ical seem to have any impact on umbil ical cord blood calprotectin concentrationscord blood calprotectin concentrations

Conclusions (2)Conclusions (2)

• Calprotectin may have a l imited role Calprotectin may have a l imited role during fetal l i fe, possibly due to the during fetal l i fe, possibly due to the lower degree of neutrophil activation in lower degree of neutrophil activation in the fetus.the fetus.

• Neutrophil activation and apoptosis seem Neutrophil activation and apoptosis seem to increase with advancing gestational to increase with advancing gestational age in the IUGR group, as indicated by age in the IUGR group, as indicated by the increasing calprotectin the increasing calprotectin concentrations with progressing concentrations with progressing pregnancypregnancy

Conclusions (3)

• Excessive inflammatory response associated with vaginal delivery may account for the higher calprotectin concentrations in the latter