Asisted Reproductive Techniques-Embryology. Oocyte Pick Up (OPU)Oocyte Pick Up (OPU)...

Asisted Reproductive Asisted Reproductive Techniques-EmbryologyTechniques-Embryology

•Oocyte Pick Up (OPU)Oocyte Pick Up (OPU)•Microinjection-İnseminationMicroinjection-İnsemination•Embryo CultureEmbryo Culture•Embryo TransferEmbryo Transfer•CryopreservationCryopreservation•PGDPGD•Assisted HatchingAssisted Hatching

•Oocyte Pick Up (OPU)Oocyte Pick Up (OPU)•Microinjection-İnseminationMicroinjection-İnsemination•Embryo CultureEmbryo Culture•Embryo TransferEmbryo Transfer•CryopreservationCryopreservation•PGDPGD•Assisted HatchingAssisted Hatching

Embryology Laboratory PracticesEmbryology Laboratory Practices

This process is carried out This process is carried out by vaginal route through a by vaginal route through a needle placed in an needle placed in an ultrasound probe.ultrasound probe.

Follicular fluid which is surrounded by cumulus cells, examined under a stereo microscope and then the oocytes collected.

Cumulus-oocyte complex Cumulus-oocyte complex maturation is evaluated maturation is evaluated before it is removed before it is removed to to incubator.incubator.

OPU (Oocyte Pick Up)OPU (Oocyte Pick Up)

MikroinjectionMikroinjection After a period of incubation at After a period of incubation at

least 2-3 hoursleast 2-3 hours,, the cumulus the cumulus cells cells are are removed by enzymatic removed by enzymatic and mechanicaland mechanical procedure. procedure.

Selected by the microinjection Selected by the microinjection process, a single sperm cell is process, a single sperm cell is injected into injected into the the oocytes.oocytes.

Microinjection procedure is Microinjection procedure is performed with the assistance performed with the assistance of micropipettes attached to an of micropipettes attached to an inverted microscope.inverted microscope.

OOocytes are controlledocytes are controlled for for fertilization after 14-16 hoursfertilization after 14-16 hours..

FFertilization rates increased ertilization rates increased from 80% to 90from 80% to 90 with the with the mmicroinjectionicroinjection..

MikroinjectionMikroinjection

Conventional In Vitro Conventional In Vitro Fertilization (IVF) İnseminationFertilization (IVF) İnsemination

Stages of embryo Stages of embryo development development (IVF-ET)(IVF-ET)

Pronükleer Evre16-18 saat sonra

2 Hücreli Evre24-27 saat sonra

4 Hücreli Evre41-44 saat

sonra

8 Hücreli Evre66-71 saat

sonra

Morula Evresi4 gün sonra

Blastosist Evresi

5 gün sonra

Embryo Transfer Embryo Transfer ProcedureProcedure Embryos Embryos areare transferred to the transferred to the

uterusuterus after oocyte collection after oocyte collection processprocess--two or three daystwo or three days..

In some patients, blastocyst transfer In some patients, blastocyst transfer can be appliedcan be applied the day of the day of 5 or 6.5 or 6.

Embryo transfer Embryo transfer is is very simple and very simple and painless procedure for thepainless procedure for the candidate candidate of mother.of mother.

Embryos were transferred to Embryos were transferred to specially produced for this process specially produced for this process by loading had abdominal by loading had abdominal cathetercatheterand leftand left into the uterus into the uterus withwith the vaginal ultrasound-guided.the vaginal ultrasound-guided.

TTransferred of embryos are ransferred of embryos are determined by the number and determined by the number and quality of embryos and the age of quality of embryos and the age of mother.mother.

AAfter the transferfter the transfer,remaining the,remaining the top top quality quality of of embryos embryos are are frozen for usefrozen for use in the futurein the future..

Top Quality Embryo Selection CriteriaTop Quality Embryo Selection Criteria

The day of 2 (after 41-44 hours) have at least 4 or 5 blastomeres

The day of 3 (after 66-71 hours) have at least 7 blastomeres

Cytoplasmic fragmentation<%20 Blastomere lack of multinükleasyon

(Van Royen et.al.1999,2001, Gerris et.al.1999)

Assisted HatchingAssisted Hatching

TThe embryo continues to grow and he embryo continues to grow and dividedivide into the uterusinto the uterus..

After a period of time to rip around layer After a period of time to rip around layer of the zona pellucidaof the zona pellucida and and implantimplant into into the uterusthe uterus..

Before being transferred the embryo, Before being transferred the embryo, the thinning zona pellucidathe thinning zona pellucida, , makes it makes it easier to holdeasier to hold the uterus. the uterus.

Specially;Specially;--Women over the age of 35Women over the age of 35--In case of a thick zona pellucidaIn case of a thick zona pellucida

-Recurrent implantation failure-Recurrent implantation failure-In f-In frozen embryo transfer.rozen embryo transfer.In our institution, the application time is In our institution, the application time is too short, reliable, and reproducible too short, reliable, and reproducible technique using a laser high pregnancy technique using a laser high pregnancy and and rates can be achieved.rates can be achieved.

What is a co-culture?What is a co-culture?

In some patients, the treatment of the previous In some patients, the treatment of the previous IVF IVF attemptsattempts,, embryo development was slow and embryo development was slow and poor poor quality can not be observed and achievequality can not be observed and achievedd repeated repeated attempts to pregnancy.attempts to pregnancy.

In this case In this case of patients for the development of better of patients for the development of better embryo, endometrial cells instead of synthetic feed embryo, endometrial cells instead of synthetic feed solution was prepared by supporting a culture medium solution was prepared by supporting a culture medium (co-culture) is used.(co-culture) is used.

Previous trials of patients with this method is applied its Previous trials of patients with this method is applied its own center than enables the development of good-own center than enables the development of good-quality embryos and pregnancy rates increase.quality embryos and pregnancy rates increase.

However, to apply this method is not used in several However, to apply this method is not used in several centers in the laboratory centers in the laboratory for for requires additional labor requires additional labor and technical support.and technical support.

How to make co-How to make co-culture? culture? The luteal phase of the menstrual The luteal phase of the menstrual

cycle of 20-23 days in the last period cycle of 20-23 days in the last period of endometrial samples are taken with of endometrial samples are taken with the assistance of Pipelle.the assistance of Pipelle.

After this mechanical and enzymatic After this mechanical and enzymatic digestiondigestion,, stromal and glandular cells stromal and glandular cells are separatedare separated in in flasksflasks and and incubated .incubated .

3 days 3 days before before OPUOPU,, cells surrounds the cells surrounds the entire surface of flasks (confluent) entire surface of flasks (confluent) fourfour-w-well (300,000 viable cells / well) ell (300,000 viable cells / well) are planted.are planted.

After the formation of PNAfter the formation of PN,, embryos are embryos are taken on these cells ataken on these cells andnd incubated incubated there until the blastocyst stagethere until the blastocyst stage..

In tIn this methodhis method,, embryos embryos areare being being extended on the endometrium extended on the endometrium likelike to to the body'sthe body's and and growth factors in the growth factors in the environment supports the environment supports the development of the embryo.development of the embryo.

In addition, toxins that can damage In addition, toxins that can damage the embryo, removed from the the embryo, removed from the environmentenvironment by the by the antioxidantsantioxidants..

Pre-implantation Genetic Pre-implantation Genetic Diagnosis(PGD)Diagnosis(PGD)

Point;Point;Prevent transmission of chromosomal Prevent transmission of chromosomal abnormalitiesabnormalitiesReduce the risk of Reduce the risk of abortabortTo select genetically healthy To select genetically healthy embryosembryos

Who Applies to PGD?Who Applies to PGD?

Quantitative chromosomal abnormalities Quantitative chromosomal abnormalities Anöploidi (Trisomies, monozamiler, nullizomiler), Anöploidi (Trisomies, monozamiler, nullizomiler),

Triploidi, Triploidi, haploidihaploidi Structural chromosomal abnormalitiesStructural chromosomal abnormalities

Deletions, microdeletions (DiGeorge, Kallman etc.)Deletions, microdeletions (DiGeorge, Kallman etc.)Translocations (Reciprocal and Robertsonian)Translocations (Reciprocal and Robertsonian)Inversion and insertionInversion and insertionSex-linked recessive diseasesSex-linked recessive diseasesDuchenne muscular dystrophy, fragile X syndrome, Duchenne muscular dystrophy, fragile X syndrome, hemophilia, Hunter syndrome, Wiskott-Aldrich hemophilia, Hunter syndrome, Wiskott-Aldrich syndrome, Lesch-Nyan syndrome, etc.syndrome, Lesch-Nyan syndrome, etc.

Single gene defectsSingle gene defects Autosomal recessive (cystic fibrosis, beta-Thalasemia, Autosomal recessive (cystic fibrosis, beta-Thalasemia,

Spinal muscular atrophy (type I), etc.Spinal muscular atrophy (type I), etc. Autosomal dominant (Myotonic dystrophy, Marfan's Autosomal dominant (Myotonic dystrophy, Marfan's

syndrome, etc.)syndrome, etc.)

Who Applies to PGD?Who Applies to PGD?

IVFIVF Recurrent implantation failureRecurrent implantation failure Advanced female ageAdvanced female age Recurrent miscarriagesRecurrent miscarriages Patients with previous Patients with previous

pregnancies, detectedpregnancies, detected aneuploidyaneuploidy

Pre-implantation Genetic Pre-implantation Genetic Diagnosis(PGD)Diagnosis(PGD)

FISHFISHDiagnosis of numerical and Diagnosis of numerical and structural chromosomal structural chromosomal abnormalitiesabnormalities

PCRPCRDiagnosis of single gene Diagnosis of single gene defectsdefects

NORMALNORMAL

2nd polar body

Day 1

Day 2

Day 3

blastomere biopsy

Day 5

blastosist biopsy

1st polar body

Day 0

Embryo Biopsy

Opening the zona pellucidaOpening the zona pellucida MechanicalMechanical Chemical (Acid Tyrode’s Chemical (Acid Tyrode’s

Solution)Solution) Laser TechnologyLaser Technology

Paternal Lymphocyte Vaccine

POİNT

With Lymphocyte Vaccine Treatment Method, 'block antibody, called antibodies that damage the developing baby in the in the uterus, suppression of the targeted cells.

Who can Apply?Who can Apply? Unexplained InfertilityUnexplained Infertility Recurrent İmplantation FailureRecurrent İmplantation Failure Recurrent AbortionsRecurrent Abortions


Necessary before the;

Candidates for ‘Dad’ analyzed of strong Hepatitis and HIV in terms. People whose tests (+) blood from, are not used to prepare the vaccine.

In cases where the mother is Rh-negative, father Rh+positive, to prevent problems in the future due to blood incompatibility with the given Rhogam.

Lymphocyte vaccine is not any harm in pregnant woman and the developing baby. Mothers whose treated with lymphocyte vaccine, there is not increase in congenital anomalies in babies or thrive.


Stem CellStem Cell

TotipotentTotipotentHücreHücre

PluripotentPluripotentHücreHücre

Progenitor kök Progenitor kök hücrelerhücreler

İnsülin-salgılayan İnsülin-salgılayan beta hücreleri beta hücreleri Sinir Sinir

hücresihücresi

Kas Kas hücresihücresi

Özerk Özerk hücrelerhücreler

Kan Hücreleri

EmbryoEmbryo

Embryonic Stem CellEmbryonic Stem Cell

Adult Stem CellAdult Stem Cell

TotipotentTotipotent

PluripotentPluripotent

Embryonic Stem CellsEmbryonic Stem Cells

Day 1Day 1 Day 2Day 2 Day 3Day 3 Day 4Day 4 Day 5Day 5

ICMICM

TrophectodermTrophectoderm


Evans ve Kaufman, 1981 (Mouse Evans ve Kaufman, 1981 (Mouse ES)ES)

Thompson , 1998 (Human ES)Thompson , 1998 (Human ES)


In lIn laboratory conditions can be improved aboratory conditions can be improved continuouslycontinuously,, without any structural change without any structural change. .

CCanan be be create any type of adult cell create any type of adult cell i inn body .body .

Like bLike bone, blood, muscle and liver tissues, such as one, blood, muscle and liver tissues, such as creating an important source in the near future creating an important source in the near future will be used for treatment purposes.will be used for treatment purposes.

Human Embryonic Stem Cell Human Embryonic Stem Cell CultureCulture

Cell SourcesCell Sources

•• After the procedure, the unwanted After the procedure, the unwanted

frozen embryos donated for research frozen embryos donated for research

purposespurposes..

Embryonic Stem Cell CultureEmbryonic Stem Cell Culture

ProcessesProcesses

• • TThe preparation of he preparation of ssupport upport

cellcell

• • Removal of zona pellucidaRemoval of zona pellucida

•• İmmune surgeryİmmune surgery

•• Direct cultureDirect culture

•• PrimaryPrimary colony production and colony production and

passagepassage

••Long-term cultureLong-term culture

• Normal karyotype

• Immunocytochemical Analysis

• SSEA-3

• SSEA-4

• TRA-1-60

• TRA-1-81

• Alkaline phosphatase expression

• Telomerase expression

• Creating a SCID feature of mice teratoma

• Normal karyotype

• Immunocytochemical Analysis

• SSEA-3

• SSEA-4

• TRA-1-60

• TRA-1-81

• Alkaline phosphatase expression

• Telomerase expression

• Creating a SCID feature of mice teratoma

NIH embryonic stem cell identification criteria



Colony MorphologyColony Morphology

Cell MorphologyCell Morphology

Autonomous Surface AntigensAutonomous Surface Antigens

Normal Karyotype

Embryonic Stem Cell CultureEmbryonic Stem Cell Culture In Vitro DifferentiationIn Vitro Differentiation

Three embryonic germ layers is developing to differentiation of cell types spontaneously or adding growth factors ( Reubinoff et al. Nature Biotechnology 2000, Schuldiner et al. PNAS 2000)

Neuron ( Reubinoff et al Nature 2001, Schuldiner et al. Brain res. 2001)

Cardiomyocyte( Kehat et al. J clin İnvest 2001, Xu et al. Circ.Res. 2002, Mummery et al. Circ. 2003)

The insulin-producing cells( Assady et al. Diabetes 2001, Odorico, Kaufmann and Thomson, Stem Cells 2001)

Blood Cells( Kaufmann et al PNAS 2001) Endothelial Cells( Levenberg et al. PNAS 2002)

In Vitro Differentiation of Human Embryonic In Vitro Differentiation of Human Embryonic Stem CellsStem Cells

Mekanik pasajlama Kistik EB Oluşumu

Kültür <10 gün

Jelatin-kaplı kültür kabı

(endoderm) (ektoderm) (mezoderm)


In vitro differentiationIn vitro differentiation

NestinNestin MAP2 a&bMAP2 a&b Troponin ITroponin ITroponin ITroponin I

Cardiomyocyte differentiation-TEM Cardiomyocyte differentiation-TEM StudyStudyErken Faz Erken Faz

Mitokondri

Z Bandı

Sarkomer

Miyofibriller


Areas of UseAreas of Use

Examination of embryonic developmentExamination of embryonic development

İn Farmakology'de tissue-specific drug testing İn Farmakology'de tissue-specific drug testing

Toxicology in the identification of new agentsToxicology in the identification of new agents

For Therapeutic UseFor Therapeutic Use

Embryonic Stem CellEmbryonic Stem Cellss

Areas of UseAreas of Use

Parkinson's and Alzheimer's disease MS Paralysis, Spinal Cord Injuries Diabet Joint and Bone damage Liver damage Muscular dystrophy Cardiac failure, Myocardial infarction Canser Hematologic diseases

Embryonic Stem CellEmbryonic Stem Cellss

Clinical Therapeutic UseClinical Therapeutic Use

Preliminary studies :Preliminary studies : AAfter transplantationfter transplantation,, uundifferentiated human embryonic stem ndifferentiated human embryonic stem

cellscells to to motor neuropathy paralyzed ratsmotor neuropathy paralyzed rats,, recovery was recovery was observed in.observed in.

Kerr DA, Llado J 2003Kerr DA, Llado J 2003 AAfter injection of ESCsfter injection of ESCs to e to experimental myocardial infarction in xperimental myocardial infarction in

ratsrats was determined improvement in contractile function and was determined improvement in contractile function and reduction of infarct size.reduction of infarct size.

Denice M. Hodgson, 2004Denice M. Hodgson, 2004 When were given Insulin-producing cells from embryonic stem

cells in diabetic mice returned to normal levels of glycemia.

Soria B, 2000 Parkinson modeli oluşturulmuş farelerde embriyonik kök hücre embriyonik kök hücre

kaynaklı dopaminerjik nöronların fonksiyon gösterdiği bulundu. kaynaklı dopaminerjik nöronların fonksiyon gösterdiği bulundu. Parkinson's model was createdParkinson's model was created in micein mice and and was found thatwas found thatss functional of dopaminergic neurons from embryonic stem cellsfunctional of dopaminergic neurons from embryonic stem cells..

Kim JH, Auerbach JM, 2002 Kim JH, Auerbach JM, 2002

The use of materials from animal sourcesThe use of materials from animal sourcesSupport systems, of human originSupport systems, of human origin(HFF)(HFF)

The immune system is the problem of The immune system is the problem of

compliancecomplianceCloning for therapeutic purposesCloning for therapeutic purposes

Genetic manipulationGenetic manipulation

Stem cell bankStem cell bank

Differentiation of protocols incompatibilityDifferentiation of protocols incompatibility

The protection of life after transplantationThe protection of life after transplantation

The risk of tumorThe risk of tumor

Ethical problemsEthical problems

The use of materials from animal sourcesThe use of materials from animal sourcesSupport systems, of human originSupport systems, of human origin(HFF)(HFF)

The immune system is the problem of The immune system is the problem of

compliancecomplianceCloning for therapeutic purposesCloning for therapeutic purposes

Genetic manipulationGenetic manipulation

Stem cell bankStem cell bank

Differentiation of protocols incompatibilityDifferentiation of protocols incompatibility

The protection of life after transplantationThe protection of life after transplantation

The risk of tumorThe risk of tumor

Ethical problemsEthical problems

Problems that need to be resolved before the Problems that need to be resolved before the clinical trial:clinical trial:

Result and DiscussionResult and DiscussionEmbryonic Stem Embryonic Stem CCells ells ccan bean be unlimited unlimited

culture under laboratory conditions,culture under laboratory conditions, th thisis

properties an important source for cell properties an important source for cell

therapy. therapy.

These cells in the near future, especially These cells in the near future, especially

by solving the existing ethical and by solving the existing ethical and

technical problems, heart disease, technical problems, heart disease,

endocrine diseases, including endocrine diseases, including

neurodegenerative and may be the neurodegenerative and may be the

source for the treatment of many other source for the treatment of many other

diseases.diseases.

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