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Asisted Reproductive Techniques-Embryology

Asisted Reproductive Techniques-Embryology. Embryology Laboratory Practices. Oocyte Pick Up (OPU) Microinjection-İnsemination Embryo Culture Embryo Transfer Cryopreservation PGD Assisted Hatching. OPU ( Oocyte Pick Up ).

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Asisted Reproductive Techniques-Embryology

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  1. AsistedReproductive Techniques-Embryology

  2. EmbryologyLaboratoryPractices • OocytePickUp (OPU) • Microinjection-İnsemination • EmbryoCulture • Embryo Transfer • Cryopreservation • PGD • AssistedHatching

  3. OPU (OocytePickUp) • This process is carried out by vaginal route through a needle placed in an ultrasound probe. • Follicular fluid which is surrounded by cumulus cells,examined under a stereo microscope andthenthe oocytes collected. • Cumulus-oocyte complex maturation is evaluated before it is removed toincubator.

  4. After a period of incubation at least 2-3 hours, the cumulus cells areremoved by enzymatic and mechanicalprocedure. Selected by the microinjection process, a single sperm cell is injected into theoocytes. Microinjection procedure is performed with the assistance of micropipettes attached to an inverted microscope. Oocytes are controlledforfertilization after 14-16 hours. Fertilization rates increased from 80% to 90with the microinjection. Mikroinjection

  5. Mikroinjection

  6. ConventionalInVitroFertilization (IVF) İnsemination

  7. Pronükleer Evre 16-18 saat sonra 2 Hücreli Evre 24-27 saat sonra Stages of embryodevelopment (IVF-ET) 4 Hücreli Evre 41-44 saat sonra 8 Hücreli Evre 66-71 saat sonra Morula Evresi 4 gün sonra Blastosist Evresi 5 gün sonra

  8. Embryo Transfer Procedure • Embryos are transferred to the uterusafter oocyte collection process-two or three days. • In some patients, blastocyst transfer can be appliedtheday of 5 or 6. • Embryo transfer is very simple and painless procedure for thecandidate of mother. • Embryos were transferred to specially produced for this process by loading had abdominal catheterandleft into the uterus with the vaginal ultrasound-guided. • Transferred of embryos are determined by the number and quality of embryos and the age of mother. • After the transfer,remainingthetop quality of embryos arefrozen for use in thefuture.

  9. Top QualityEmbryoSelectionCriteria • Theday of 2(after 41-44 hours) have at least 4 or 5 blastomeres • Theday of 3 (after 66-71 hours) have at least 7 blastomeres • Cytoplasmicfragmentation<%20 • Blastomerelack of multinükleasyon (Van Royen et.al.1999,2001, Gerris et.al.1999)

  10. AssistedHatching • The embryo continues to grow and divideinto the uterus. • After a period of time to rip around layer of the zonapellucidaandimplant into the uterus. • Before being transferred the embryo, the thinning zonapellucida, makes it easier to holdtheuterus. • Specially; -Women over the age of 35 -In case of a thick zonapellucida -Recurrentimplantationfailure -In frozen embryo transfer. In our institution, the application time is too short, reliable, and reproducible technique using a laser high pregnancy andrates can be achieved.

  11. What is a co-culture? • In some patients, the treatment of the previous IVF attempts, embryo development was slow andpoor quality can not be observed and achieved repeated attempts to pregnancy. • Inthiscaseof patients for the development of better embryo, endometrial cells instead of synthetic feed solution was prepared by supporting a culture medium (co-culture) is used. • Previous trials of patients with this method is applied its own center than enables the development of good-quality embryos and pregnancy rates increase. • However, to apply this method is not used in several centers in the laboratory forrequires additional labor and technical support.

  12. How tomakeco-culture? • The luteal phase of the menstrual cycle of 20-23 days in the last period of endometrial samples are taken with the assistance of Pipelle. • After this mechanical and enzymatic digestion, stromal and glandular cells are separated in flasksandincubated . • 3 days beforeOPU, cells surrounds the entire surface of flasks (confluent) four-well (300,000 viable cells / well) are planted. • After the formation of PN, embryos are taken on these cells and incubated there until the blastocyst stage. • In this method, embryos are being extended on the endometrium like to the body'sandgrowth factors in the environment supports the development of the embryo. • In addition, toxins that can damage the embryo, removed from the environmentbytheantioxidants.

  13. Pre-implantationGeneticDiagnosis(PGD) Point; • Prevent transmission of chromosomal abnormalities • Reduce the risk of abort • Toselectgeneticallyhealthyembryos

  14. WhoAppliesto PGD? • Quantitativechromosomalabnormalities Anöploidi (Trisomies, monozamiler, nullizomiler), Triploidi, haploidi • Structuralchromosomalabnormalities Deletions, microdeletions (DiGeorge, Kallmanetc.) Translocations (ReciprocalandRobertsonian) Inversionandinsertion Sex-linkedrecessivediseases Duchennemusculardystrophy, fragile X syndrome, hemophilia, Huntersyndrome, Wiskott-Aldrichsyndrome, Lesch-Nyansyndrome, etc. • Single gene defects Autosomalrecessive (cysticfibrosis, beta-Thalasemia, Spinalmuscularatrophy (type I), etc. Autosomal dominant (Myotonicdystrophy, Marfan'ssyndrome, etc.)

  15. WhoAppliesto PGD? IVF • Recurrentimplantationfailure • Advanced femaleage • Recurrentmiscarriages • Patients with previous pregnancies, detectedaneuploidy

  16. NORMAL Pre-implantationGeneticDiagnosis(PGD) • FISH Diagnosis of numerical and structural chromosomal abnormalities • PCR Diagnosis of single gene defects

  17. Oocyte / EmbryoBiopsy Stages Day 1 Day 2 2nd polar body Day 3 Day 5 1st polar body blastosist biopsy blastomere biopsy Day 0

  18. EmbryoBiopsy Openingthe zona pellucida • Mechanical • Chemical (AcidTyrode’sSolution) • LaserTechnology

  19. 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.

  20. Paternal Lymphocyte Vaccine Who can Apply? • UnexplainedInfertility • RecurrentİmplantationFailure • RecurrentAbortions

  21. Paternal Lymphocyte Vaccine Necessary before the; • Candidates for ‘Dad’ analyzedof strong Hepatitis and HIV in terms. People whosetests (+) blood from, are not used to prepare the vaccine. • In cases where themother 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 anomaliesin babies or thrive.

  22. Totipotent Hücre Pluripotent Hücre Progenitor kök hücreler Kan Hücreleri İnsülin-salgılayan beta hücreleri Özerk hücreler Kas hücresi Sinir hücresi StemCell Embryo EmbryonicStemCell AdultStemCell

  23. Day 5 Day 3 Day 4 Day 1 Day 2 Pluripotent Totipotent EmbryonicStemCells

  24. Embryonic Stem Cells Trophectoderm ICM Evans ve Kaufman, 1981 (Mouse ES) Thompson , 1998 (Human ES)

  25. In laboratory conditions can be improved continuously, without any structural change. Can be create any type of adult cell inbody . Like bone, blood, muscle and liver tissues, such as creating an important source in the near future will be used for treatment purposes. EmbryonicStemCells

  26. CellSources Human Embryonic Stem Cell Culture • After the procedure, the unwanted frozen embryos donated for research purposes.

  27. Processes Embryonic Stem Cell Culture • The preparation of support cell • Removal of zona pellucida •İmmunesurgery •Directculture • Primarycolony production and passage •Long-termculture

  28. Embryonic Stem Cell Culture NIH embryonic stem cell identification criteria • Normal karyotype • ImmunocytochemicalAnalysis • SSEA-3 • SSEA-4 • TRA-1-60 • TRA-1-81 • Alkaline phosphataseexpression • Telomeraseexpression • Creating aSCID feature of mice teratoma

  29. Embryonic Stem Cell Culture ColonyMorphology Normal Karyotype AutonomousSurfaceAntigens CellMorphology

  30. Embryonic Stem Cell CultureInVitroDifferentiation • 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)

  31. In Vitro Differentiation of Human Embryonic Stem Cells Kistik EB Oluşumu Mekanik pasajlama Kültür <10 gün Jelatin-kaplı kültür kabı (endoderm) (ektoderm) (mezoderm)

  32. Invitrodifferentiation Embryonic Stem Cell Culture Nestin MAP2 a&b Troponin I

  33. Erken Faz Erken Faz Sarkomer Z Bandı Mitokondri Cardiomyocytedifferentiation-TEM Study Miyofibriller

  34. Areas of Use • Examination of embryonicdevelopment • İn Farmakology'detissue-specificdrugtesting • Toxicology in the identification of new agents • ForTherapeuticUse Embryonic Stem Cell Culture

  35. Areas of Use • Parkinson's and Alzheimer's disease • MS • Paralysis, SpinalCordInjuries Diabet • Jointand Bone damage • Liverdamage • Muscular dystrophy • Cardiacfailure, Myocardialinfarction • Canser • Hematologicdiseases Embryonic Stem Cells

  36. ClinicalTherapeuticUse Preliminarystudies : • After transplantation,undifferentiated human embryonic stem cellstomotor neuropathy paralyzed rats, recovery was observed in. Kerr DA, Llado J 2003 • After injection of ESCsto experimental myocardial infarction in ratswas determined improvement in contractile function and reduction of infarct size. Denice M. Hodgson, 2004 • Whenwere 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 kaynaklı dopaminerjik nöronların fonksiyon gösterdiği bulundu. Parkinson's model was createdin miceandwas found thats functional of dopaminergic neurons from embryonic stem cells. Kim JH, Auerbach JM, 2002 Embryonic Stem Cells

  37. Problems that need to be resolved before the clinical trial: • The use of materials from animal sources • Support systems, of human origin(HFF) • Theimmunesystem is the problem of compliance • Cloningfortherapeuticpurposes • Geneticmanipulation • Stemcell bank • Differentiation of protocolsincompatibility • The protection of life after transplantation • The risk of tumor • Ethicalproblems

  38. ResultandDiscussion • Embryonic Stem Cells can beunlimited culture under laboratory conditions,thisproperties an important source for cell therapy. • These cells in the near future, especially by solving the existing ethical and technical problems, heart disease, endocrine diseases, including neurodegenerative and may be the source for the treatment of many other diseases.

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