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ASSISTED REPRODUCTION TECHNICS

ASSISTED REPRODUCTION TECHNICS. Inseminations :. by husband -AIH, by donor-AID intravaginal-impotention, hypospadiasis, retrograde ejaculation, vaginismus cervical - OAT, cervical defects intrauterine - OAT, negative penetration test, idiopathic sterility. Indications to insemination.

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ASSISTED REPRODUCTION TECHNICS

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  1. ASSISTED REPRODUCTION TECHNICS

  2. Inseminations : by husband-AIH, by donor-AID intravaginal-impotention, hypospadiasis, retrograde ejaculation, vaginismus cervical - OAT, cervical defects intrauterine - OAT, negative penetration test, idiopathic sterility

  3. Indications to insemination • idiopathic sterility • congenital defects • retrograde ejaculation • sperm’s hypowolemy • OAT • azoospermia • sexual disorder

  4. Insemination- conditions • non- obstructed Fallopian tubes • monitoring of ovulation • induction of ovulation • bacteriological state of vagine, cervix, sperm • min. 1-5 mln sperm cells with progressive motility in 1 ml of sperm

  5. Preparation of sperm • swim up method • filtration in Percoll gradient TARGET: • separation of sperm cells from sperm plasma • selection and increase number of sperm cells with good morphology and motility • contaminations removal /dead sperm cells, bacterium/ • stimulation of capacitation

  6. Insemination performing • 1-3 times in the cycle /optimum before and after ovulation / • USG monitoring • ovulation induction - Clostilbegyt , HMG • verification /HSG , Echovist-test / • resignation after 6-10 unsuccessful inseminations /classification to laparoscopy or IVF/

  7. Efficacy • the highest : AID retrograde ejaculation cervix defects • the lowest : OAT endometriosis male infertility treated with AIH- 7% of pregnancies pro patient / 2,1% pro cycle / disturbation of ovulation-AIH-29% pregnancies pro patient /11% pro cycle/

  8. AID - conditions • male interfility - azoospermia, examination of urinary sediment after ejaculation, biopsy of testes • OAT after unsuccessful AIH and resignation ICSI • transsexualismus • risk of infections and genetic disorders transmission • multiple, unsuccessful IVF or ICSI

  9. AID technic • frozen sperm • sperm from sperm bank • collection during maximally 6 month • 1-3 times in the cycle

  10. Advantages of AID • patient’s safety • anonymous of donor • accessibility of sperm

  11. IVF - indications • absolute :- absent or inoperable tubal obstruction • relative : - tubal obstruction - periadnexal adhesions - idiopathic infertility - multiple, unsuccessful inseminations - endometriosis - male factor - PCO - immunologic infertility - genetic defects - early menopause - oocyte’s donation

  12. Course of IVF • hormonal stimulation - CC, CC + HMG, GnRHa + HMG (SP, LP, Ultra SP, Ultra LP) • monitoring stimulation - USG, E2 • ovulation indication - HCG (Biogonadyl, Pregnyl, Profasi) • punction • preparation of oocytes, sperm cells • insemination and incubation of oocytes in 5% CO2 amd temp. 370 C • evaluation fertilization after 18 hours (2PN) • embryo transfer after 48 hours in st. 4-8 blastomers • freezing supernumerary embryons • suplementation of luteal phase

  13. Assisted Reproduction Technics • male’s factor conditioned - failures - 60-80% • fertilizations - 20-30% inseminated oocytes • lack of fertilizations - 30% /a group with good reproduction’s potential/

  14. Microassisted Fertilization - MAF • facilitation of syngamy by mechanical or chemical dissection of zona pellucida • injection sperms into perivitelline space • injection single sperm cell into oocyte’s cytoplasm

  15. In Vitro Fertilization - IVF • classic • micromanipulations : ICSI SUZI PZD AZH • ZIFT /PROST/ • TET

  16. Partial Zona Dissection - PZD • make possible fusion of sperm cells with olemma and fertilization • mankaments of method: - high percent of oocytes with polispermic fertilization - high percent of non-fertilized oocytes

  17. Subzonal sperm insertion - SUZI • injection of sperm cells /5-15/ under oocyte’s zona pellucida • sperm cells - after capacitation - in the beginning acrosomal reaction • application: - severe oligoastenozoospermia -preceding IVF procedures - without fertilization pregnancy/cycle - 19%, pregnancy/transfer - 27% Polispermic fertilizations - 50%

  18. Intracytoplasmatic Sperm Injection - ICSI • Preparation of sperm: -separation of sperm cells by centrifugation in Percoll gradient -ejaculate with single sperm cells - washing and centrifugation + multiple swim-up method • Preparation of oocytes: - oocyte’s denudation from corona radiata cells / enzymatic and mechanic method/ - oocyte’ s incubation in the 60 IU/ml hialuronidaze’s solution - aspiration into the pipete (diameter of oocyte) - washing in Earle, BM1 HEPES medium

  19. Intracytoplasmatic Sperm Injection - ICSI • Microscopic assessment of oocyte - untouched structure - first polar body - maturityof oocyte: 80% oocytes - MII 20% oocytes -GV/Germinal Vesicle/ GVBD/Germinal Vesicle Braekdown/ MI /Metafase I/ MI+co- culture with Vero line cells - maturity

  20. Intracytoplasmatic Sperm Injection - ICSI • Microinstruments: - injection pipet - external diameter = 7 um - internal diameter = 5 um - holding pipet - external diameter = 60 um - internal diameter = 20 um

  21. Intracytoplasmatic Sperm Injection - ICSI • Methods: - microscope picture with Hoffman’s contrast -micromanipulators -microdrops: with oocytes, with sperm cells -PVP/poliwinylopirolidon/- slowness of sperm cells’ motility -environmental conditions: temp., pH, mineral oil SPERM CELLS: the best kinetic and morphologic parameters OOCYTES: immobilization, positioning

  22. Intracytoplasmatic Sperm Injection - ICSI • Efficiency: 60-70% fertilizations • Failure: - lack of motility sperm cells - injection sperm cells with round heads - oocytes with cytoplasm degeneration - oocyte lesion during procedure - complete lack of fertilization after ICSI - 3% • Risk: congenital defects - 2,7% , chromosomal anomalies - 0,5%

  23. Micromanipulation • ICSI - the most often • PZD - partial zona dissection • SUZI - subzonal sperm insertion • AZH - assisted zona hatching

  24. Micromanipulation -indications • lowered sperm parameters • < 500 000 motility serm cells in the ejaculate • lack of fertilization in preceding IVF procedures or fertilization lower than 5% cells /right sperm parameters/ • obstruction azoospermia

  25. ICSI - course • identical introduction like in IVF procedure • different preparation of oocytes (cleaning from granulosa cells) • micromanipulator’s introduction 1 sperm cell into cytoplasm of mature oocyte

  26. GIFT - conditions • minimally 1 non-obstruction Fallopian tube and ovary • regular uterine • correct sperm

  27. Others • ZIFT /PROST/ - laparoscopic zygote transfer into ampulla of the uterine tube in 2PN stage • TET - laparoscopic embryo transfer into ampulla of the uterine tube

  28. /Testicular Sperm Extraction - TESETesticular Sperm Aspiration - TESA/ • Conditions azoospermia: -dysfunction of testicular tubules fertilization - 60% pregnancies - 30%

  29. Micro-Epidydymal Sperm Aspiration - MESA • application : azoospermia: - lack of deferent duct - obstruction of deferent ducts

  30. FERTILIZATION OF PRECURSOR CELLS OR IMMATURE SPERM CELLS • spermatide injection • spermatide nucleous injection RISK OF DEVELOPMENTAL ABNORMALITIES

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