1. ASSISTED REPRODUCTION TECHNICS

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

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

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

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

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

7. Efficacy l the highest : AID retrograde ejaculation cervix defects l 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 l male interfility - azoospermia, examination of urinary sediment after ejaculation, biopsy of testes l OAT after unsuccessful AIH and resignation ICSI l transsexualismus l risk of infections and genetic disorders transmission l multiple, unsuccessful IVF or ICSI

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

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

11. IVF - indications l absolute : - absent or inoperable tubal obstruction l 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 l hormonal stimulation - CC, CC + HMG, GnRHa + HMG (SP, LP, Ultra SP, Ultra LP) l monitoring stimulation - USG, E 2 l ovulation indication - HCG (Biogonadyl, Pregnyl, Profasi) l punction l preparation of oocytes, sperm cells l insemination and incubation of oocytes in 5% CO 2 amd temp. 37 0 C l evaluation fertilization after 18 hours (2PN) l embryo transfer after 48 hours in st. 4-8 blastomers l freezing supernumerary embryons l suplementation of luteal phase

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

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

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

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

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

18. Intracytoplasmatic Sperm Injection - ICSI l Preparation of sperm: -separation of sperm cells by centrifugation in Percoll gradient -ejaculate with single sperm cells - washing and centrifugation + multiple swim-up method l 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 l 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 l 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 l 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 l Efficiency: 60-70% fertilizations l 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% l Risk : congenital defects - 2,7%, chromosomal anomalies - 0,5%

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

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

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

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

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

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

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

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