ASSISTED REPRODUCTION TECHNICS Inseminations by husbandAIH by donorAID

ASSISTED REPRODUCTION TECHNICS

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

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

Insemination- conditions l l l 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

Preparation of sperm swim up method l filtration in Percoll gradient TARGET: l l l 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

Insemination performing l l l 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/

Efficacy l the highest : l the lowest : AID retrograde ejaculation cervix defects 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/

AID - conditions l l l 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

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

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

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

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

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

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

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

Partial Zona Dissection - PZD l l 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

Subzonal sperm insertion - SUZI l l l 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%

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, BM 1 HEPES medium

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/ l MI+co- culture with Vero line cells - maturity

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

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. , p. H, mineral oil SPERM CELLS: the best kinetic and morphologic parameters OOCYTES: immobilization, positioning

Intracytoplasmatic Sperm Injection - ICSI l l 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% l Risk: congenital defects - 2, 7% , chromosomal anomalies - 0, 5%

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

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

ICSI - course l l l 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

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

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

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