ASSISTED REPRODUCTION ASSISTED REPRODUCTION

TECHNICSTECHNICS

Inseminations :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 inseminationIndications to insemination

idiopathic sterility congenital defects retrograde ejaculation sperm’s hypowolemy OAT azoospermia sexual disorder

Insemination- conditionsInsemination- 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

Preparation of spermPreparation 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

Insemination performingInsemination 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/

EfficacyEfficacy

the highest : AID

retrograde ejaculation

cervix defects the lowest : OAT

endometriosismale 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 - conditionsAID - 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

AID technicAID technic

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

Advantages of AIDAdvantages of AID

patient’s safety anonymous of donor accessibility of sperm

IVF - indicationsIVF - 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

Course of IVFCourse 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

Assisted Reproduction Assisted Reproduction TechnicsTechnics

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 - MAFMicroassisted 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

In Vitro Fertilization - IVFIn Vitro Fertilization - IVF

classic micromanipulations : ICSI

SUZI

PZD

AZH ZIFT /PROST/ TET

Partial Zona Dissection - PZDPartial 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

Subzonal sperm insertion - SUZISubzonal 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

fertilizationpregnancy/cycle - 19%, pregnancy/transfer - 27%

Polispermic fertilizations - 50%

Intracytoplasmatic Sperm Intracytoplasmatic Sperm Injection - ICSIInjection - 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

Intracytoplasmatic Sperm Intracytoplasmatic Sperm Injection - ICSIInjection - ICSI

Microscopic assessment of oocyte

- untouched structure

- first polar body

- maturityof oocyte: 80% oocytes - MII20% oocytes -GV/Germinal Vesicle/

GVBD/Germinal Vesicle Braekdown/

MI /Metafase I/MI+co- culture with Vero line cells - maturity

Intracytoplasmatic Sperm Intracytoplasmatic Sperm Injection - ICSIInjection - ICSI

Microinstruments:

- injection pipet

- external diameter = 7 um

- internal diameter = 5 um

- holding pipet

- external diameter = 60 um

- internal diameter = 20 um

Intracytoplasmatic Sperm Intracytoplasmatic Sperm Injection - ICSIInjection - 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

Intracytoplasmatic Sperm Intracytoplasmatic Sperm Injection - ICSIInjection - 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%

MicromanipulationMicromanipulation

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

Micromanipulation -Micromanipulation -indicationsindications

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

ICSI - courseICSI - 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

GIFT - conditionsGIFT - conditions

minimally 1 non-obstruction Fallopian tube and ovary

regular uterine correct sperm

OthersOthers

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

TET - laparoscopic embryo transfer into ampulla of the

uterine tube

/Testicular Sperm Extraction - TESE/Testicular Sperm Extraction - TESETesticular Sperm Aspiration - TESA/Testicular Sperm Aspiration - TESA/

Conditions

azoospermia:-dysfunction of testicular tubules

fertilization - 60%

pregnancies - 30%

Micro-Epidydymal Sperm Micro-Epidydymal Sperm Aspiration - MESAAspiration - MESA

application :

azoospermia:

- lack of deferent duct

- obstruction of deferent ducts

FERTILIZATION OF PRECURSOR FERTILIZATION OF PRECURSOR CELLS OR IMMATURE SPERM CELLS OR IMMATURE SPERM CELLSCELLS

spermatide injection spermatide nucleous injection

RISK OF DEVELOPMENTAL ABNORMALITIES