assisted reproduction technics. inseminations : l by husband-aih, by donor-aid l...
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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