Assisted Reproductive Techniques IUI (intrauterine insemination) AIH (artificial insemination by husband) AID (artificial insemination by donor) IUI (intrauterine insemination) AIH (artificial insemination by husband) AID (artificial insemination by donor) GIFT (gamet intrafallopian transfer) GIFT (gamet intrafallopian transfer) IVF (in vitro fertilization) ZIFT (zygote intrafallopian transfer) PROST (pronuclear stage intrafallopian transfer) IVF-ET (in vitro fertilization and embryo transfer) ICSI (intracytoplasmic sperm injection) IVF (in vitro fertilization) ZIFT (zygote intrafallopian transfer) PROST (pronuclear stage intrafallopian transfer) IVF-ET (in vitro fertilization and embryo transfer) ICSI (intracytoplasmic sperm injection)

Indications to IUI Cervical factor Cervical factor Chronic anovulation (COH-PCOS) Chronic anovulation (COH-PCOS) Male factor Male factor Immunologic disorders Immunologic disorders Endometriosis Endometriosis Idiopatic infertility Idiopatic infertility

IUI Proceeding Proceeding - Ovulation stimulation - Sperm preparation (>1-4·10 6 /ml) - Artificial insemination Efficacy (depended on indications and stimulation protocol) Efficacy (depended on indications and stimulation protocol) –10 – 30% pregnancies per cycle –40 – 60% accumulated no improvement after 4 cycles CONCLUSION: unjustified more than 4 correct IUI

Sperm preparation (IUI, IVF) gradient Sperm liquefaction Prepare the „gradient” Stratification on gradient Centrifugation Again centrifugation in EBSS ART semen % Silica 80% Silica

Ovulation stimulation for IUI Clomiphene citrate Clomiphene citrate –50 – 250 mg p.o., day 5-9 Clomiphene citrate + hMG (FSH) Clomiphene citrate + hMG (FSH) –50 – 250 mg p.o., day 5-9 –75 IU from day 9 hMG (FSH) hMG (FSH) –75 – 150 IU from day (3) 5 Aim Aim –growth 1-3 follicles to 18mm. When E 2 250–300 pg/ml/follicle IU hCG is administered to cause ovulation

Basic indications to IVF Partial or complete tubal obliteration Partial or complete tubal obliteration Chronic anovulation (COH-PCOS) Chronic anovulation (COH-PCOS) Male factor Male factor Immunologic disorders Immunologic disorders Endometriosis Endometriosis Idiopatic infertility Idiopatic infertility

Indications to ICSI Indications to ICSI with sperm from ejaculate O, A, T, OAT <1-4·10 6 /ml after preparation <5% normal forms failure of classic IVF (no fertilization) Indications to ICSI with sperm from ejaculate O, A, T, OAT <1-4·10 6 /ml after preparation <5% normal forms failure of classic IVF (no fertilization) Indications to MESA azoospermia (obstruction of ejaculatory ducts- obstructive azoospermia) Indications to MESA azoospermia (obstruction of ejaculatory ducts- obstructive azoospermia) Indications to TESE azoospermia (patency of ejaculatory ducts- nonobstructive azoospermia) Indications to TESE azoospermia (patency of ejaculatory ducts- nonobstructive azoospermia)

IVF-ET (classic) Ovulation stimulation Ovulation stimulation Sperm preparation Sperm preparation Collecting the oocytes (under ultrasound control) Collecting the oocytes (under ultrasound control) Oocytes maturity assessment Oocytes maturity assessment Oocytes insemination Oocytes insemination Fertilization assessment (16-24h) Fertilization assessment (16-24h) Embryo culture to 4 (48h) - 8 (72h) Embryo culture to 4 (48h) - 8 (72h) blastomers stage or to blastocyst stage (120h) Embryo transfer (ET) Embryo transfer (ET) Embryo cryopreservation Embryo cryopreservation

ICSI Ovulation stimulation Ovulation stimulation Sperm preparation Sperm preparation Collecting the oocytes (under ultrasound control) Collecting the oocytes (under ultrasound control) Oocytes maturity assessment Oocytes maturity assessment Intracytoplasmic sperm injection Intracytoplasmic sperm injection Fertilization assessment (16-24h) Fertilization assessment (16-24h) Embryo culture to 4 (48h) - 8 (72h) blastomers stage or to blastocyst stage (120h) Embryo culture to 4 (48h) - 8 (72h) blastomers stage or to blastocyst stage (120h) Embryo transfer (ET) Embryo transfer (ET) Embryo cryopreservation Embryo cryopreservation

Ovulation stimulation for IVF (COH – controlled ovarian hyperstimulation) Short protocoł aGnRH from day 1 hMG (FSH) 150–300IU from day 3 Short protocoł aGnRH from day 1 hMG (FSH) 150–300IU from day 3 Long protocoł aGnRH from day 20 previous cycle hMG (FSH) 150–300 IU from day 3 Long protocoł aGnRH from day 20 previous cycle hMG (FSH) 150–300 IU from day 3 Aim growth some follicles. When dominant follicle is >18mm and 2 other at least 16 mm and E2 >1000pg/ml but 18mm and 2 other at least 16 mm and E2 >1000pg/ml but <5000pg/ml (OHSS risk), IU hCG is administered to cause oocytes maturity)

IVF - ICSI (skuteczność) IVFICSI ICSI MESA ICSI TESE Fertilizations50%65%60%55% Cells divisions 90%95% Pregnancies per cycle 15-25%25-35%35-45%25-35%

IVF - ICSI (e) IVF - ICSI (e ffectiveness ) Implantation percentage when 1 embryo is transferred in stage 4 – 8 blastomers is 12,5 – 17,5% Implantation percentage when 1 embryo is transferred in stage 4 – 8 blastomers is 12,5 – 17,5% About 60% of embryos goes to stage of 4 blastomers (and far?) About 60% of embryos goes to stage of 4 blastomers (and far?) Pregnancies percentage per cycle (patients < 40) Pregnancies percentage per cycle (patients < 40) –Less than 7 oocytes - 13% –More than 7 oocytes - 29% Effectiveness of 1 mikrosurgery is equal with cumulative efficacy of 5 IVF trials

Cryopreservation Freezing and storage Freezing and storage –Embryos Stage 2 pronucleus Stage 2 pronucleus Stage 2-4 blastomers Stage 2-4 blastomers Stage blastocyst Stage blastocyst –Oocytes and ovarian tissue Benefits Benefits –Low cost, no OHSS, possibility of more „aggresive” ovulation stimulation in first cycle Effectiveness - 10 – 20% pregnancies per cycle

Preparation to cryo-ET Natural cycle (indication is growth the ovarian follicle) alternatively supplement therapy with estrogens and progestagens Natural cycle (indication is growth the ovarian follicle) alternatively supplement therapy with estrogens and progestagens Controlled cycle aGnRH with supplement estrogens and progestagens therapy Controlled cycle aGnRH with supplement estrogens and progestagens therapy

Complications and potential risk of ART Complications Complications –OHSS Rare cardio- pulmonary failure, renal failure, DIC... Rare cardio- pulmonary failure, renal failure, DIC... –Multiple pregnancy (5 – 40% !) Prematurity and preterm labours (to 98%), PIH (25%), bleeding (35%), anemia (15%), isthmocervical insufficiency (15%) Prematurity and preterm labours (to 98%), PIH (25%), bleeding (35%), anemia (15%), isthmocervical insufficiency (15%) Strategies: Transfer of 1-2 embryos; multiembryo transfer and consecutive embrioreduction or leaving this problem for obstetricians and neonatologists

Complications and potential risk of ART Potential risk Potential risk –Ovarian cancer Increased risk of serous carcinomas, low malignancy (high grade) Increased risk of serous carcinomas, low malignancy (high grade) More frequent after Clomiphene citrate More frequent after Clomiphene citrate No confirmation in large randomised clinical trials !!! No confirmation in large randomised clinical trials !!! –Theoretical risk of hormonosensitive neoplasm (breast, endometrium) –Genetic defects transfer Male infertility (AZF, delY...) Male infertility (AZF, delY...) Besides no risk of malformations was confirmed (but too short observations) – 2,2–2,7% Besides no risk of malformations was confirmed (but too short observations) – 2,2–2,7%

IVM & IVC IVM (in vitro maturation) IVM (in vitro maturation) –OHSS prevention –In vitro culture of ovarian follicles from antral to developed follicle– IVF – IVC – ET Multiple pregnancy prevention- IVC (in vitro culture) Multiple pregnancy prevention- IVC (in vitro culture) –In vitro culture of embryos to blastocyst stage (the best one for implantation) – 40-60% of pregnancies (blastocyst –sequential media) when compare to 12,5 – 17,5% (embryo in the stage of blastomers) –Culture the embryos to this stage make some problems. About % of embryos in vitro goes to blastocyst stage. –IVC gives the possibility of reliable evaluation the embryos quality.

Preimplantation diagnostic Indications Indications Age > 35 (?) Age > 35 (?) Previous child with chromosome abnormalities Previous child with chromosome abnormalities Carrier of genetic defects Carrier of genetic defects –Aneploidies (e.g. Down syndrome) –Monogenic disorders (np. fibrocystic disease) –X-linked inheritance (hemophilia) (important child sex) Sampling Sampling –Blastomers biopsy Methods Methods –PCR (polymerase chain reaction) –FISH (fluorescent in-situ hybridization)

Conditions to start ART IUI IUI –Woman Vaginal bacteriological examination Vaginal bacteriological examination –Man 3-7 days of sexual abstinence 3-7 days of sexual abstinence IVF IVF –Woman Vaginal bacteriological examination hormonal profile Cervical canal explore with a probe (?) Hysteroscopy (?) –Man 3-7 days of sexual abstinence Sperm bacteriological examination prophylactic antibiotic therapy (?)