Limits and Interfaces in Science Humboldt Kolleg Sao

Limits and Interfaces in Science Humboldt Kolleg Sao Paulo, Brasil 28. 30. November 2009 Reproductive Medicine: an update T. Cordes and K. Diedrich Dept. of Obstetrics & Gynecology University of Schleswig-Holstein, Campus Lübeck, Germany

City of Lübeck

City of Lübeck . . . around christmas time

Milestones in reproductive medicine • 1960 • 1970 • 1980 • 1990 • 2000 - ovarian stimulation with clomifene and gonadotrophins - radioimmunoassay - secretion, synthesis, mechanism of Gn. RH a. gonadotrophins - in vitro fertilisation - cryopreservation - recombinant gonadotrophins - preimplantation genetic diagnosis - intracytoplasmatic sperm injection (ICSI) - Gn. RH-antagonists and gonadotrophins - in vitro maturation of oocytes - embryonic stemcells - SET (single embryo transfer) - vitrification

Once upon a time… Birth after reimplantation of a human embryo Steptoe P. C. / Edwards R. G. Lancet 2 (1978): 366 07/78 Louise Brown was born

Number of infertile couples in Germany: approx. 15 20% of all couples (1. 2 – 1. 6 Millions)

Probability of successfull infertility treatment after: Dor et al. , 1996 cause of infertility pregnancy rate ovarian 57. 7% tubal 63. 3% male 71. 4%

Children born after ART until 2006 birth rate per cycle worlwide: 3. 9 Mio 26% in Germany : (1982 -2006) 148. 000 21% after in vivo fertilization 24%

New Developments in Reproductive Medicine • Ovarian stimulation: Gn. RH antagonists and long acting FSH • Elective single embryotransfer (e. SET) • Blastocyst transfer • Preimplantation genetic diagnosis and screening • In vitro maturation • Cryopreservation and vitrification

History of ovarian stimulation • 1970 Clomifen h. MG • 1980 Gn. RH-agonist / h. MG • 1990 rec. FSH Gn. RH-antagonist / h. MG or rec. FSH • 2000 long acting FSH

Efficiancy of ovarian stimulation with h. MG endogeneous LH surge (progesterone): 22% (Stanger et al. 1985) oocyte quality embryo quality pregnancy rate incidence after down regulation: <2%

Gn. RH agonist and antagonist protocol LHRH-agonist: daily injection/ depot/ nasal spray 8 OPU d -14 HCG 6 4 „long protocol“ Ampoules HMG ET 2 0 -16 -14 -12 -10 -8 Menses -6 -4 -2 0 2 6 8 10 12 14 16 17 day of cycle Cetrorelix 8 d 6 HCG 6 „Lübeck protocol“ 4 OPU 4 ET Ampoules Gonadotropins 2 Menses 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 day of cycle

Gonadotrophins & Gn. RH antagonist

Does the probability of live birth after ovarian stimulation and IVF depend on the type of Gn. RHanalogue used? A systematic review and meta-analysis G Griesinger, J Collins, B Tarlatzis, P Devroey, K Diedrich, EM Kolibianakis

LIVE BIRTH Odds ratio: 0. 859 Rate difference p=0. 085 2. 7%

Hospital admission due to OHSS RR : 0. 47 ~ 2 times less risk to be admitted due to OHSS with Gn. RH antagonists

Advantages of Gn. RH antagonists - fits into the normal cycle („the patients like it“) - less side effects in comparison to the long protocol: 1. 2. 3. 4. Ø cysts Ø hormonal withdrawl less OHSS simple - no significant difference in the probability of live birth between Gn. RH agonists and antagonists

long acting FSH follicle aspiration after 36 h FSH-CTP 10000 IE h. CG 1 2 3 4 5 6 7 8 9 10 11 12 13 14 …. Gn. RH-Antagonist LF 10 mm LF 14 mm LF 17 mm

New Developments in Reproductive Medicine • Ovarian stimulation: Gn. RH antagonists and long acting FSH • Elective single embryotransfer (e. SET) • Blastocyst transfer • Preimplantation genetic diagnosis and screening • In vitro maturation • Cryopreservation and vitrification

Children after ART: 1997 2002 IVF ICSI Total % Singleton 11455 12096 23551 59. 83 Twin 6782 6553 13335 33. 87 Triplet 1228 1221 2449 6. 22 Quadruplet 23 8 31 0. 08 Total 19488 19878 39366 100 DIR 2001

Problems of multiple pregnancies • pregnancy related diseases • prematurity • increase of neonatal morbidity and mortality • costs

Aims 1. To avoid multiple pregnancies 2. Improve the pregnancy and life birth rate Solution: Transfer of one selected embryo

Embryo selection > 30% Implantation < 5%

Pregnancy rate after elective single embryo transfer (e. SET) and elective double embryo transfer (e. DET) e. SET = 40. 3% pregnancy rate 1% gemini e. DET = 44% pregnancy rate 32% gemini Gerris, 2005 ESHRE

Prison sentence up to three years or financial penalty for § 1, Abs. 1, Nr. 3 „a person transfering more than 3 embryos to the womb in the course of one treatment cycle“ § 1, Abs. 1, Nr. 5 „a person fertilizing more oocytes than he or she intends to tranfer in the course of one treatment cycle“

Germany vs. Sweden No. of embryos / ET after IVF and ICSI, 2004 Deutschland Schweden 1 embryo 11. 6% 67. 4% 2 embryos 61. 3% 32. 5% 3 embryos 27. 1% 0. 1% Andersen et al. , 2008

Germany vs. Sweden: Multiple pregnancies after ART born children (IVF) / ET 18. 3% 26. 4% gemini / ET 20. 7% 5. 6% triplets / ET 1. 1% 0. 0% DIR 2004; Andersen et al. , 2008

New Developments in Reproductive Medicine • Ovarian stimulation: Gn. RH antagonists and long acting FSH • Elective single embryotransfer (e. SET) • Blastocyst transfer • Preimplantation genetic diagnosis and screening • In vitro maturation • Cryopreservation and vitrification

Blastocyst

Blastocyst culture Cochrane , 2007

Preimplantation Genetic Diagnosis Indication: High risk of genetic diseases. Preimplantation Genetic Screening (PGS) Aneuploidie Screening improves the pregnancy rate and abortion rate especially in older women (? ? )

PGS: a metaanalysis Mastenbroek et al. 2008, Hum. Reprod. 23

Präimplantationsscreening ASRM Committee Report, 2008 A careful review of the published studies of this technique led the ASRM Practice Committee to conclude that the available evidence does not support the use of PGS to improve live-birth rates in patients with advanced maternal age, previous implantation failure, recurrent pregnancy loss, or to reduce miscarriage rate in patients with recurrent pregnancy loss related to aneuploidy at this time.

New Developments in Reproductive Medicine • Ovarian stimulation: Gn. RH antagonists and long acting FSH • Elective single embryotransfer (e. SET) • Blastocyst transfer • Preimplantation genetic diagnosis and screening • In vitro maturation • Cryopreservation and vitrification

First oocyte maturation in vitro The Lancet 1965 „Oocytes from antral follicles can finalize their meiotic maturation in vitro in 24 – 48 hours“ R. G. Edwards et al. Edwards RG et al. 1965

Physiological basics of IVM Early oocyte retrieval from antral follicles before selection and atresia. . . day 3 day 8 - 12 d>10 mm

Development of IVM 1983 Veeck First birth after IVM 1991 Cha IVM on immature oocyte extracted by using ovarian biopsy during a cesarean section resulted in healthy twins 2000 Cha birth of 20 healthy children after IVM 2003 Mikkelsen birth of 33 healthy children after IVM today >300 children after IVM

Therapeutic indications for IVM • PCOS • high responder with a risk for OHSS • normo cyclic patients • cryopreservation of oocytes (oncology) • low responder • implantation failure Mikkelsen et al. 2003, Smitz 2005, von Otte 2005

Treatment protocol Estradot 100 first Examination: basic ultrasound and hormone level Exam. 2, 3, … 4 days ultrasound and hormone level of low dose h. MG („priming“ with 75 IE h. MG/day) Tag 1 2 Menses primordial follicle ≥ 12 mm and E ≥ 7 mm 10. 000 IE h. CG 3 4 5 6 7 8 36 h later aspiration of small antral follicles 9 Crinone 8% ♂ Male: sperms Embryo transfer 2 -3 days after ovum aspiration 10 …. IVM embryo oocyte maturation (24 h) (2 - 3 days) culture Fertilization via ICSI

In vitro maturation of oocytes number of patients 140 Metaphase II 57% 2 pronuclei after in vitro maturation and ICSI 47% pregnancies 30 v. Otte 2007

New Developments in Reproductive Medicine • Ovarian stimulation: Gn. RH antagonists and long acting FSH • Elective single embryotransfer (e. SET) • Blastocyst transfer • Preimplantation genetic diagnosis and screening • In vitro maturation • Cryopreservation and vitrification

Methods of cryopreservation slow cooling vitrification

Vitrification vs slow cooling V I T R I F I C A T I O N 37°C Slow freezing (equilibrated) H 2 O Temp Degrees C total dehydratation 5 35 H 2 O Room temperature 0. 3 – 0. 5°C/min > 25000°C/min weak dehydratation Weak dehydratation 80 196 Storage 10 20 30 Temps (min)

Slow cooling (Whittingham et al. , Science 1972, Willmut et al. , Life Science 1972) Vitrification (Rall and Fahy 1985) 2 sec. - 0. 3°C/min - 50. 000°C/min

In contrast to slow-rate freezing protocols, during vitrification the entire solution remains unchanged and the water does not precipitate, so no ice crystals are formed.

Clin. pregnancies / ET after cryo transfer (1996 2004) Cryo transfer Clin. pregnancy / ET Abortion rate after cryo transfer 67, 257 15. 5 % 21. 64 % German IVF Index 2004

Lübeck Results (till 01/2007) survival rate n=752 n=155 pregnancy rate

Future of Reproductive Medicine • new embryo protection law (european? ) • improvement of pregnancy rate by the elective single embryo transfer (morphological criteria) • avoid multiple pregnancies • vitrification • in vitro maturation • Fertility preservation for women with cancer • aim of infertility treatment: simple, safe, comfortable, successful and cheap

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