1. Limits and Interfaces in Science
Humboldt­Kolleg
Sao Paulo, Brasil
28.­30.
November 2009
RReepprroodduuccttiivvee MMeeddiicciinnee::
aann uuppddaattee
T. Cordes and K. Diedrich
Dept. of Obstetrics & Gynecology
University of Schleswig-Holstein, Campus Lübeck, Germany

2. City of Lübeck

3. City of Lübeck
... around christmas time

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

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

6. Number of infertile couples in
Germany:
approx. 15 ­20%
of all couples
(1.2 – 1.6 Millions)

7. Probability of successfull infertility treatment
after: Dor et al., 1996
cause of infertility pregnancy rate
ovarian 57.7%
tubal 63.3%
male 71.4%

8. Children born after ART until 2006
birth rate
per cycle
worlwide: 3.9 Mio 26%
in Germany : 148.000
21%
(1982-2006)
after in vivo fertilization 24%

9. New Developments in
Reproductive Medicine
• Ovarian stimulation: GnRH­antagonists
and long acting FSH
• Elective single embryotransfer (eSET)
• Blastocyst transfer
• Preimplantation genetic diagnosis and screening
• In­vitro­maturation
• Cryopreservation and vitrification

10. History of ovarian stimulation
• 1970 Clomifen
hMG
• 1980 GnRH-agonist / hMG
• 1990 recFSH
GnRH-antagonist / hMG or recFSH
• 2000 long acting FSH

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

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

14. Gonadotrophins & GnRH-antagonist

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

16. Citation Year 0,1 0,2 0,5 1 2 5 10
Effect Lower Upper PValue
Albano 2000 ,829 ,444 1,547 ,556
European 2000 ,748 ,519 1,078 ,119
Olivennes 2000 ,799 ,336 1,901 ,612
North American 2001 ,777 ,470 1,284 ,324
Middle East 2001 ,973 ,604 1,568 ,910
Akman 2001 ,760 ,177 3,263 ,712
Hohmann 2003 ,929 ,398 2,169 ,865
Martinez 2003 1,569 ,307 8,011 ,587
Franco 2003 ,545 ,065 4,562 ,573
Hwang 2004 1,105 ,347 3,526 ,866
Sauer 2004 1,067 ,334 3,409 ,913
Xavier 2005 ,845 ,288 2,484 ,760
Loutradis 2005 ,704 ,271 1,827 ,469
Malmusi 2005 1,000 ,257 3,888 1,000
Marci 2005 10,358 ,533201,451 ,062
Cheung 2005 1,550 ,242 9,940 ,642
Check 2005 1,818 ,518 6,382 ,347
Barmat 2005 ,651 ,262 1,621 ,356
Bahceci 2005 ,839 ,436 1,613 ,598
Badrawi 2005 ,803 ,320 2,015 ,640
Schmidt 2005 1,000 ,181 5,533 1,000
Lee 2005 ,696 ,229 2,114 ,522
FixedCombined (22) ,859 ,722 1,021 ,085
Favor agonists Favor antagonists
Odds ratio:0.859
p=0.085
LIVE BIRTH
Rate difference
2.7%

17. Hospital admission due to OHSS
Citation Year Rate1 Rate2 PValue 0,01 0,1 1 10 100
Effect Lower Upper
Albano 2000 ,00 ,01 ,20 ,16 ,01 3,91
Badrawi 2005 ,04 ,04 1,00 1,00 ,15 6,82
Bahceci 2005 ,04 ,07 ,49 ,62 ,15 2,49
European 2000 ,01 ,02 ,07 ,33 ,10 1,17
Lee 2005 ,07 ,10 ,72 ,73 ,13 4,04
Middle East 2001 ,02 ,05 ,07 ,34 ,10 1,17
North American 2001 ,01 ,02 ,76 ,76 ,13 4,46
Olivennes 2000 ,02 ,05 ,25 ,34 ,05 2,35
RandomCombined (8) ,01 ,47 ,27 ,84
Favor agonists Favor antagonists
RR : 0.47
~ 2 times less risk to be admitted due to OHSS with GnRH antagonists

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

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

20. New Developments in
Reproductive Medicine
• Ovarian stimulation: GnRH-antagonists and long acting FSH
• Elective single embryotransfer (eSET)
• Blastocyst transfer
• Preimplantation genetic diagnosis and screening
• In-vitro-maturation
• Cryopreservation and vitrification

21. Children after ART: 1997-2002
DIR 2001
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

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

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

25. Embryo selection
> 30% < 5%
Implantation

26. Pregnancy rate after
elective single embryo transfer (eSET) and
elective double embryo transfer (eDET)
eSET = 40.3% pregnancy rate
1% gemini
eDET = 44% pregnancy rate
32% gemini
Gerris, 2005
ESHRE

27. 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“

28. 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

29. 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

30. New Developments in
Reproductive Medicine
• Ovarian stimulation: GnRH-antagonists and long acting FSH
• Elective single embryotransfer (eSET)
• Blastocyst transfer
• Preimplantation genetic diagnosis and screening
• In-vitro-maturation
• Cryopreservation and vitrification

31. Blastocyst

32. Cochrane , 2007
Blastocyst culture

33. 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 (??)

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

35. 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.

36. New Developments in
Reproductive Medicine
• Ovarian stimulation: GnRH-antagonists and long acting FSH
• Elective single embryotransfer (eSET)
• Blastocyst transfer
• Preimplantation genetic diagnosis and screening
• In-vitro-maturation
• Cryopreservation and vitrification

37. 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

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

39. 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

40. 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

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

42. 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

43. New Developments in
Reproductive Medicine
• Ovarian stimulation: GnRH-antagonists and long acting FSH
• Elective single embryotransfer (eSET)
• Blastocyst transfer
• Preimplantation genetic diagnosis and screening
• In-vitro-maturation
• Cryopreservation and vitrification

44. Methods of cryopreservation
slow cooling
vitrification

45. --55
377°°CC
TTeemmpp DDeeggrreeeess CC
--880
--119966
V I T R I F I C A T I O N Slow freezing
RRoooomm
tteemmppeerraattuurree
(equilibrated)
0.3 – 0.5° C/min
TTeemmppss ((mmiinn))
Vitrification vs slow cooling
total dehydratation
10 20 30
H2O
> 25000°C/min
Storage
Storage
H2O
Storage
weak dehydratation
Weak dehydratation
--355

46. Slow cooling
(Whittingham et al., Science 1972,
Willmut et al., Life Science 1972)
- 0.3°C/min
Vitrification
(Rall and Fahy 1985)
2 sec.
- 50.000°C/min

47. In contrast ttoo ssllooww--rraattee ffrreeeezziinngg
pprroottooccoollss,, dduurriinngg vviittrriiffiiccaattiioonn tthhee eennttiirree
ssoolluuttiioonn rreemmaaiinnss uunncchhaannggeedd aanndd tthhee
wwaatteerr ddooeess nnoott pprreecciippiittaattee,, ssoo nnoo
iiccee ccrryyssttaallss aarree ffoorrmmeedd..

48. Clin. pregnancies / ET
after cryo transfer
(1996-2004)
Cryo transfer 67,257
Clin. pregnancy / ET 15.5 %
Abortion rate after cryo transfer 21.64 %
German IVF Index 2004

49. Lübeck Results
(till 01/2007)
survival rate pregnancy rate
Slow-cooling Vitrifikation
100%
80%
60%
40%
20%
0%
59%
91%
Slow-cooling Vitrifikation
50%
40%
30%
20%
10%
0%
19%
34%
n=752 n=155

51. 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

52. Aachen
Berlin
Bonn
Bremen
Dresden
Düsseldorf
Duisburg
Erlangen
Frankfurt
Freiburg
Giessen
Göttingen
Greifswald
Halle
Hamburg
Hannover
Heidelberg
Innsbruck
Homburg
Jena
Kiel
Köln
Leipzig
Lübeck
Magdeburg
Mainz
Marburg
München
Münster
Rostock
Tübingen
Ulm
Würzburg
usw.
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www.fertiprotekt.de
Universitäres Kinderwunschzentrum Lübeck UKL
Thank you!
tim.cordes@uk-sh.de