1. The Endometrium and Frozen Embryo Transfer
Robert F Casper MD
University of Toronto, Division of Reproductive Sciences, Lunenfeld-Tanenbaum Research Institute
and
TRIO Fertility, Toronto

2. Disclosures
Scientific Advisory Boards:
Ferring, EMD-Serono and Merck

3. Learning Objectives
At the end of this presentation, the participants will be able to:
Discuss ultrasound monitoring of the endometrium
Describe hormonal endometrial preparation for FET
Compare methods available to determine optimal timing of embryo transfer

4. Human Embryo Freezing
First report of human embryo freezing and pregnancy by Trounson and Lohr in 1983
Natural cycles or hMG stimulated cycles used for FET

5. Hormonal Preparation
First reports of hormonal replacement for endometrial preparation in donor egg cycles around 1985
GnRH agonist downregulation and hormonal replacement for FET in 1991
Increasing doses of E2 to mimic natural follicular phase
Endometrial biopsy to determine if endometrium in phase in the cycle prior to FET

6. Muasher et al, Hum Reprod 1991

7. Ultrasound and Endometrium
Stimulation protocols for IVF in most programs in the 1980s used CC and hMG
In 1989, we reported a correlation between endometrial thickness and pregnancy rates in IVF
In 1990, we published the possible importance of endometrial pattern
Gonen et al, Fertil Steril 1989
Gonen and Casper, JIVFET 1990

8. Ultrasound and Endometrium
We observed that few pregnancies occurred with endometrial thickness below 6 mm
We later showed that a triple line pattern was more favorable for implantation than a hyperechogenic pattern

10. Ultrasound and Endometrium
In 1990, we also published the observation of an adverse effect of CC on the endometrium
Basis for the development of letrozole for ovulation induction
Gonen and Casper, Hum Reprod 1990

11. h
Natural Cycle
CC Cycle
Gonen and Casper, Hum Reprod 1990

12. Ultrasound and Endometrium
We hypothesized that ultrasound measurement of endometrial thickness and pattern could be used to determine when to add progesterone for FET or DEP
We also hypothesized that a simple fixed dose regimen of E2 could be used for endometrial preparation
Shapiro et al, 1993

13. Ultrasound for Endometrium
12 women enrolled; 6 with POF, 4 with Turner’s syndrome, 2 with surgical menopause
Maintained on 2 mg E2 while waiting for a volunteer donor <35 y from the IVF program
Once a donor identified, E2 dose increased to 4 mg daily or 8 mg daily
Daily ultrasounds done until favorable endometrium developed (>6 mm and triple line pattern)
Shapiro et al, 1993

14. Ultrasound and Endometrium
Favorable endometrium on U/S achieved in a mean of 5 days for 8 mg E2 and in 7 days for the 4 mg daily dose
On day of oocyte retrieval 100 mg P4 in oil IM daily
Up to three day 2 embryos transferred on day 3 of progesterone
Pregnancy rate 42% overall and 62.5% if a favorable endometrium was present
Shapiro et al, 1993

15. Conclusion
Endometrium >6 mm and triple line pattern on U/S could predict implantation in donor egg cycles
Favorable endometrium can be achieved with different doses and time periods of estrogen
Larger doses resulted in quicker growth
Unlike endometrial biopsy, ultrasound monitoring of endometrial development is non-invasive, inexpensive and can be done in the cycle of interest
Shapiro et al, 1993

16. Nine studies (8263 cycles) NC vs HT (OR 1.2, 95% CI 0.86–1.6)
Meta-analysis of Endometrial Preparation for FET Clinical Pregnancy Rates
Five studies (1965 cycles) NC vs modified NC (OR 0.91, 95% CI 0.74–1.1)
Nine studies (8263 cycles) NC vs HT (OR 1.2, 95% CI 0.86–1.6)
Five studies (2789 cycles) NC vs HT with GnRH agonist (OR 0.82, 95% CI 0.67–1.0)
Three studies (631 cycles) HT vs HT with GnRH agonist (OR 0.77, 95% CI 0.44–1.4)
Groenewoud et al, Hum Reprod 2014

17. Summary
No difference in clinical pregnancy or live birth rates between any of the endometrial preparation protocols for FET
Hormone replacement cycles with or without GnRH agonist suppression
Natural cycles or hCG triggered natural cycles with or without luteal support
Groenewoud et al, Hum Reprod 2014

18. Conclusion
Use simplest, least intrusive protocol for endometrial preparation
Single ultrasound for endometrial thickness and pattern
Natural progesterone and ET after 4 or 6 days for day3 or day5 embryos, respectively

19. Endometrial Wave Studies

20. Sub-endometrial Uterine Contractions
Fanchin R et al, Hum Reprod 1998

21. Uterine Contractions and Pregnancy Rate
Fanchin R et al, Hum Reprod 1998

22. Intramuscular vs Vaginal Progesterone?

23. Is Intramuscular Progesterone Better than Vaginal?
USA mainly intramuscular P4
Europe and Canada mainly vaginal P4
Studies with fresh ET where there is a CL and endogenous P4 present showed no difference although results inconsistent
No randomized studies in FET or Donor egg cycles where there is no CL

24. Is Intramuscular Progesterone Better than Vaginal?
Hypothesis that depot effect of IM P4 and continuous exposure may be better than vaginal P4
Serum conc of P4 greater with IM than vaginal
Vaginal route of administration results in discreet peaks of P4 with low serum P4 levels
But EB studies showed increased endometrial concentrations of P4

25. Is Intramuscular Progesterone Better than Vaginal?
RCT of 34 women undergoing frozen blastocyst transfers at TRIO Fertility
Randomized to receive vaginal progesterone (200 mg micronized P4 three times daily) or intramuscular P4 (50 mg P4 in oil) once daily
Primary outcome measure number of endometrial waves on day 5 of progesterone
Women in either group could choose preferred P4 administration starting the day after the FET
Hershko-Klement et al (submitted)

26. Is Intramuscular Progesterone Better than Vaginal?
No difference between groups in number of women with 3 or more contractions on day before FET
There was an association between sub-endometrial wave frequency and pregnancy
Hershko-Klement et al (in preparation)

28. Number of sub-endometrial waves and pregnancy

29. IM vs Vaginal P4
If ≥ 3 contractions, IM progesterone given that evening and wave study repeated before the embryo thawed
In most cases, contractions diminished in number and FET done
If still ≥ 3 contractions, FET cancelled

30. Atosiban in IVF
180 women undergoing ICSI with top quality embryos Randomized to atosiban or placebo intravenously Day 3 embryo transfer (median 3 embryos) Difficult embryo transfers were excluded
Moraloglu et al, Reprod Biomed Online 2010

31. Atosiban in IVF
Moraloglu et al, Reprod Biomed Online 2010

32. Atosiban RCT in IVF
800 general IVF patients in China, Hong Kong and Vietnam
Randomized to receive atosiban (n=400) or saline (n=400) on day of embryo transfer
Maximum of two to four day 3 embryos transferred
Atosiban (or saline) IV bolus 30 min prior to fresh embryo transfer followed by IV infusion for 3 hours
No difference in any pregnancy rate parameter (live birth rate 39.8% vs 38%)
Ng et al, Hum Reprod 2014

33. Individualized Embryo Transfer

34. Endometrial Receptivity Array (ERA)
Miravet-Valenciano et al, Curr Opin Obstet Gynecol, 2015

35. Preliminary Study using ERA
25 control women (0 or 1 previous failed cycle)
EB on LH+7 in natural cycles or day 6 of P4 in HRT cycles
88% of EB were receptive on ERA
85 patients with RIF (≥ 3 previous failed cycles)
74.1% biopsies were receptive on ERA
25.9% biopsies were non-receptive on ERA
Ruiz-Alonso et al, Fertil Steril 2013

36. Dating vs ERA
Diaz-Gimeno et al Fertil Steril 2013

38. Individualized Embryo Transfer
Failed pregnancy with good quality embryos for transfer in at least 2 cycles
Mock FET cycle with EB on day 6 or 7 of progesterone
Standard endometrial dating (Noyes criteria)
Adjust frozen embryo transfer in next cycle according to delay in cycle dating if present
Gomaa et al, RBMO, 2015

39. Individualized Embryo Transfer
80 women with mean age 37 years
Mean of 2.4 previous embryo transfers with top quality embryos
Biopsies in 26% of women were out of phase by 3 to 5 days (delayed)
Progesterone administration extended by up to 3 days (FET on day 9 of progesterone) in women with out of phase endometrium
Gomaa et al, RBMO, 2015

40. Individualized Embryo Transfer
Women with endometrium in phase and no change in transfer day had pregnancy in 16/71 cycles (22.5%)
Women with out of phase endometrium and delayed transfer had pregnancy in 8/22 cycles (36.4%)
Gomaa et al, RBMO, 2015

41. Endometrial Dating Study
130 women with regular cycles
Proven fertility and mean age 35 years
Endometrial biopsy randomized for various times in the luteal phase
Urine LH tests for mid-cycle surge
Endometrial biopsies cut into 3 consecutive thin H&E sections and read by 3 experienced pathologists blinded to day of biopsy
Murray et al, Fertil Steril, 2004

42. Endometrial Dating (Noyes Criteria)
Murray et al, Fertil Steril, 2004

44. Clinical FET Suggestions
Use simplest E2 protocol for endometrial preparation
Can add progesterone when ultrasound shows favorable endometrial thickness and pattern
No difference in vaginal or IM progesterone
FET after 6 days of progesterone for blastocysts
Can prolong progesterone if endometrium delayed on biopsy (ERA or Noyes)
Do wave study on day before FET
If more than 2 waves per minute, add IM progesterone (50 mg) that evening