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

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

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

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

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

Muasher et al, Hum Reprod 1991

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

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

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

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

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

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

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

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

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

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

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

Endometrial Wave Studies

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

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

Intramuscular vs Vaginal Progesterone?

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

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

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)

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)

Number of sub-endometrial waves and pregnancy

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

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

Atosiban in IVF Moraloglu et al, Reprod Biomed Online 2010

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

Individualized Embryo Transfer

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

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

Dating vs ERA Diaz-Gimeno et al Fertil Steril 2013

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

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

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

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

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

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