Intramuscular progesterone versus 8% Crinone vaginal gel for luteal phase support for day 3 cryopreserved embryo transfer

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1 Intramuscular progesterone versus 8% Crinone vaginal gel for luteal phase support for day 3 cryopreserved embryo transfer Daniel J. Kaser, M.D., a Elizabeth S. Ginsburg, M.D., a Stacey A. Missmer, Sc.D., a,b,c Katharine F. Correia, M.A., a and Catherine Racowsky, Ph.D. a a Department of Obstetrics, Gynecology, and Reproductive Biology and b Department of Medicine, Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School; and c Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts Objective: To compare outcomes after intramuscular progesterone (IMP) or 8% Crinone vaginal gel for luteal support for day 3 cryopreserved embryo transfer (CET). Design: Retrospective cohort study with multivariable analysis. Setting: Academic medical center. Patient(s): All autologous and donor egg in vitro fertilization and intracytoplasmic sperm injection patients who had a day 3 CET from January 1, 2008, to April 30, 2011, with luteal support using mg/d IMP or 8% Crinone twice daily, initiated 3 days before the CET. Intervention(s): None. Main Outcome Measure(s): Implantation rate, clinical pregnancy, and live birth rates per CET. Result(s): IMP (n ¼ 440) and Crinone (n ¼ 298) recipients were similar for all demographic characteristics and cycle parameters assessed. Although implantation rates did not differ significantly between the two groups (Crinone vs. IMP: 19.6% vs. 30.4%), women supplemented with Crinone had significantly lower rates of clinical pregnancy (36.9% vs. 51.1%) and live birth (24.4% vs. 39.1%) compared with those on IMP. Conclusion(s): We observed that day 3 CET cycles with 8% Crinone luteal support had a 44% and 49% lower odds of clinical pregnancy and live birth, respectively, compared with those with IMP support. Further studies are required to identify the optimal timing and dose of 8% Crinone vaginal gel for use in CET cycles. (Fertil Steril Ò 2012;98: Ó2012 by American Society for Reproductive Medicine.) Key Words: Crinone vaginal gel, intramuscular progesterone, luteal phase support, frozen, pregnancy Discuss: You can discuss this article with its authors and with other ASRM members at fertstertforum.com/kaserdj-crinone-luteal-phase-support-intramuscular-progesterone/ Use your smartphone to scan this QR code and connect to the discussion forum for this article now.* * Download a free QR code scanner by searching for QR scanner in your smartphone s app store or app marketplace. Progesterone is required for implantation and maintenance of early pregnancy until the luteoplacental shift, which occurs at 8 10 weeks' gestation. It is the standard of care to provide exogenous P to women undergoing fresh and cryopreserved (CETs) embryo transfers. In fresh IVF cycles, luteal support is necessary because endogenous P is decreased due to GnRH down-regulation and disruption of mural granulosa cells at oocyte retrieval (1 4). In comparison, there is minimal endogenous P production in Received May 18, 2012; revised and accepted August 7, 2012; published online September 6, D.J.K. owns stock in Merck and Company and Cardinal Health. E.S.G. has nothing to disclose. S.A.M. has nothing to disclose. K.F.C. has nothing to disclose. C.R. is a board member of the American Society for Reproductive Medicine. Reprint requests: Catherine Racowsky, Ph.D., Division of Reproductive Medicine, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA ( cracowsky@partners.org). Fertility and Sterility Vol. 98, No. 6, December /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. CET and donor cycles, so pharmacologic support is necessary (5). The preferred route of administration for P in CET cycles is an area of active research. The current criterion standard is intramuscular progesterone (IMP). Oral P is available but undergoes extensive hepatic metabolism, may cause significant sedation, and has limited bioavailability to achieve secretory endometrium (6). Intravaginal formulations such as 8% Crinone gel or other vaginal inserts are reported to have better patient satisfaction due to limited systemic absorption and ease of use (7). Although good-quality data 1464 VOL. 98 NO. 6 / DECEMBER 2012

2 Fertility and Sterility support the equivalence of Crinone and IMP for fresh in vitro fertilization (IVF) cycles (7 13), the most effective P for CET cycles remains to be determined (14 20). The objective of the present study was to assess the efficacy of IMP versus Crinone for P support in CET cycles by comparing implantation and pregnancy rates between the two treatment groups. MATERIALS AND METHODS This study was approved by the Partners' Healthcare Institutional Review Board. Experimental Design All autologous and egg donor CET cycles with day 1 or day 3 freeze and day 3 embryo transfer performed at Brigham and Women's Hospital from January 1, 2008, to April 30, 2011, were reviewed for the type of P used for luteal phase support. Day 3 ET accompanied by day 3 cryopreservation of supernumary embryos is standard in our program. Patient characteristics, CET cycle parameters, post-thaw embryo survival and morphology, and clinical outcomes were compared between those supplemented with 25 mg IMP for 1 day, then increasing to 50 mg/d (locally compounded at one of two pharmacies: Village Fertility, Waltham, Massachusetts, or Freedom Fertility, Byfield, Massachusetts) and those supplemented with micronized P in 90 mg bioadhesive vaginal gel once per day for 1 day and then twice daily (8% Crinone; Watson Pharmaceuticals) until 10 weeks gestational age. Cycles were excluded for the following reasons: day 2 or day 5 cryopreservation, day 5 transfer, biopsied embryos, no luteal phase support (i.e., natural cycle), IMP dose other than mg/d, and P formulations other than IMP or Crinone. Clinical Protocols Standard controlled ovarian hyperstimulation, insemination, and intracytoplasmic sperm injection (ICSI) protocols were used for fresh autologous and donor egg cycles as previously described (21). Pronuclear zygotes and day 3 embryos were then cryopreserved as outlined below. In preparation for CET, a GnRH agonist was started on cycle day 21 of the prior cycle for pituitary down-regulation, with a combined oral contraceptive lead-in for patients with oligoovulation or polycystic ovary syndrome. Baseline serum testing was performed on cycle day 2. In patients with serum P <3 ng/ml, 3 mg estradiol acetate (Estrace; Warner Chilcott) by mouth twice daily was used to achieve a target endometrial echocomplex (EEC) of R7 mm. In patients who did not achieve the 7 mm target thickness, estradiol acetate was continued for an additional week. If the EEC still remained <7 mm after an additional week of oral estrogen, the cycle was usually canceled and a different estrogen priming protocol prescribed (e.g., 1 2mgE 2 per vagina twice daily or 0.3 mg transdermal patch every other day). Progesterone was started in 7.5% of cycles (55/738) with EEC <7 mm (6.7% of Crinone, 7.9% of IMP). Luteal P with IMP or Crinone was initiated 3 days before transfer (i.e., the day of transfer was the fourth day of P for patients receiving day 3 thawed embryos as well as for those receiving day 1 thawed embryos cultured to day 3). The type of P used for luteal support was left to the discretion of the clinician and did not represent a change in institutional protocol during the study period. Supplemental P was continued until 10 weeks' gestation or until a negative serum quantitative hcg. Day 3 ETs were routinely performed with the use of a Wallace catheter (Marlow/Cooper Surgical); difficult transfers were done with a Sureview catheter (Smiths-Medical). Laboratory Protocols Two-pronuclear zygotes and day 3 embryos were cryopreserved with the use of Liebo's one-step slow-freeze protocol (22). Embryos were washed in HTFþHEPES buffer, supplemented with 10% human serum albumin, and allowed to equilibrate at room temperature for minutes before being transferred to 1.5 mol/l 1,2-propanediol cryoprotectant (PG solution) for a further minutes. Straws (0.25 ml; IMV-ZA475 or MT19040/0010; Origio) were loaded with PG solution, followed by air, the embryos in PG solution, air, and then a column of the 1.08 mol/l sucrose. Sealed straws were placed in a controlled rate Biocool freezing machine (FTS), in an ethanol bath at 6.0 C and seeded with supercooled forceps. Cooling at a rate of 0.4 C per minute was carried out to a final temperature of 40.0 C. Straws were then plunged into liquid nitrogen for storage. For thawing, straws were allowed to sit at room temperature for 2 minutes before mixing the contents. The straws were then warmed at 37 C for 3 minutes, followed by a 1-minute incubation at room temperature. Expelled embryos were then serially washed at room temperature in HTFþHEPES supplemented with 10% human serum albumin and incubated at room temperature for 10 minutes before being transferred into growth media (G1.5 [Vitrolife] or Global [Lifeglobal]). Post-thaw viability, morphology, and cell stage were recorded. Our standardized protocol for number of embryos to thaw was applied which, for autologous embryos, takes into consideration the patient's age, the number of embryos to transfer for a fresh cycle, and availability of thawed embryos with blastomere survival of R50%; for donor cycles, two embryos were transferred, with the lead embryo having at least four cells and the other with at least two cells. Day 1 embryos were cultured in G1.5 or Global medium until day 3 for transfer, and cleavage-stage embryos were incubated for a minimum of 2 hours before ET. Number of embryos transferred was determined by the number and morphology of available embryos, patient age at embryo cryopreservation, and clinical history. Outcome Variables Female patient demographics, including patient age at cryopreservation and transfer, body mass index (BMI) of patient or embryo recipient, day 3 FSH, parity, number of prior failed cycles and spontaneous abortions, endometrial thickness on day of mapping, percentage of cycles with assisted hatching, uterine-factor infertility, use of a gestational carrier, and outcome in corresponding fresh cycle were compared. A prior failed cycle was defined as any fresh or frozen cycle that did not result in a live birth. Uterine factors included VOL. 98 NO. 6 / DECEMBER

3 ORIGINAL ARTICLE: ASSISTED REPRODUCTION adenomyosis, exposure to diethylstilbestrol, fibroids without prior myomectomy, intrauterine synechiae, or a unicornuate system. In cycles derived from donor eggs, the age of the egg donor at freeze was used for patient age, and the age of embryo recipient was used for uterine age. The proportion of embryos with R50% percent blastomere survival was recorded. Because more than one embryo was typically frozen per straw, it was not possible to track every embryo individually from cryopreservation to thaw. However, it was possible to determine the number of embryos thawed with R50% blastomere survival. Other variables analyzed were postthaw embryo morphology according to cell number, fragmentation, and symmetry as previously described (23). Briefly, a fragmentation score of 0%, 1% 9%, 10% 25%, 26% 50%, or >50% was assigned to each embryo. Symmetry scores corresponded to perfect symmetry, moderate asymmetry, and severe asymmetry. A good-quality embryo was defined as having R7 cells on day 3 without regard to fragmentation or symmetry. Lead cell number was defined as the number of blastomeres in the embryo transferred with the most number of cells. Standard clinical outcomes of implantation rate, clinical pregnancy and live birth rates were calculated. Implantation rate was defined as the number of fetal heart beats on 6-week ultrasound divided by the total number of embryos transferred. Clinical pregnancy rate was defined as the proportion of transfers that resulted in at least one intrauterine gestational sac. Spontaneous abortion referred to loss of a clinical pregnancy before 20 weeks' gestation. Live birth rate was defined as the number of transfers that resulted in live birth. Delivery outcome was missing for 11 cycles that had documented clinical pregnancies but unknown final outcome; those cycles were not included in the analysis of live birth. Statistical Analyses Statistical analyses were performed with the use of Statistical Analysis Software version 9.2 (SAS Institute). Effect estimates, 95% confidence intervals (CIs), and two-sided Wald P values were obtained from generalized estimating equations to account for the correlation between multiple cycles from the same woman after adjusting for patient age at embryo cryopreservation and day of cryopreservation a priori (because cryopreservation at the pronuclear stage at our institution typically occurs preferentially for good-prognosis patients at high risk for ovarian hyperstimulation syndrome [OHSS]). The binomial distribution with logit link, yielding odds ratios (ORs), was applied for all outcomes except implantation rate. The Poisson distribution with log link was used to evaluate implantation rate (outcome of number of fetal hearts at 6 weeks as described above, offset by the number of embryos transferred), yielding relative risks (RRs). Additional variables tested for as potential confounders included uterine factor diagnosis, assisted hatching, obesity, parity, uterine age at transfer, use of a gestational carrier, number of embryos thawed, total number of cells transferred, CET difficulty (easy, some difficulty, major difficulty), mean fragmentation and symmetry scores of transferred embryos, endometrial thickness on day of mapping, and outcome in corresponding fresh cycle (live birth, transfer but no live birth, no transfer). Because the effect estimate for P group did not change by >10% from the base model with the addition of any one of the potential confounders, none were retained in the final model (24). Final models therefore adjusted for patient age at embryo cryopreservation and day of embryo cryopreservation (day 1 vs. day 3). Of the 25 gestational carrier cycles, all but one was supported with IMP. Therefore, a subanalysis of the full study population was performed excluding gestational carrier cycles, to consider confounding by indication. Additional subanalyses were performed restricting the included cycles to those with a day 3 freeze and those derived from autologous versus donor oocytes. The exclusion of an effect estimate of 1.0 from the confidence interval as well as complementarily the Wald two-sided P values of <.05 were considered to be statistically significant. RESULTS Patient Demographics and Cycle Parameters Four hundred forty day 3 CET cycles with IMP luteal support and 298 cycles with Crinone luteal support were identified during the study period. Patient demographics were similar between the two P groups for all characteristics assessed (Table 1). Moreover, assisted hatching was similarly utilized TABLE 1 Patient demographics for day 3 cryopreserved embryo transfer cycles supported with intramuscular progesterone (IMP) versus Crinone. IMP (n [ 440) 8% Crinone (n [ 298) Age at embryo cryopreservation (y) Age at transfer (y) Parity (53.0) 162 (54.4) R1 207 (47.0) 136 (45.6) No. prior failed cycles a 0 81 (18.4) 55 (18.5) R1 359 (81.6) 243 (81.5) No. prior SAB (63.4) 203 (68.1) R1 161 (36.6) 95 (31.9) Day 3 FSH (miu/ml) BMI (kg/m 2 ) of embryo recipient No. obese embryo recipients b 61 (14.2) 60 (20.6) Primary diagnosis Unexplained 83 (19.3) 67 (22.8) Male factor 108 (25.1) 62 (21.1) Tubal factor 34 (7.9) 35 (11.9) Anovulation 56 (13.0) 46 (15.6) Endometriosis 21 (4.9) 20 (6.8) Diminished reserve 84 (19.5) 57 (19.4) Uterine factor c 15 (3.5) 5 (1.7) Gestational carrier 24 (5.6) 1 (0.3) Other 5 (1.2) 1 (0.3) Note: Values represent n (%) or mean SD. BMI ¼ body mass index; SAB ¼ spontaneous abortion. a Failed cycle refers to any fresh or frozen cycle without live birth. b Obesity defined as BMI R30 kg/m 2. c Uterine factors include adenomyosis, exposure to diethylstilbestrol, fibroids without prior myomectomy, intrauterine synechiae, or a unicornuate system. Kaser. IMP vs. Crinone for cryopreserved cycles. Fertil Steril VOL. 98 NO. 6 / DECEMBER 2012

4 Fertility and Sterility TABLE 2 Day 3 post-thaw embryo survival, embryo quality, and transfer characteristics for cryopreserved embryo transfer cycles supported with intramuscular progesterone (IMP) versus Crinone. IMP (n [ 440) 8% Crinone (n [ 298) No. embryos thawed No. cells pre-thaw No. viable cells post-thaw No. embryos with R50% survival Total no. cells transferred Lead cell no. at transfer No. good-quality embryos transferred a No. embryos transferred Note: Values represent mean SD. a Good-quality embryo was defined as R7 cells on day 3 without regard to fragmentation or symmetry. Kaser. IMP vs. Crinone for cryopreserved cycles. Fertil Steril in both groups (37.7% vs. 38.9%), endometrial thickness on the day of mapping was equivalent ( vs mm), and there was no difference in the percentage of cycles having day 3 cryopreservation (88.4% vs. 86.2% [IMP vs. Crinone, respectively]). The percentage of cycles with cryopreserved embryos derived from donor oocytes was likewise similar (IMP 21.3% vs. Crinone 16.1%). Day 3 Embryo Survival, Morphology, and Transfer Characteristics Post-thaw survival, embryo quality, and number of embryos transferred were similar between the IMP and Crinone groups (Table 2). Distribution of embryos according to morphology scores was similar as well (IMP: 88.3% of embryos had <10% fragmentation, 31.7% perfect symmetry; Crinone: 88.1% of embryos had <10% fragmentation, 29.2% perfect symmetry). The two groups did not differ regarding type of catheter used for ET. The most common type of catheter used was the Wallace catheter (IMP 96.1% vs. Crinone 96.6%). CET difficulty also was similar between groups: 2.1% of IMP and 2.0% of Crinone transfers were classified as having major difficulty; 11.8% of IMP and 9.1% of Crinone transfers were classified as having some difficulty; and 86.1% of IMP and 88.9% of Crinone cycles were classified as having an easy transfer. Clinical Outcomes The odds of a CET resulting in a clinical pregnancy were significantly lower for the Crinone group (36.9% vs. 51.1%, adjusted OR [AOR] 0.56, CI ; P¼.0002; Table 3). No significant differences in the incidence of spontaneous abortion were observed between the two groups (11.5% for Crinone vs. 10.2% for IMP, AOR 1.13, CI ; P¼.61). Correspondingly, the odds of a CET resulting in a live birth were also significantly lower for the Crinone group (24.4% vs. 39.1%, AOR 0.51, CI ; P<.0001). The significantly lower odds of live birth in the Crinone group persisted when additional adjusting was performed for uterine factor diagnosis, assisted hatching, obesity, parity, uterine age at transfer, use of a gestational carrier, number of embryos thawed, total number of cells transferred, CET difficulty, mean fragmentation and symmetry scores of transferred embryos, endometrial thickness on day of mapping, and outcome in corresponding fresh cycle (AOR 0.51, CI ; P¼.0001). Subanalysis of Day 1 Versus Day 3 Cryopreserved Embryos Embryos were cryopreserved on two days during the study period: on day 1 (n ¼ 93) and day 3 (n ¼ 645) after oocyte retrieval. The day of embryo cryopreservation was included a priori in the base model, because day 1 cryopreservation preferentially occurs for good-prognosis (e.g., high OHSS risk) patients in our practice. When analysis was restricted to only cycles with day 1 embryo cryopreservation, the live birth rates were 29.3% (12/41) for Crinone and 51.0% (26/51) for IMP (AOR 0.40, CI ; P¼.03). When only cycles with day 3 embryo cryopreservation were analyzed, the odds of live birth were also significantly lower for Crinone than for IMP (23.6% [60/254] vs. 37.5% [143/381], TABLE 3 Clinical outcomes from day 3 cryopreserved embryo transfer cycles supported with intramuscular progesterone (IMP) versus Crinone. Clinical outcome IMP (n [ 440) 8% Crinone (n [ 298) Effect estimate (95% CI) P value a Implantation rate b ( ).39 Biochemical pregnancy 51 (11.6) 39 (13.1) 1.08 ( ).73 Clinical pregnancy 225 (51.1) 110 (36.9) 0.56 ( ) <.001 Spontaneous abortion 44 (10.2) 34 (11.5) 1.13 ( ).61 Live birth c 169 (39.1) 72 (24.4) 0.51 ( ) <.0001 Note: Values represent n (%) or mean SD. CI ¼ confidence interval. a Generalized estimating equations were used to account for correlations between multiple cycles from the same woman. A binomial distribution and logit link were applied for all outcomes except implantation rate. A Poisson distribution and log link were used to study the number of fetal hearts at 6 weeks, offset by the number of embryos transferred. Models were adjusted a priori for patient age at embryo freezing and day of embryo cryopreservation (1 vs. 3). Two-sided Wald P values also were calculated from these models. b Implantation rate was defined as number of fetal heart beats at ultrasound R6 weeks after transfer divided by number of embryos transferred. c Delivery outcome was missing for 11 cycles that had documented clinical pregnancies but unknown final outcome; those cycles were not included in the analysis of live birth. The odds of live birth were unchanged when the full model was applied (IMP 39.1% vs. Crinone 24.4%, OR 0.51, CI ; P¼.0001) after adjusting for patient age at embryo freezing, day of embryo cryopreservation (1 vs. 3), and the following potential confounders: uterine factor diagnosis, assisted hatching, obesity, parity, gestational carrier cycles, CET difficulty level (easy, some difficulty, major difficulty), outcome from corresponding fresh cycle (live birth, transfer but no live birth, no transfer), uterine age at transfer, number of embryos thawed, total number of cells transferred, mean fragmentation score of transferred embryos, mean symmetry score of transferred embryos, and endometrial thickness at mapping. Kaser. IMP vs. Crinone for cryopreserved cycles. Fertil Steril VOL. 98 NO. 6 / DECEMBER

5 ORIGINAL ARTICLE: ASSISTED REPRODUCTION AOR 0.53, CI ; P¼.0004). Delivery outcome was missing for 11 cycles that had documented clinical pregnancies but unknown final outcome; those cycles were not included in the analysis of live birth. Subanalysis of Autologous Versus Donor Embryos The association between P type and delivery persisted when the analysis was confined to autologous cycles only or to donor cycles only. Specifically, the live birth rate among autologous cycles using IMP versus Crinone, respectively, was 37.8% versus 25.9% (AOR 0.59, CI ; P¼.003), and the live birth rate among donor cycles using IMP versus Crinone, respectively, was 44.1% versus 16.7% (AOR 0.29, CI ; P¼.02). Subanalysis Excluding Gestational Carrier Cycles Because all but one of the carriers was treated with IMP, the association between P support and cycle outcome was also analyzed after excluding all gestational carrier cycles so as to consider confounding by indication for treatment. The clinical outcomes were consistent with the main analysis: Cycles with Crinone support had lower odds of clinical pregnancy (AOR 0.61, CI ; P¼.004) and live birth (AOR 0.55, CI ; P¼.0007). DISCUSSION The principal finding of this study was that CET cycles using IMP for P support were associated with higher pregnancy and delivery rates than those using Crinone vaginal gel. Specifically, when administered three days before a day 3 CET, women who received Crinone had 44% lower odds of clinical pregnancy and 49% lower odds of live birth, compared with women who received IMP. Although our results are derived exclusively from CET cycles, they may be relevant to fresh donor oocyte cycles in which the recipient uterus requires similar hormonal priming. To our knowledge, this is the first study to address the efficacy of IMP and Crinone for CET cycles with the use of multivariable analysis and is the only one to report a statistical difference in clinical outcomes. Earlier studies addressing the use of Crinone in prepared cycles are limited. There are two prospective trials that showed equivalence of the two formulations in donor oocyte programs, although both were underpowered (14 16). Gibbons et al. (14, 16) randomized 54 patients to 90 mg 8% Crinone twice daily and 18 patients to 100 mg IMP once daily from 3 days before embryo transfer to 10 weeks' gestation. Those authors reported no differences in implantation or live birth rates, with power to detect a 50% difference in pregnancy rates. Jobanputra et al. (15, 16) prospectively followed 44 patients on 90 mg 8% Crinone once daily and 42 patients on 100 mg IMP once daily from 3 days before transfer to 8 weeks of pregnancy. Again, no differences in implantation or ongoing pregnancy rates were found, with power to detect a 25% difference in pregnancy rates. Three retrospective studies that address this question in either donor oocytes or CET are small, used nonparametric statistical analysis, and have only been presented in abstract form (17 19). There are several publications that compare pregnancy rates in frozen and donor cycles supported with IMP or other types of vaginal P (suppositories, rings, and inserts), but only one reports on live birth as an outcome (25 27). The authors of a recent meta-analysis concluded that there is insufficient evidence from these trials to be able to identify one particular intervention for endometrial preparation that clearly improves the treatment outcome for women receiving embryo transfers with either frozen embryos or embryos derived from donated oocytes (28). In the present study, luteal-phase support with Crinone was associated with lower rates of clinical pregnancy and live birth than IMP support. These findings raise the question as to whether IMP, at least at the current doses and schedules, more suitably prepares the endometrium for implantation than Crinone in cycles in which there is minimal endogenous P production. Unlike a fresh embryo transfer, in which one or more corpora lutea secrete endogenous P, the endometrium in a CET or donor oocyte cycle relies on exogenous supplementation for support of secretory endometrium. Differences in pharmacokinetics, target tissue concentrations, and time to steady state between IMP and vaginal micronized P may affect endometrial receptivity in CET and donor cycles (14, 29). Histologic studies comparing midluteal endometrial biopsies from patients on IMP with those on vaginal P have yielded conflicting results; some report more endometrial maturation delay and glandular-stromal asynchrony in patients on vaginal P (29, 30), whereas others report the opposite finding (31). There has also been concern that vaginal P establishes a supraphysiologic concentration of P at the level of the endometrium via a first uterine pass effect (30). Early luteinization and advancement of endometrial gland maturity may occur as a result of inappropriately high levels of P (31). It is possible that vaginal P at the standard time and dose used in CET cycles produces an endometrium that is out of phase with embryonic development. Synchrony between the development of a blastocyst and the endometrium is critical to successful apposition, adhesion and invasion of the trophoblast. The importance of timing for initiation of luteal-phase P was demonstrated in two randomized controlled trials of IMP and Crinone in fresh IVF-ET cycles from our institution. In the first study, when P was begun 1 day after oocyte retrieval, Propst et al. (32) reported a lower live birth rate in the Crinone group (24.5% vs. 39.4%, OR 2.00, CI ). In a follow-up study, when Crinone was initiated 24 hours later (i.e., 2 days after oocyte retrieval), Yanushpolsky et al. (7) demonstrated equivalent live birth rates (45.2% for Crinone vs. 42.2% for IMP, OR 1.1, CI ). The authors reconcile these disparate results by citing the known higher endometrial concentration following Crinone supplementation as a possible explanation for premature endometrial advancement and impaired receptivity. It is reasonable to hypothesize that the same holds true for CET; that is, the higher endometrial concentration of P following Crinone treatment may cause premature closure of the implantation window. Perhaps if Crinone were initiated 2 days before CET, instead of 3 days before as in our current protocol, pregnancy rates may be similar to those with IMP VOL. 98 NO. 6 / DECEMBER 2012

6 Fertility and Sterility There are several limitations to the present study. Patients were not randomized to the type of luteal support, and therefore unidentified confounding may have biased the results. However, across more than a dozen variables considered critically as potential confounders, none altered the observed associations. Therefore, it is unlikely that an unknown factor exists of sufficient prevalence and strength of association with both the clinically chosen P support and the cycle outcome. Owing to the retrospective design, it was not possible to correlate P type and outcomes with endometrial biopsies from a mock cycle. Future sufficiently powered randomized studies with large sample sizes and careful consideration of enrollment bias should readdress the optimal timing and dose of 8% Crinone vaginal gel in CET cycles. To perform a noninferiority trial of IMP and Crinone in this setting, assuming a 37% live birth rate following CET in women <35 years old, as published by the Society for Assisted Reproduction Clinic Outcome Reporting System, 366 patients would need to be enrolled into each arm to demonstrate a 10% noninferiority margin with 80% power and.025 alpha. Ideally, in such a study, serum and tissue concentrations of P would be determined, along with analysis of potential molecular and ultrastructural markers of the implantation window. Our findings indicate that IMP should be used for luteal-phase support in day 3 CET cycles until further research has been performed to establish equivalence with 8% Crinone vaginal gel. REFERENCES 1. DiLuigi AJ, Nulsen JC. Effects of gonadotropin-releasing hormone agonists and antagonists on luteal function. Curr Opin Obstet Gynecol 2007;19: Smitz J, Devroey P, Camus M, Deschacht J, Khan I, Stasessen C, et al. 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