Abstract
STUDY QUESTION: What is the optimal timing of progesterone exposure to achieve live births for day-6 blastocysts in artificial frozen-thawed embryo transfer (FET) cycles? SUMMARY ANSWER: Live birth rates (LBRs) were comparable between day-6 and day-7 progesterone supplementation for day-6 blastocyst transfers in artificial FET cycles. WHAT IS KNOWN ALREADY: In artificial FET cycles, embryo-endometrium synchrony is primarily determined by the duration of progesterone exposure. Although blastocyst transfer is conventionally performed on day six of progesterone supplementation, the optimal duration of progesterone exposure remains uncertain. Available evidence suggests that the window of implantation may be relatively flexible for day-5 blastocysts, whereas day-6 blastocysts may have a distinct and potentially narrower window of implantation. However, prospective data evaluating different durations of progesterone exposure for blastocyst transfer-particularly for day-6 blastocysts-remain limited. STUDY DESIGN SIZE DURATION: This open-label randomized controlled trial (RCT) included 338 women undergoing FET in artificial cycles who had exclusively day-6 blastocysts available for transfer at enrolment. These blastocysts originated either from IVF/ICSI cycles with exclusive day-6 blastocyst formation or from previous embryo transfer attempts in which only day-6 blastocysts remained cryopreserved. Participants were randomized prior to progesterone initiation to undergo blastocyst transfer on 6 or 7 days of progesterone supplementation. Recruitment occurred between September 2022 and May 2024, with final follow-up completed in March 2025. PARTICIPANTS/MATERIALS SETTING METHODS: A total of 338 participants were randomized equally to undergo blastocyst transfer on either Day 6 or Day 7 of progesterone supplementation. The primary outcome was LBR. Secondary outcomes included singleton and twin LBR, biochemical and clinical pregnancy, pregnancy loss, good birth outcome, and maternal and neonatal outcomes. The primary analysis followed the intention-to-treat (ITT) principle, with a per-protocol (PP) analysis also performed. Post hoc analyses included multivariable regression and subgroup analyses. MAIN RESULTS AND THE ROLE OF CHANCE: In the ITT analysis, the LBR was 45.0% (76/169) in the day-6 progesterone group and 40.2% (68/169) in the day-7 group (absolute difference, -4.7% [95% CI, -15.3% to 5.8%]; adjusted relative ratio (RR), 0.90 [95% CI, 0.70-1.14], P = 0.367). Rates of biochemical pregnancy, clinical pregnancy, pregnancy loss, and good birth outcome were comparable between groups. No differences were observed in maternal and neonatal outcomes. PP analysis yielded results consistent with the ITT analysis. Exploratory post hoc subgroup analyses identified a significant interaction between blastocyst expansion stage and treatment group (P for interaction = 0.004). Among early blastocyst transfers, the LBR was significantly lower in the day-7 progesterone group compared with the day-6 group (36.8% vs 53.8%; absolute difference, -17.1% [95% CI, -30.3% to -3.8%]; adjusted RR, 0.69 [95% CI, 0.51-0.94], P = 0.018). In contrast, among late blastocyst transfers, the LBR tended to be higher in the day-7 group (46.8% vs 31.7%; absolute difference, 15.0% [95% CI, -1.9% to 32.0%]; adjusted RR, 1.47 [95% CI, 0.95-2.28], P = 0.082), although this difference did not reach statistical significance. LIMITATIONS REASONS FOR CAUTION: This study was open-label, which may have introduced performance bias; however, embryologists were blinded to group allocation, and all outcomes were objective, minimizing the risk of detection bias. Subgroup analyses were conducted post hoc, and the sample size may have been insufficient to detect differences within these subgroups. Accordingly, these findings should be interpreted with caution. In addition, the study was not powered to assess potential differences in treatment effects among specific subpopulations, such as patients with polycystic ovary syndrome, endometriosis, or repeated implantation failure. WIDER IMPLICATIONS OF THE FINDINGS: Based on this trial, LBRs were comparable between transfers performed on six versus seven days of progesterone exposure for day-6 blastocysts in artificial FET cycles. Exploratory subgroup findings, including those related to blastocyst expansion stage, are hypothesis-generating and require confirmation in future adequately powered RCTs. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Natural Science Foundation of Guangdong Province, China (2025A1515010073). No conflicts of interest are declared for all authors. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry (No. ChiCTR2200062901). URL: https://www.chictr.org.cn/bin/project/edit?pid=175020. TRIAL REGISTRATION DATE: 23 August 2022. DATE OF FIRST PATIENT’S ENROLMENT: 12 September 2022.