Recent clomiphene citrate exposure does not impact subsequent clinical outcomes in single euploid frozen embryo transfer cycles.

Do infertile couples who recently utilized clomiphene citrate (CC) for ovulation induction or ovarian stimulation (<90 days previously) followed by a single euploid embryo transfer (SEET) have lower implantation potential compared with patients who were not exposed to CC within 90 days before embryo transfer (ET)? There does not appear to be an association between recent CC exposure and lower implantation potential in patients who undergo a frozen embryo transfer (FET) of euploid embryos. Clomiphene has been found to be associated with lower pregnancy rates when compared against other ovarian stimulation medications. The majority of published research about the effects of CC on implantation potential suggest an anti-estrogenic effect on the endometrium. Quality evidence and information about utilization of CC and its effect on implantation potential after euploid ETs is lacking in the literature. A retrospective cohort study with propensity score matching was carried out. We included all patients that underwent an autologous SEET from September 2016 to September 2022 at a single academic-private ART center. The study group included patients that had utilized CC during either ovulation induction cycles and/or controlled ovarian stimulation at least 90 days before FET. A propensity score-matched control group of patients that were unexposed to CC within 90 days prior to SEET was used for comparisons. The primary outcome was positive pregnancy test (defined as a positive serum β-hCG measured 9 days after ET), with other outcomes including clinical pregnancy, ongoing pregnancy, biochemical pregnancy loss, and clinical pregnancy loss rates per SEET. Multivariate regression analyses fitted with generalized estimating equations were utilized to analyze if there was an association between CC utilization and IVF outcomes. Furthermore, the study evaluated the cumulative effect of CC and endometrial receptivity in vivo and subsequent IVF outcomes. A total of 593 patients with utilization of CC in <90 days before ET were compared with 1779 matched controls. Positive pregnancy test rates were comparable among the control group and the CC exposed groups, respectively (74.3% versus 75.7%, P = 0.79), as were clinical pregnancy (64.0% versus 65.0%, P = 0.60), ongoing pregnancy (51.8% versus 53.2%, P = 0.74), biochemical pregnancy loss (15.7% versus 14.03%, P = 0.45), and clinical pregnancy loss rates were also comparable among cohorts (17.1% versus 18.1%, P = 0.71). No association was found between utilization of clomiphene and lower implantation rates (adjusted odds ratio 0.95, 95% CI 0.76-1.18). Also, no differences were observed in sub-analyses based on multiple CC utilization periods. Finally, no association was found between the number of consecutive cumulative clomiphene cycles and sub-optimal IVF outcomes. The study has inherent bias that originated from its retrospective design. Serum levels of CC were not measured and sample size for the sub-analyses was small. There does not appear to be an association between recent CC exposure and lower implantation potential in patients who undergo a FET of euploid embryos. This finding remains consistent, even in patients who undergo multiple, consecutive clomiphene cycles prior to ET. There were no long-term effects of CC on endometrial development and clinical characteristics examined in this study. Patients that utilized CC medication prior to a SEET cycle for either ovarian stimulation or ovulation induction, can be assured that there is no evidence of a residual effect of recent CC administration that could jeopardize their pregnancy probability. No funding was received for the realization of this study. A.C. is advisor and/or board member of Sema4 (stakeholder in data) and Progyny. The other authors have no conflicts of interest to declare. N/A.

Hernandez-Nieto C ，Lee J ，Alkon-Meadows T ，Soto-Cossio L ，Sandler B ，Mukherjee T ，Copperman A ... -  《-》

Late follicular phase progesterone elevation during ovarian stimulation is not associated with decreased implantation of chromosomally screened embryos in thaw cycles.

What is the impact of a late follicular phase progesterone elevation (LFPE) during controlled ovarian hyperstimulation (COH) on embryonic competence and reproductive potential in thaw cycles of preimplantation genetic testing for aneuploidy (PGT-A) screened embryos? Our study findings suggest that LFPE, utilizing a progesterone cutoff value of 2.0 ng/ml, is neither associated with impaired embryonic development, increased rate of embryonic aneuploidy, nor compromised implantation and pregnancy outcomes following a euploid frozen embryo transfer (FET) cycle. Premature progesterone elevation during COH has been associated with lower pregnancy rates due to altered endometrial receptivity in fresh IVF cycles. Also, increased levels of progesterone (P) have been suggested to be a marker for ovarian dysfunction, with some evidence to show an association between LFPE and suboptimal embryonic development. However, the effect of LFPE on embryonic competence is still controversial. Retrospective cohort analysis in a single, academic ART center from September 2016 to March 2020. In total, 5244 COH cycles for IVF/PGT-A were analyzed, of those 5141 were included in the analysis. A total of 23 991 blastocysts underwent trophectoderm biopsy and PGT analysis. Additionally, the clinical IVF outcomes of 5806 single euploid FET cycles were evaluated. Cohorts were separated in two groups: Group 1: oocytes retrieved from cycles with normal P levels during ovulation trigger (P ≤ 2.0 ng/ml); Group 2: oocytes retrieved after cycles in which LFPE was noted (P > 2.0 ng/ml). Extended culture and PGT-A was performed. Secondly, IVF outcomes after a single euploid FET were evaluated for each cohort. Four thousand nine hundred and twenty-five cycles in Group 1 were compared with 216 cycles on Group 2. Oocyte maturity rates, fertilization rates and blastulation rates were comparable among groups. A 65.3% (n = 22 654) rate of utilizable blastocysts was found in patients with normal P levels and were comparable to the 62.4% (n = 1337) observed in those with LFPE (P = 0.19). The euploidy rates were 52.8% (n = 11 964) and 53.4% (n = 714), respectively, albeit this difference was not statistically significant (P = 0.81). Our multivariate analysis was fitted with a generalized estimating equation (GEE) and no association was found with LFPE and an increased odds of embryo aneuploidy (adjusted odds ratio 1.04 95% CI 0.86-1.27, P = 0.62). A sub-analysis of subsequent 5806 euploid FET cycles (normal P: n = 5617 cycles and elevated P: n = 189 cycles) showed no differences among groups in patient's BMI, Anti-Müllerian hormone (AMH), endometrial thickness at FET and number of prior IVF cycles. However, a significant difference was found in patient's age and oocyte age. The number of good quality embryos transferred, implantation rate, clinical pregnancy rate, ongoing pregnancy rate, multiple pregnancy rate and clinical pregnancy loss rates were comparable among groups. Of the registered live births (normal P group: n = 2198; elevated P group: n = 52), there were no significant differences in gestational age weeks (39.0 ± 1.89 versus 39.24 ± 1.53, P = 0.25) and birth weight (3317 ± 571.9 versus 3 266 ± 455.8 g, P = 0.26) at delivery, respectively. The retrospective nature of the study and probable variability in the study center's laboratory protocol(s), selected progesterone cutoff value and progesterone assay techniques compared to other ART centers may limit the external validity of our findings. Based on robust sequencing data from a large cohort of embryos, we conclude that premature P elevation during IVF stimulation does not predict embryonic competence. Our study results show that LFPE is neither associated with impaired embryonic development nor increased rates of aneuploidy. Embryos obtained from cycles with LFPE can be selected for transfer, and patients can be reassured that the odds of achieving a healthy pregnancy are similar to the embryos exposed during COH cycles to physiologically normal P levels. No funding was received for the realization of this study. Dr A.B.C. is advisor and/or board member of Sema 4 (Stakeholder in data), Progyny and Celmatix. The other authors have no conflicts of interest to declare. NA.

Hernandez-Nieto C ，Lee JA ，Alkon-Meadows T ，Luna-Rojas M ，Mukherjee T ，Copperman AB ，Sandler B ... -  《-》

What is the reproductive potential of day 7 euploid embryos?

What is the rate of euploidy and the reproductive potential of embryos biopsied after 6 days of development? Embryos biopsied after 6 days of development have higher rates of aneuploidy; however, day 7 euploid embryos selected at transfer can achieve acceptable pregnancy rates and live birth (LB) outcomes. Recent publications have shown promising treatment results after euploid day 7 embryo transfers (ETs), albeit these studies were limited by small sample sizes. Whereas the current clinical standard has been to discard embryos that do not reach expansion by day 6 of development, the lack of robust data surrounding the clinical utility of day 7 embryos warrants further evaluation. Retrospective cohort analysis in a single, academic in vitro fertilization (IVF) center from January 2012 to March 2018. A total of 25 775 embryos underwent trophectoderm (TE) biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Additionally, the clinical IVF outcomes of 3824 single, euploid frozen embryo transfer (FET) cycles were evaluated. Cohorts were segregated by day of TE biopsy following oocyte retrieval (day 5, day 6 or day 7). PGT-A was performed to identify embryonic ploidy rates. Secondly, IVF and LB outcomes after single, euploid FET were evaluated for each cohort. A total of day 5 (n = 12 535), day 6 (n = 11 939) and day 7 (n = 1298) embryos were included in the study analysis. The rate of embryo euploidy was significantly lower in day 7 blastocysts compared to day 5 or day 6 cohorts (day 7 = 40.5%; day 5 = 54.7%; day 6 = 52.9%; (P < 0.0001)). After adjusting for age, anti-Müllerian hormone, BMI, embryo quality and number of embryos biopsied, there was a significant association between aneuploidy and embryos biopsied on day 7 when compared to day 5 biopsied embryos (OR = 1.34, CI 95% 1.09-1.45, P = 0.001) and day 6 biopsied embryos (OR = 1.26, CI95% 1.07-1.16, P < 0.001).A sub-analysis of subsequent 3824 single, euploid FET cycles (day 5: n = 2321 cycles; day 6: n = 1381 cycles; and day 7: n = 116 cycles) showed significant differences among cohorts in implantation, clinical pregnancy, LB and clinical loss rates. There was a significant decrease in the odds of implantation, clinical pregnancy and LB, but no association with clinical loss or multiple pregnancy rates in patients who utilized day 7-biopsied embryos during treatment. The retrospective nature of the study and potential variability in the study center's laboratory protocol(s) compared to other reproductive treatment centers may limit the external validity of our findings. Additionally, patients who transferred euploid embryos, biopsied on day 7 of development due to an absence of day 5 or day 6 counterparts, may have introduced selection bias in this study. Embryonic developmental stage, morphological grade and ploidy status are paramount factors affecting ET selection and implantation potential. This study reveals that embryos ineligible for TE biopsy on day 5 or day 6 of development may benefit from extended culture to day 7. Our study demonstrates patient benefit when extended culture to day 7 of development is routinely performed for embryos failing to meet biopsy criteria by day 5 or 6. No funding was received for the realization of this manuscript. Dr Alan Copperman is Advisor or Board Member of Sema 4 (Stake holder in Data), Progyny and Celmatix. This retrospective analysis was approved by an Institutional Review Board (WIRB PRO NUM: 20161791; Study Number: 1167398).

Hernandez-Nieto C ，Lee JA ，Slifkin R ，Sandler B ，Copperman AB ，Flisser E ... -  《-》

Is there an optimal window of time for transferring single frozen-thawed euploid blastocysts? A cohort study of 1170 embryo transfers.

Is there an optimal window of time when the transfer of single frozen-thawed euploid blastocysts is associated with a maximal live birth rate (LBR)? Performing a single frozen-thawed euploid blastocyst transfer at 160 ± 4 h post-hCG trigger in modified-natural frozen-thawed embryo transfer (FET) cycles was independently associated with a higher LBR as compared to transfers outside this window; however, in natural FET cycles, LBRs were comparable across a wider range of time intervals. There is compelling evidence for maintaining embryo-endometrial synchrony to optimize clinical outcomes following FETs, which could potentially be achieved by matching the transfer time of an embryo post-ovulation to its developmental age post-oocyte retrieval. For modified-natural cycles, ovulation is widely accepted to occur ∼40 h following the hCG trigger, whilst ovulation following spontaneous LH surge onset is thought to vary from 24 to 56 h. This is a multicentered retrospective cohort study analyzing 1170 single frozen-thawed euploid blastocyst transfers following trophectoderm biopsy and preimplantation genetic testing (PGT) between May 2015 and February 2019. Limiting the analysis to single euploid embryo transfers allowed for a more accurate estimation of the endometrial synchrony factor by controlling for the developmental stage of the embryo (full blastocyst or more advanced) and its genetic composition. LBR per FET was the primary outcome measure. Patients underwent natural or gonadotrophin-induced preparation of the endometrium, with serial serum oestradiol, LH and progesterone measurements. Optimally timed transfers were predefined as those conducted 120 ± 4 h post-ovulation since biopsy and subsequent cryopreservation of full blastocysts which is usually performed at 116-124 h post-oocyte retrieval. This was considered the equivalent of 160 ± 4 h post-hCG trigger in modified-natural cycles (n = 253), as ovulation was assumed to occur ∼40 h after the hCG trigger. For natural cycles (n = 917), this was also considered to be, on average, 160 ± 4 h post the spontaneous LH surge. Thus, study groups were determined as those with optimal timing or not, and additional exploratory and subgroup analyses were performed, varying the time window in terms of onset and width, both overall and per endometrial preparation protocol. Statistical analysis was performed using the generalized estimating equations (GEE) framework to control for the clustered nature of the data while adjusting for potential confounders. Overall, LBRs were significantly higher when the transfer had been performed at 160 ± 4 h post-hCG trigger or LH surge onset compared to when it had been performed outside this window (44.7% vs 36.0%; P = 0.008). A multivariable regression GEE model including the cycle type (natural versus modified-natural), previtrification embryo quality (top versus good quality), embryo stage (fully hatched versus hatching or earlier blastocyst), vitrification day (D5 versus D6) and survival rate (>90% versus <90%) as covariates, confirmed that, overall, embryo transfers conducted 160 ± 4 h post-hCG trigger or LH surge onset (the assumed equivalent of 120 ± 4 h post-ovulation) were associated with a significantly higher LBR (relative risk (RR) 1.21, 95% CI 1.04-1.41). Subgroup exploratory analyses per endometrial preparation protocol demonstrated that these findings were primarily present in the modified-natural cycle group (RR 1.52, 95% CI 1.15-1.99), whilst the natural cycle group showed comparable LBRs across a wider range of time intervals. Moreover, the overall LBR for the natural group (36.8%; 95% CI 33.7-39.9%) was lower than that of the modified-natural group (41.3%; 95% CI 35.4-47.1%), suggesting that there likely remains a greater potential to further optimize the timing of natural cycle embryo transfers. As with all retrospective studies, the presence of residual unknown bias cannot be excluded. Additionally, patients included in this study were a selected group who underwent PGT for specific reasons and hence the results obtained might not be directly applicable to the general population or embryos that have not undergone embryo biopsy. Furthermore, the criteria utilized to interpret hormonal data from natural cycles were specifically adopted for the present study and need to be validated in further studies. The results of this study highlight the significance of embryo-endometrial synchrony for the optimization of frozen embryo transfer outcome. However, it also clearly supports that the implantation window is in most cases wide and the achievement of live birth is possible with relatively high success rates even outside the optimal window of 160 ± 4 h post-trigger for modified-natural cycles and across a range of time intervals for natural cycles. Additionally, this study suggests that implantation rates could be further optimized in natural cycles by improving methods of assessing embryo-endometrial synchrony. C. V. is supported by a National Health and Medical Research Council Early Career Fellowship (GNT1147154). No other funding was received for this study and there are no competing interests. N/A.

An BGL ，Chapman M ，Tilia L ，Venetis C ... -  《-》

Hatching status before embryo transfer is not correlated with implantation rate in chromosomally screened blastocysts.

Do the reproductive outcomes from the transfer of fully hatched (FH) blastocysts differ from those of not fully hatched (NFH) blastocysts? Biochemical pregnancy rate (BPR), implantation rate (IR), live birth rate (LBR) and early pregnancy loss (EPL) rate are similar in FH and NFH single euploid blastocyst embryo transfers. The use of extended culture and PGS often leads to transfer of an embryo that is well developed and frequently FH from the zona pellucida. Without the protection of the zona, an FH embryo could be vulnerable to trauma during the transfer procedure. To date, no other study has evaluated the reproductive competence of an FH blastocyst transfer. The retrospective study included 808 patients who underwent 808 cycles performed between September 2013 and July 2015 at a private academic IVF center. Of these, 436 cycles entailed transfer of a NFH blastocyst (n = 123 fresh transfer, n = 313 frozen/thawed embryo transfer (FET)) and 372 cycles entailed transfer of an FH blastocyst (n = 132 fresh, 240 FET). Fresh and FET cycles and associated clinical outcomes were considered separately. LBR was defined as the delivery of a live infant after 24 weeks of gestation. Trophectoderm biopsies were performed on Day 5 (d5) or 6 (d6) for embryos meeting morphology eligibility criteria (set at ≥3BC). Morphologic grading was determined using a modified Gardner-Schoolcraft scale prior to transfer. A single euploid embryo was selected for transfer per cycle on either the morning of d6, for fresh transfers or 5 days after progesterone supplementation for patients with transfer in an FET cycle. Embryos were classified as NFH (expansion Grade 3, 4 or 5) or FH (expansion Grade 6) cohorts. The main outcome measure was IR. In the fresh transfer group, IR was similar between NFH and FH cycles (53.7% versus 55.3%, P = 0.99, odds ratio (OR) 0.9; 95% confidence interval (CI) 0.6-1.5). Secondary outcomes were also statistically similar between groups: BPR (65.9% versus 66.7%, OR 1.0; 95% CI: 0.6-1.6), LBR (43.1% versus 47.7%, P = 0.45, OR 1.2; 95% CI: 0.7-1.9) and EPL rate (22.8% versus 18.2%, OR 1.3; 95% CI: 0.7-2.4). After adjusting for age, BMI, endometrial thickness at the LH surge and oocytes retrieved in a logistic regression (LR) model, the hatching status remained not associated with IR (P > 0.05). In the FET cycles, IR was similar between NFH and FH cycles (62.6% versus 61.7%, OR 1.0; 95% CI: 0.7-1.5). Secondary outcomes were similar between groups: BPR (74.1% versus 72.9%, respectively, OR 1.1; 95% CI: 0.7-1.6), LBR (55.0% versus 50.0%, OR 0.8; 95% CI: 0.6-1.1) and EPL rate (18.9% versus 22.9%, respectively, OR 0.8; 95% CI: 0.5-1.2). After adjusting for age, BMI, endometrial thickness at the LH surge and oocytes retrieved in an LR model, the hatching status was not shown to be associated with implantation (P > 0.05). Limitations include the retrospective design and data from a single institution. Additionally, the study was limited to patients that developed high-quality blastocysts suitable for biopsy. The results suggest that FH embryos are not more fragile or less likely to implant when compared to NFH counterparts. We found no evidence of altered IR or other clinical outcomes in the transfer of FH euploid embryos. JG is funded by MSTP grant T32 GM007280 (NIH). No additional funding was received. There are no conflicts of interest to declare..

Rodriguez-Purata J ，Gingold J ，Lee J ，Whitehouse M ，Slifkin R ，Briton-Jones C ，Copperman A ，Sandler B ... -  《-》