Is elevated late-follicular phase progesterone (EP) associated with a deleterious impact on embryo quality (EQ) and cumulative live birth rates (LBRs)? EP was associated with a decrease in embryo utilization and cumulative LBRs. Ovarian stimulation promotes the production of progesterone (P) which adversely affects IVF pregnancy outcomes. However, evidence regarding a potential association between EP an EQ is lacking. A retrospective analysis of all GnRH antagonist down-regulated ICSI cycles followed by a fresh embryo transfer (ET) between 2010 and 2015 was performed. The sample was stratified according to the following P levels on the day of ovulation triggering: ≤0.50, 0.51-1.49 and ≥1.50 ng/ml. The primary outcomes were embryo utilization rates (number of embryos transferred or cryopreserved) and cumulative LBR, defined as the occurrence of the first live-birth after either the fresh or one of the subsequent frozen ET. Overall, 3400 cycles were included in the analysis, using multivariable regression to account for potential confounding. Female age and the number of oocytes retrieved increased significantly with increasing serum P values. Utilization rates decreased linearly as P increased for Day 3 embryos (72.3, 63.0 and 45.4%, respectively), while for Day 5 embryos only the EP group was associated with a significant decrease (48.8, 47.8 and 38.8%, respectively). EP was also associated with decreased fresh and cumulative LBRs. The main limitations of this study were its retrospective nature and the fact that it was restricted to GnRH antagonist cycles. These results raise the question whether EP may also be associated with a decrease in cumulative pregnancy outcomes by increasing embryo wastage. Further studies may evaluate the potential benefit of additional measures besides the freeze-all strategy to avoid this issue, such as lowering the stimulation dose or applying a step-down protocol. None.

STUDY QUESTION What is the effect of artificial shrinkage by laser-induced collapse before vitrification on the implantation potential after transfer of vitrified–warmed blastocysts? SUMMARY ANSWER The artificial shrinkage by laser-induced collapse did not significantly increase the implantation rate per transferred collapsed blastocyst (37.6%) compared with non-collapsed blastocysts (28.9%) [odds ratio (OR): 1.48, 95% confidence interval (CI): 0.78–2.83]. WHAT IS KNOWN ALREADY Retrospective studies have demonstrated that artificial shrinkage of the blastocyst prior to vitrification can have a positive effect on blastocyst survival after warming. A recent study found a similar survival rate but higher implantation rate for collapsed blastocysts. So far, no randomized controlled trial has been conducted to investigate the implantation potential of collapsed blastocysts. STUDY DESIGN, SIZE, DURATION Prospective randomized trial. Patients were recruited from December 2011 until April 2014 and warming cycles were included until July 2014. Patients were randomized in the fresh cycle if blastocysts were available for vitrification and were allocated to the study or control arm according to a computer-generated list. In the study group, blastocysts underwent laser-induced collapse before vitrification. In the control group, blastocysts were vitrified without collapsing. PARTICIPANTS/MATERIALS, SETTING, METHODS In total, 443 patients signed informed consent and 270 patients had blastocysts vitrified. One-hundred and thirty-five patients were allocated to the study group and 135 to the control group. Sixty-nine patients from the study group and 69 from the control group returned for at least one warming cycle in which 85 and 93 blastocysts were warmed in the first cycle, respectively. Primary outcome was implantation rate per embryo transferred in the first warming cycle. Secondary outcomes were survival and transfer rates, blastocyst quality after warming, clinical pregnancy rate and implantation rate per warmed blastocyst. Blastocysts were vitrified–warmed one by one using closed vitrification and one or two blastocysts were transferred per warming cycle. MAIN RESULTS AND THE ROLE OF CHANCE We calculated that the group sample sizes of 80 embryos in the collapse group and 80 embryos in the control group were needed to achieve 80% power to detect a difference between the group proportions of +20% with P < 0.05. In the study group, 69 first warming cycles resulted in 69 transfers with 1.2 blastocysts (n = 85) transferred. In the control group, an average of 1.3 blastocysts (n = 83) were transferred in 67 out of 69 warming cycles. Implantation rates per embryo transferred in the first warming cycle were not different between both groups (38 versus 29%, OR: 1.48; 95% CI: 0.78–2.83), neither was the implantation rate per warmed embryo (38 versus 26%, OR: 1.74; 95% CI: 0.92–3.29). When all warming cycles were considered (n = 135 in each group), survival rate after collapse was significantly higher compared with the control group (98.0 versus 92.0%, OR: 4.25; 95% CI: 1.19–15.21). Furthermore, a higher percentage of high-quality blastocysts (36.3 versus 23.5%, OR: 1.86; 95% CI: 1.12–3.08) and hatching blastocysts (19.2 versus 5.4%, OR: 4.18; 95% CI: 1.84–9.52) were found compared with the control group. LIMITATIONS, REASONS FOR CAUTION The study lasted more than 2.5 years since fewer patients than expected returned for a warming cycle because of the high ongoing pregnancy rates in the fresh IVF/ICSI cycle. WIDER IMPLICATIONS OF THE FINDINGS Although no significant higher implantation rate was found after collapse, the better survival and post-warm embryo quality convinced us to recognize a clinical benefit of artificial shrinkage and to implement it in routine vitrification practice. STUDY FUNDING/COMPETING INTERESTS None. TRIAL REGISTRATION NUMBER NCT01980225, www.clinicaltrials.gov. The first patient was included November 2011 and the study was registered October 2013.

How reliable are cleavage stage and trophectoderm (TE) biopsies compared to inner cell mass (ICM) biopsies? The reliability of TE biopsy compared to ICM biopsy is almost perfect, but only substantial between cleavage stage biopsy and ICM biopsy. One of the prevailing reasons for implantation failure is presumed to be chromosomal aneuploidy in human preimplantation embryos. Preimplantation genetic testing for aneuploidies (PGT-A) has been introduced into assisted reproduction in an effort to increase pregnancy rates. Increasing evidence indicates that genetic results obtained following blastomere or TEbiopsy may not accurately reflect the true genetic status of the embryo due to the presence of embryonic mosaicism, and therefore the reliability of PGT is highly controversial. This was an observational descriptive study, performed in a private infertility centre from August 2016 to January 2017. The mean female age was 33.9 years, ranging from 24 to 46 years, and the mean number of biopsied embryos per couple was 2.2 (range 1-7 embryos). Blastomere biopsies had been performed at cleavage stage on Day 3 (D3) due to the turnover time of genetic testing and the inability to cryopreserve embryos in accordance with the local law governing ART. To confirm the genetic results in embryos not chosen for transfer, additional biopsies of the TE at blastocyst stage (BLASTO-TE) as well as of the ICM (BLASTO-ICM) were performed on D5. Only surplus blastocysts, which had not been selected for transfer and were not cryopreserved in accordance with the law governing ART, had been included. Comparison of all biopsies (D3/BLASTO-ICM/BLASTO-TE) per embryo demonstrated that 50 (59.5%) out of 84 embryos showed concordance in all three results (= full concordance). Thirty-four (40.4%) embryos had at least two discordant results between the three biopsies, regardless of whether the embryo diagnosis (aneuploid/euploid) was discordant or not, or in aneuploid embryos, whether the chromosomal patterns were inconsistent. Nine (= 10.7%) embryos had complete discordance between all three biopsies. False positive results between D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM were 26.4%/30.2% and 7.5%, respectively, while the Kappa agreement between the different approaches was 0.647, 0.553 and 0.857, respectively. Therefore the reliability of D3/BLASTO-TE, D3/BLASTO-ICM and BLASTO-TE/BLASTO-ICM can be interpreted as substantial, as moderate and as almost perfect. The limitation of this study is the possible bias in the concordance/discordance rate because embryos that had been selected for transfer did not undergo biopsy on D5. The obvious discordance between the three different approaches for PGT-A underlines the limitations of genetic testing and highlights the importance of ongoing research in order to improve the accuracy of PGT-A results. Until then reproductive specialists will continue to make challenging decisions on whether to transfer or discard an embryo in light of current evidence questioning the reliability of genetic results. This study was supported by Igenomix. The funder provided support in the form of salary for R.C. The co-author R.C. is an employee of Igenomix. She participated in the blinded analysis of the samples; however the final data collection and statistical analysis of the results, as well as the decision to publish, was taken by B.L, I.E. and H.F. The authors B.L., I.E., A.L., A.B., A.A., N.D. and H.F. have no competing interests. The funder did not have any additional role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The commercial affiliation of R.C. did not play any role in the study. This study was approved by the Ethics Committee of IVIRMA Middle East Fertility Clinic, Abu Dhabi, UAE (Research Ethics Committee IVI-MEREFA009a/2017).

Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analyzed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after ICSI were compared in three different assays. First, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at day 1. Secondly, embryo development up to day 3 was analyzed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analyzed by immunostaining on day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older subfertile / infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.