Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.

These findings provide foundational evidence for future investigation of 2% O as the preferred O tension for extended culture of human embryos.

OBJECTIVE: As the human embryo traverses the utero-tubal junction late on day 3, it is exposed to a step-down in O 2 tension from 5-7% in the Fallopian tube to 2% in the uterus. The question therefore arises whether the IVF culture system should mimic this progressively hypoxic environment. The present study tested the hypothesis that sequential exposure first to 5% O 2 from days 1 to 3, and then to 2% O 2 from days 3 to 5, improves blastocyst yield and quality compared to continuous exposure to 5% O 2 .DESIGN: Randomized controlled trial of sibling embryos. MATERIALS AND METHODS: Donated sibling embryos (n¼203), blocked on pronuclei (PN) status (2PN vs. 3PN), were randomized to either 5% O 2 from days 1 to 5 (5-5% Group; n¼102) or 5% O 2 from days 1 to 3 and then 2% O 2 to day 5 (5-2% Group; n¼101). Stage and grade were assessed on day 5 by embryologists blinded to Group; usable blastocysts were those meeting freezing criteria. Cell counts of embryos from 3PNs were obtained by nuclear staining; 2PNs were used for ongoing gene expression studies. Odds ratios (OR) with 95% CI were calculated using generalized estimating equations to account for correlations among embryos from the same woman, adjusted a priori for oocyte age and source (autologous vs. donor), IVF vs. ICSI, PN number, fresh vs. thawed embryo and % goodquality embryo (GQE) on day 3. Differences in mean cell number and proportions of developmental stages were analyzed by independent t-tests and chi-square, respectively. Based on data comparing atmospheric (20%) vs. 5% O 2, this study was powered to detect a 16.5% difference in day 5 GQE (a¼0.05, b¼0.80). 1 RESULTS: The percentage of day 3 GQEs did not differ between groups (5-5% O 2 vs. 5-2% O 2 : 28.4% vs. 30.7%; OR 1.14 [CI¼0.74-1.76]; P¼0.76). Embryos in the 5-2% Group were less likely to arrest at cleavage and more likely to blastulate (Table ). Notably, in the 5-2% Group, there was a two-fold increase in the odds of conversion to a usable blastocyst ; P¼0.02), yet blastocysts had fewer cells (Table ).CONCLUSIONS: These findings support our hypothesis that blastocyst yield and quality may be superior when O 2 tension is reduced from 5% to 2% on day 3, thereby recapitulating the O 2 environment in vivo. Further studies are warranted to confirm whether these preliminary findings translate into a paradigm shift for extended culture in clinical IVF, and to investigate the significance of lower cell counts in blastocysts cultured in 2% O 2 , particularly as related to the 'quiet hypothesis' for embryo metabolism. 2 References: