Trophectoderm cells of human mosaic embryos display increased apoptotic levels and impaired differentiation capacity: a molecular clue regarding their reproductive fate?

STUDY QUESTION: Are there cell lineage-related differences in the apoptotic rates and differentiation capacity of human blastocysts diagnosed as euploid, mosaic, and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A) based on concurrent copy number and genotyping analysis? SUMMARY ANSWER: Trophectoderm (TE) cells of mosaic and aneuploid blastocysts exhibit significantly higher levels of apoptosis and significantly reduced differentiation capacity compared to those of euploid blastocysts. WHAT IS KNOWN ALREADY: Embryos diagnosed as mosaic after PGT-A can develop into healthy infants, yet understanding the reasons behind their reproductive potential requires further research. One hypothesis suggests that mosaicism can be normalized through selective apoptosis and reduced proliferation of aneuploid cells, but direct evidence of these mechanisms in human embryos is lacking. Additionally, data interpretation from studies involving mosaic embryos has been hampered by retrospective analysis methods and the high incidence of false-positive mosaic diagnoses stemming from the use of poorly specific PGT-A platforms. STUDY DESIGN, SIZE, DURATION: Prospective cohort study performing colocalization of cell-lineage and apoptotic markers by immunofluorescence (IF). We included a total of 64 human blastocysts donated to research on Day 5 or 6 post-fertilization (dpf) by 43 couples who underwent in vitro fertilization treatment with PGT-A at IVI-RMA Valencia between September 2019 and October 2022. A total of 27 mosaic blastocysts were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study consisted of two phases: Phase I (caspase-3, n = 53 blastocysts): n = 13 euploid, n = 22 mosaic, n = 18 aneuploid. Phase II (terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), n = 11 blastocysts): n = 2 euploid, n = 5 mosaic, n = 4 aneuploid. Following donation for research, vitrified blastocysts were warmed, cultured until re-expansion, fixed, processed for IF, and imaged using confocal microscopy. For each blastocyst, the following cell counts were conducted: total cells (DAPI+), TE cells (GATA3+), inner cell mass (ICM) cells (GATA3-/NANOG+), and apoptotic cells (caspase-3+ or TUNEL+). The incidence of apoptosis was calculated for each blastocyst by dividing the number of caspase-3+ cells (Phase I) or TUNEL+ cells (Phase II) by the number of TE or ICM cells. Statistical analysis was performed according to data type and distribution (P < 0.05 was considered statistically significant). MAIN RESULTS AND THE ROLE OF CHANCE: Phase I: Mosaic blastocysts displayed a similar number of total cells (49.6 ± 15 cells at 5 dpf; 58.8 ± 16.9 cells at 6 dpf), TE cells (38.8 ± 13.7 cells at 5 dpf; 49.2 ± 16.2 cells at 6 dpf), and ICM cells (10.9 ± 4.2 cells at 5 dpf; 9.7 ± 7.1 cells at 6 dpf) compared to euploid and aneuploid blastocysts (P > 0.05). The proportion of TE cells retaining NANOG expression increased gradually from euploid blastocysts (9.7% = 63/651 cells at 5 dpf; 0% = 0/157 cells at 6 dpf) to mosaic blastocysts (13.1% = 104/794 cells at 5 dpf; 3.4% = 12/353 cells at 6 dpf) and aneuploid blastocysts (27.9% = 149/534 cells at 5 dpf; 4.6% = 19/417 cells at 6 dpf) (P < 0.05). At the TE level, caspase-3+ cells were frequently observed (39% = 901/2310 cells). The proportion of caspase-3+ TE cells was significantly higher in mosaic blastocysts (44.1% ± 19.6 at 5 dpf; 43% ± 16.8 at 6 dpf) and aneuploid blastocysts (45.9% ± 16.1 at 5 dpf; 49% ± 15.1 at 6 dpf) compared to euploid blastocysts (26.6% ± 16.6 at 5 dpf; 17.5% ± 14.8 at 6 dpf) (P < 0.05). In contrast, at the ICM level, caspase-3+ cells were rarely observed (1.9% = 11/596 cells), and only detected in mosaic blastocysts (2.6% = 6/232 cells) and aneuploid blastocysts (2.5% = 5/197 cells) (P > 0.05). Phase II: Consistently, TUNEL+ cells were only observed in TE cells (32.4% = 124/383 cells). An increasing trend was identified toward a higher proportion of TUNEL+ cells in the TE of mosaic blastocysts (37.2% ± 21.9) and aneuploid blastocysts (39% ± 41.7), compared to euploid blastocysts (23% ± 32.5), although these differences did not reach statistical significance (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: The observed effects on apoptosis and differentiation may not be exclusive to aneuploid cells. Additionally, variations in aneuploidies and unexplored factors related to blastocyst development and karyotype concordance may introduce potential biases and uncertainties in the results. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate a cell lineage-specific effect of aneuploidy on the apoptotic levels and differentiation capacity of human blastocysts. This contributes to unravelling the biological characteristics of mosaic blastocysts and supports the concept of clonal depletion of aneuploid cells in explaining their reproductive potential. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by grants from Centro para el Desarrollo Tecnológico Industrial (CDTI) (20190022) and Generalitat Valenciana (APOTIP/2019/009). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Should embryo rebiopsy be considered a regular strategy to increase the number of embryos available for transfer?

PURPOSE: To investigate whether embryo rebiopsy increases the yield of in vitro fertilization (IVF) cycles. METHODS: Retrospective study including 18,028 blastocysts submitted for trophectoderm biopsy and preimplantation genetic testing for aneuploidy (PGT-A) between January 2016 and December 2021 in a private IVF center. Out of the 517 embryos categorized as inconclusive, 400 survived intact to the warming procedure, re-expanded, and were suitable for rebiopsy. Of them, 71 rebiopsied blastocysts were transferred. Factors affecting the probability of obtaining an undiagnosed blastocyst and clinical outcomes from blastocysts biopsied once and twice were investigated. RESULTS: The overall diagnostic rate was 97.1%, with 517 blastocysts receiving inconclusive reports. Several blastocyst and laboratory features, such as the day of the biopsy, the stage of development, and the biopsy methodology, were related to the risk of obtaining an inconclusive diagnosis after PGT-A. A successful diagnosis was obtained in 384 of the rebiopsied blastocysts, 238 of which were chromosomally transferable. A total of 71 rebiopsied blastocysts were transferred, resulting in 32 clinical pregnancies [(clinical pregnancy rate (CPR)=45.1%], 16 miscarriages [(miscarriage rate (MR)=41%], and, until September 2020, 12 live births [(live birth rate (LBR)=23.1%]. A significantly lower LBR and higher MR were obtained after transferring rebiopsied blastocysts compared to those biopsied once. CONCLUSION: Although an extra round of biopsy and vitrification may cause a detrimental effect on embryo viability, re-analyzing the test-failure blastocysts contributes to increasing the number of euploid blastocysts available for transfer and the LBR.

Elevated serum progesterone does not impact euploidy rates in PGT-A patients.

PURPOSE: Some women undergoing stimulated cycles have elevated serum progesterone (P) on the day of ovulation trigger, but its effect on embryo quality is unclear. We analyze embryo quality among patients with high and low serum P undergoing preimplantation genetic testing for aneuploidy (PGT-A). METHODS: This retrospective study included 1597 patients divided into two groups by serum P values: < 1.5 ng/mL or ≥ 1.5 ng/mL. A gonadotrophin-releasing hormone (GnRH) antagonist protocol was established for each patient. Serum P levels were measured on the day of triggering. Propensity score matching and Poisson regression were done. Age, body mass index, and ovarian sensitivity index were also compared. RESULTS: Elevated serum P was not significantly associated with euploid embryo rate or other embryo-quality variables evaluated in our study. Age was the only variable associated with euploidy rate (per MII oocyte, P < 0.001; per biopsied embryo, P = 0.008), embryo biopsy rate (P < 0.001), absolute number of euploid embryos (P = 0.008), and top-quality embryo rate (P = 0.008). Categorical variables decreased in value for every year of increased age in patients with high serum P. CONCLUSIONS: Elevated serum P did not affect the number of euploid and good-quality embryos for transfer in GnRH antagonist intracytoplasmic sperm injection (ICSI) cycles. Contrary to the clear influence of premature P elevation on endometrial receptivity based on literature, our results may help to tip the balance towards the absence of a negative effect of P elevation on embryo competence. More studies are needed to fully understand the effect of P elevation on reproductive outcomes.

Prediction of embryo survival and live birth rates after cryotransfers of vitrified blastocysts.

RESEARCH QUESTION: Which pre-vitrification parameters are the most predictive of survival and live birth in vitrified-warmed blastocyst transfer cycles? DESIGN: A retrospective study including 11,936 warmed blastocysts. Pre-vitrification morphological parameters analysed for blastocysts included day of vitrification; blastocyst expansion degree; trophoectoderm grade (A, B and C); and inner cell mass grade (A, B and C). Univariate and multivariate generalized estimating equations models were used to analyse survival, clinical pregnancy and live birth rate. A stepwise regression analysis was conducted to select and classify by order which outcomes were the most predictive. RESULTS: The odds of survival increased almost twice for blastocysts with lower expansion degree (OR 1.92; 95% CI 1.37 to 2.69; P < 0.001) and by about 50% for blastocysts vitrified on day 5 (OR 1.56; 95% CI 1.27 to 1.89; P < 0.001). Multivariate generalized estimating equations model showed that trophectoderm grade followed by the day of vitrification were the most significant predictors of live birth. The odds of live birth increased nearly three times for blastocysts with trophectoderm graded as A compared with those with trophectoderm graded as C (OR 2.85; 95% CI 2.48 to 3.27; P < 0.001), and double for blastocysts vitrified on day 5 compared with those vitrified on day 6 (OR 2.22; 95% CI 1.97 to 2.49; P < 0.001). The odds of live birth also increased in higher expansion degree blastocysts. CONCLUSIONS: Blastocysts vitrified on day 5 and those with higher trophoectoderm grade should be given priority when warming.