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.

The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Mitochondrial DNA content decreases during in vitro human embryo development: insights into mitochondrial DNA variation in preimplantation embryos donated for research.

OBJECTIVE: To assess the mitochondrial DNA (mtDNA) load and variation in human oocytes and during preimplantation embryo development using specimens donated for research. DESIGN: Prospective cohort study. SETTING: Not applicable. PATIENTS: A total of 50 in vitro fertilization patients and 11 oocyte donors whose specimens were obtained between July 2017 and July 2018. INTERVENTIONS: None. MAIN OUTCOME MEASURES: All specimens were separately collected. Quantitative polymerase chain reaction was performed with SurePlex DNA Amplification System (Illumina). Primers for the adenosine triphosphate 8 mitochondrial gene and the ß-actin were used. Data were statistically analyzed by analysis of variance with the Scheffé multiple pairwise comparison for categorical variables and by linear regression for numerical variables. RESULTS: Human metaphase II (MII) oocytes had significantly more total mtDNA copy number than day 3 embryos, and day 3 embryos had more total and per-cell mtDNA copy number than aneuploid blastocysts. There was a significant decrease in mtDNA content associated with failed-fertilized oocytes compared to noninseminated metaphase II oocytes. CONCLUSIONS: During preimplantation development, before implantation, human embryos undergo a significant decrease in total mtDNA content and no increase in mtDNA content at the blastocyst stage. Oocytes need to carry a correct threshold of mitochondrial load in the oocyte in order to successfully fertilize. An active degradation of mtDNA before implantation occurs after fertilization takes place. These findings could be used to improve knowledge about the best embryo culture conditions and would serve as a basis for further studies addressing again the use of mtDNA content as an embryo viability marker.

Variables associated with mitochondrial copy number in human blastocysts: what can we learn from trophectoderm biopsies?

OBJECTIVE: To study the potential variables that affect the mitochondrial DNA (mtDNA) content of trophectoderm (TE) cells in blastocysts that have undergone TE biopsy. DESIGN: Observational retrospective single-center analysis. SETTING: University-affiliated private in vitro fertilization center. PATIENT(S): A total of 465 consecutive preimplantation genetic screening (PGS) cycles of 402 women undergoing preimplantation genetic testing. INTERVENTION(S): Trophectoderm biopsy performed on blastocysts of women undergoing preimplantation genetic testing-aneuploidy (PGT-A). MAIN OUTCOME MEASURE(S): The mtDNA content in trophectoderm cells. RESULT(S): We checked the possible influence of patient characteristics, ovarian stimulation variables, embryo morphology, and embryo culture conditions on mtDNA values. Of all the analyzed variables, some such as body mass index (BMI), serum progesterone (P4), aneuploidy, and trophectoderm quality had an effect on mtDNA content in blastocysts. Body mass index had a small but positive effect on the mtDNA copy number; as the BMI values increased, the probability of women producing blastocysts with an mtDNA content above the median increased by 6%. For P4 serum concentration, an increase in P4 lowered the probability of blastocysts having values above the median by 39%. Embryo-associated variables such as TE quality and aneuploidy status appeared to affect the mtDNA copy number. For the aneuploid blastocysts, the probability of being above the median increased by 42%. Finally, blastocysts with poor quality TE had more chances of carrying higher mtDNA values. CONCLUSION(S): Summarizing, larger quantities of mtDNA in blastocysts are associated with the condition of aneuploidy and low quality TE, as well as being from women with high BMI values. Understanding the biological meaning of mtDNA content in human blastocysts and what factors may interfere with their values is fundamental. Other key gaps, such as whether a correlation exists between mtDNA content and mitochondrial mass and biogenesis in human TE cells, and whether this correlation can be extended to the inner cell mass, need to be further addressed. These questions are currently being investigated.

Collapse of blastocysts is strongly related to lower implantation success: a time-lapse study.

STUDY QUESTION: Is there an association between blastocyst collapse patterns and implantation potential? SUMMARY ANSWER: Embryos that exhibit collapse are as likely to hatch as those that do not, but are less likely to implant and should not be replaced if alternatives are available. WHAT IS KNOWN ALREADY: Studies of blastocyst collapse in different species of mammals have found that most blastocysts, that experience consecutive weak contractions, hatch successfully whereas those that exhibit strong contractions or collapse, fail to hatch. STUDY DESIGN, SIZE, DURATION: Retrospective cohort study. Seven hundred and fifteen transferred blastocysts were analyzed from July 2012 to May 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: University-affiliated infertility center. Participant patients were recipients of oocyte donation and autologous IVF cycles (n = 460). Embryo development was analyzed with a time-lapse imaging system. Variables studied included blastocyst collapse (defined as the separation of ≥50% of the surface of the trophectoderm of the blastocyst from the zona pellucida), kinetic variables, embryo morphology, implantation and clinical pregnancy rates. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 139 blastocysts presented collapse (19.4%), of these 8 presented 2 collapses and 2 presented 3 collapses. The timings of embryo cleavages and the time taken to reach the morula stage and blastulation were significantly shorter in embryos with collapse than in those without. Implantation rates were compared in cycles where either all or none of the embryos replaced, implanted so that implantation data were known for every embryo. Among 408 embryos without collapse and 94 embryos with at least one collapse (94) the implantation rates were 48.5% (95% confidence intervals (CI) 46.7-53.4%) and 35% (95% CI 25.3-44.9%), respectively. The percentage of embryos that hatched was similar in both groups 28.7 and 31%, respectively. LIMITATIONS, REASONS FOR CAUTION: The retrospective nature of the study limits its potential value. WIDER IMPLICATIONS OF THE FINDINGS: Study the collapse pattern of the blastocyst, may assist selection of the blastocysts most likely to implant and increase IVF/ICSI success rates.