Meiotic Errors Research Articles

Meiotic instability and irregular chromosome pairing underpin heat-induced infertility in bread wheat carrying the Rht-B1b or Rht-D1b Green Revolution genes.

Mutations in the Rht-B1a and Rht-D1a genes of wheat (Triticum aestivum; resulting in Rht-B1b and Rht-D1b alleles) cause gibberellin-insensitive dwarfism and are one of the most important elements of increased yield introduced during the 'Green Revolution'. We measured the effects of a short period of heat imposed during the early reproductive stage on near-isogenic lines carrying Rht-B1b or Rht-D1b alleles, with respect to the wild-type (WT). The temperature shift caused a significant fertility loss within the ears of Rht-B1b and Rht-D1b wheats, greater than that observed for the WT. Defects in chromosome synapsis, reduced homologous recombination and a high frequency of chromosome mis-segregation were associated with reduced fertility. The transcription of TaGA3ox gene involved in the final stage of gibberellic acid (GA) biosynthesis was activated and ultra-performance liquid chromatography-tandem mass spectrometry identified GA1 as the dominant bioactive GA in developing ears, but levels were unaffected by the elevated temperature. Rht-B1b and Rht-D1b mutants were inclined to meiotic errors under optimal temperatures and showed a higher susceptibility to heat than their tall counterparts. Identification and introduction of new dwarfing alleles into modern breeding programmes is invaluable in the development of climate-resilient wheat varieties.

O-078 Mosaic embryos – are they accurately detected by PGT-A?

Abstract Study question Following quantitative NGS for PGT-A, do current thresholds for mosaicism calling give a true reflection of the presumed mitotic origin of mosaic errors? Summary answer A large proportion of embryo mosaicism diagnosed following quantitative NGS-based PGT-A does not correlate with a true and expected mitotic origin, showing instead meiotic signatures. What is known already Mosaicism is a true biological phenomenon that can affect ongoing embryo development. However, the reproductive potential of mosaic embryos in assisted reproduction varies greatly across publications and is not fully understood. While the transfer of mosaic embryos could result in healthy live births, embryos containing meiotic errors almost never result in healthy live births. Since the clinical implications of meiotic and mitotic errors are very different, it is critical to accurately differentiate between the two. However, diagnosing mosaicism using current NGS-based PGT-A is not always accurate due to technical and biological limitations which may misclassify mosaic embryos. Study design, size, duration Follow-up analysis was carried out on 159 embryos previously categorised as mosaic by NGS-based PGT-A. To identify the origin of mosaic calls, SNP genotyping and karyomapping were performed on the same amplified DNA used for the clinical NGS analysis. Trophectoderm biopsy samples were obtained from embryos on days 5,6 or 7 of development from a single IVF centre between 2018-2022. For all PGT-A tested embryos, the reported mosaicism rate at the IVF centre was 12%. Participants/materials, setting, methods Samples were analysed using PGTai from CooperSurgical with a 30-80% CNV mosaicism range. Patients with mosaic embryos consented to karyomapping follow-up using sibling embryo reference DNA. The origin of mosaic events was defined by SNP inheritance patterns for the affected chromosome. Meiotic errors were defined by the presence of both haplotypes from one parent for trisomy, and the absence of a parental haplotype for monosomy. Mitotic errors were inferred following a result of biparental inheritance. Main results and the role of chance A total of 154 diploid embryos identified as mosaic by NGS-based PGT-A were re-analysed by SNP-genotyping. Of these, 30.5% (n = 47) displayed at least one error of meiotic origin following SNP genotyping. Among the 154 embryos, 189 mosaic chromosomal abnormalities were identified. After SNP-genotyping, 30.7% of chromosomal abnormalities (n = 58) showed evidence of meiotic signatures: 52 whole-chromosome (24 gains, 28 losses), and 6 segmental errors (5 gains and 1 loss). Of 73 segmental errors detected in total, only 8.2% of them (n = 6) were identified to be of meiotic origin. Following PGT-A, 7.8% of embryos (n = 12/154) displayed ≥3 chromosomal abnormalities in the mosaic range. Of these embryos, 58.3% (n = 7/12) displayed one or more meiotic aneuploidies. Overall, embryos showing multiple mosaic events were significantly more likely to have a least one error of meiotic origin than embryos containing one abnormality (McNemar’s test p-value= 1.171e-14). Among mosaic calls above 50% CNV, 56.7% (n = 55/97) showed evidence of meiotic origin. In contrast, but importantly, in the low-moderate mosaicism range <50% CNV, 3.3% of errors (3/92) also displayed meiotic signatures, including two whole chromosome errors (chr21 gain at 32% CNV and chr13 loss at 46% CNV), and one segmental error (dup(6)(pter-p12.1) at 33% CNV). Limitations, reasons for caution Since karyomapping relies on recombination events to identify meiotic trisomy, the true origin of intermediate copy-number changes cannot always be identified. Therefore, errors without recombination may be missed, potentially underestimating meiotic gains, especially segmentals. The absence of a parental haplotype cannot entirely rule-out a mitotic error from a biopsy sample. Wider implications of the findings Quantitative NGS methodologies are unable to accurately distinguish the origin of mosaic errors in PGT-A samples. Since the clinical impact of meiotic and mitotic errors differs greatly, this limitation could explain mosaic embryos’ reduced viability. Reliable identification of meiotic aneuploidies in presumed mosaic embryos is likely to improve clinical outcomes. Trial registration number not applicable

O-302 Triploid conceptions are predominantly caused by female meiosis II errors and their risk increases with advancing maternal age

Abstract Study question What are the incidence and origin of ploidy abnormalities in embryos derived from 2 pronuclei (2PN) zygotes, and is their risk linked to maternal age? Summary answer While embryo haploidy is usually due to male meiosis errors, triploidy is mainly caused by female meiosis II errors, whose incidence increases with maternal age. What is known already Normal fertilization is denoted by the appearance of 2PN 16-18 hours after insemination. Deviation from 2PN is considered evidence of abnormal fertilization and ploidy anomalies. Nonetheless, even 2PN embryos can be diagnosed with ploidy abnormalities during preimplantation genetic testing (PGT). The incidence of 3PN has already been linked to advanced maternal age. However, no conclusive and well-powered studies have yet investigated the incidence, origin, and maternal age correlation of genetically-diagnosed ploidy abnormalities. A targeted NGS-based approach that simultaneously analyzes copy number and genotyping data provides a comprehensive ploidy status assessment and helps address numerous basic and clinical research questions. Study design, size, duration Retrospective study including 96,660 trophectoderm biopsies analyzed between 2020 and 2022 using a targetedNGS-based PGT platform. A total of 1,063 embryos carrying haploidy or triploidy were used to investigate maternal age correlation, and parental/meiotic origin of the anomaly. Of these, 57 embryos were from concomitant PGT-M (PGT for monogenic disorders) cycles where also parental DNA was available. These trios (embryo-parents) were used to independently estimate parental/meiotic contribution to the ploidy abnormality and genome-wide recombination events. Participants/materials, setting, methods Targeted-NGS’s accuracy in genotyping and copy number (CN) detection were previously validated on triploid cell lines and multifocal blastocyst biopsies with known ploidy status derived from abnormally fertilized oocytes. Parental and meiotic origins were estimated using two independent approaches. First, they were inferred using sex chromosomes CN from 1,063 embryos with altered ploidy status. Secondly, they were directly calculated using genotyping data from 57 trios (embryo and parents) from PGT-M cycles. Main results and the role of chance The prevalence of ploidy abnormalities in 2PN-derived embryos was 1.1% (n = 1,063/96,660), with triploids accounting for 83.0% (n = 882/1,063) and haploids for 17.0% (n = 181/1,063). Remarkably, the incidence of ploidy anomalies is positively correlated with maternal age (OR = 1.046 per year; p < 0.001). Triploidy showed strong correlation with age (OR = 1.059 per year; p < 0.001), while haploidy did not (OR = 0.96 per year; p = 0.1). Based on sex chromosomes CN analysis, the extra haploid set of triploid embryos was almost completely of maternal origin (94.6%; 95%CI:93.0-96.1), with male errors accounting for only 5.4% (95%CI:4.0-7.1). On the other hand, haploid embryos were the result of paternal errors in 97.8% of cases (95%CI:94.4-99.4), with the missing haploid set being of maternal origin in the remaining 2.2% (95%CI:0.6-5.6). In terms of triploidy’s meiotic origin, two-thirds of the errors occurred during MII (95%CI:63.4-69.8), while one-third occurred during MI (95%CI:30.2-36.5). An independent method using genotyping data of 57 trios confirmed the predominance of paternal error in haploidy (n = 12/14) and the exclusively maternal origin of all embryonic triploidies (n = 43/43). The extra haploid set resulted from an error during MI in 27.9% (n = 12/43) and during MII in 72.1% (n = 31/43) of cases. Interestingly, 16.3% of triploids (n = 7/43) showed no genome-wide recombination events. Limitations, reasons for caution Despite the uniquely large sample size, the inference based on sex chromosomes CN suffers the limitations of the modeling assumptions (independence between parental and meiotic errors), which require further validation. Due to the low prevalence of paternal errors, clinical correlation with male factor could not be investigated with sufficient power. Wider implications of the findings Thanks to the exceedingly high sample size, this is the first study to reveal an increased risk of triploid conception with advancing female age (76% higher at age 40 than at age 30), providing meaningful insights for patients counseling. Importantly, the meiotic origin of ploidy anomalies in embryos were unveiled. Trial registration number not applicable

P-742 Universal genome-wide haplotyping-based preimplantation genetic testing: a systematic seven-year experience at a single center

Abstract Study question To record the outcome and clinical benefit of universal genome-wide haplotyping-based preimplantation genetic testing (PGT). Summary answer Universal PGT allows for standardization of protocols and reduces turnaround time. Apart from accurate genetic diagnosis, it also provides chromosome information, improving embryo selection strategies. What is known already Developments in genome-wide technologies leveraged the implementation of universal PGT that combines haplotyping and copy number typing across the whole genome. Universal PGT provides diagnosis for any familial monogenic disorder (PGT-M) or structural rearrangement (PGT-SR), and allows for concurrent aneuploidy screening (PGT-A) in PGT-M/SR cycles, all using the same generic protocol and the same biopsy. For this reason, various universal PGT approaches have been developed in the last 2-3 years and are increasingly making their way to the clinic worldwide. Study design, size, duration Universal PGT as a first-tier test was implemented at UZ Leuven mid-2014, using in-house developed algorithm (siCHILD/haplarithmisis). This is a retrospective analysis of universal PGT data, collected between 1 January 2015 and 31 December 2022. Data on PGT indication, PGT cycles and biopsy results was extracted. Implantation rate (IR; with IU/EU fetal sac) per embryo transfer was calculated from frozen embryo transfer (FET) cycles for patients with known result by the end of 2021. Participants/materials, setting, methods By the end of 2022, 718 couples have successfully finalized preclinical PGT workup. At the time of writing, 573 couples (80%) have further proceeded with PGT cycles. In total, 1264 PGT cycles were performed with day-3 (D3) cleavage-stage (n = 3344) or trophectoderm (TE) biopsies (n = 1524). For 403 patients with known outcome from 954 FET cycles, IR per embryo transfer was calculated per biopsy type and embryo ranking, considering genome-wide chromosome information. Main results and the role of chance Couples were referred to PGT due to various autosomal dominant (AD, 65%), autosomal recessive (AR, 14%) and X-linked disorders (XL, 10%). Various PGT-M indications encompassed >200 genes in total, but almost 20% of couples enrolled due to inherited BRCA1/BRCA2 mutations. PGT-SR was indication in 7% of cases, mainly for familial microduplications/deletions (64%). For all PGT cycles, approximately 15% and 18% of all performed D3 and TE biopsies, respectively, were scored as abnormal largely due to meiotic errors in the embryo. All unaffected embryos were further ranked as R1 (euploid) or R2 (mosaic), and the number of R2 embryos was expectedly higher after D3 biopsies, compared to TE biopsies (250/1063, 23.6% vs 37/510, 7.3%, p<0.001). In accordance with inheritance mode, the number of unaffected embryos suitable for transfer was significantly lower in AD PGT cycles, compared to AR (32.3% (1116/3455) vs 44.0% (262/596) ; p<0.0001)). For 403 patients with FET cycles, IR was higher in R1 vs R2 embryos after D3 biopsy (231/590, 39.2% vs 30/159, 18.9 %, p <0.0001), whereas no difference was observed between R1 and R2 embryos after TE biopsy, although low number of R2 embryos after TE biopsy was transferred (85/142, 59.9% vs 7/12, 58.3%, p=ns). Limitations, reasons for caution The analyzed data is derived from a single center and the clinical outcome may not reflect the experience of other IVF/PGT centers due to confounding factors, such as PGT indication, used PGT technology and IVF lab practice. Wider implications of the findings The current dataset provides a comprehensive overview on general trends and outcome of universal PGT practice. Apart from accurate genetic diagnosis, universal PGT provides information on chromosomes, which improves embryo selection for transfer. This in turn impacts clinical counselling, interpretation and reporting of the results, and embryo transfer policies. Trial registration number Not applicable

O-060 Single-cell DNA sequencing reveals that current bulk DNA sequencing methods lead to an underestimation of chromosomal mosaicism in human blastocysts

Abstract Study question What is the incidence of chromosomal mosaicism in trophectoderm and inner cell mass from good quality human blastocysts after NGS-based single-cell molecular karyotyping? Summary answer Single-cell analysis showed that almost all human blastocysts are mosaic, involving low level mosaicism and/or abnormal cells resulting from reciprocal error events. What is known already Chromosome segregation in early human embryos is considered to be error prone. While meiotic errors affect all cells within embryos, mitotic errors lead to chromosomal mosaicism, the presence of cytogenetically different cells within an embryo. The reported incidence of mosaicism in blastocysts after bulk DNA sequencing of multicellular trophectoderm (TE) biopsies performed for preimplantation genetic testing for aneuploidy (PGT-A) varies between 2-19%. However, bulk DNA analysis can only assess net average chromosome gains or losses with a detection limit of 20-30%, and it makes distinguishing true mosaicism from technical artefact problematic. Study design, size, duration Observational study in human good quality surplus embryos which were donated for research purposes (CCMO, NL82597.000.22). The embryos were thawed and cultured until the blastocyst stage. From the blastocysts with at least a morphology grade 3BB, the ICM was dissociated from the TE. From 55 embryos, both samples were successfully disaggregated into single cells and manually placed in 384 well plates. For the cytogenetic analysis, a validated method for single-cell molecular karyotyping was used (scKaryo-seq). Participants/materials, setting, methods Embryos with at least two cytogenetically different cells were considered mosaic. To investigate if bulk DNA sequencing would have detected this mosaicism, we performed an in-silico reanalysis on our single-cell data. For each embryo, TE cells with the same mitotic abnormality were quantified. Next, we determined the number of mosaic embryos in which these abnormalities were present in at least 20% of TE cells, as a cut-off for what PGT-A methods are expected to detect. Main results and the role of chance On average 42% of the total number of cells that could be isolated per embryo were successfully karyotyped, giving a total of 1057 karyotyped cells (522 normal, 535 abnormal) from 55 embryos. Six embryos (11%) were normal, four (7%) were uniformly abnormal and 45 (82%) were mosaic. From these, 14 (26%) embryos were aneuploid mosaic with cytogenetically different abnormal cells and 31 (56%) were diploid-aneuploid mosaic with normal and abnormal cells. Here cytogenetically different abnormal cells were also frequently observed, with products of reciprocal events detected in 20 embryos. The in-silico reanalysis was performed on 38 embryos with a mosaic TE. It predicted that only 11% of the mitotic abnormalities observed in TE cells through scKaryo-seq would have been detected by bulk DNA sequencing. In 19 out of 38 embryos at least one mitotic abnormality affected more than 20% of the TE cells, meaning that only 50% of the embryos would have been recognized as mosaic with bulk DNA analysis. Bulk DNA sequencing methods used for PGT-A lead to an underestimation of mosaicism, since low-level mosaicism that affects a few cells within an embryo and products of reciprocal events without a net gain or loss remain undetected. Limitations, reasons for caution Isolation and cytogenetic analysis of viable single cells at the blastocyst stage is technically challenging and only 42% of the cells per embryo could be successfully karyotyped. Furthermore, it is unknown to what extent in vitro culture conditions and the freezing-thawing process influenced our findings. Wider implications of the findings These single cell observations support the notion that mosaicism is a common biological phenomenon in human blastocysts. Any selective mechanism eliminating embryos or cells with chromosomal abnormalities is still not fully active at this stage. The underestimation of mosaicism by bulk DNA analysis has potential implications for current PGT-A practices Trial registration number not applicable

P-002 SARS-CoV-2 tropism spectrum for human testicular cells

Abstract Study question In human testicular cells, how are SARS-CoV-2 receptors and their priming proteases for the viral spike (S) protein distributed spatially? Summary answer Human testicular tissue expressed both the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) receptors and proteases investigated, key factors for cellular susceptibility to SARS-CoV-2 entry/infection. What is known already Male reproductive function is altered on numerous levels by SARS-CoV-2 and coronavirus disease. Angiotensin-converting enzyme 2 (ACE2) is the main SARS-CoV-2 host membrane receptor but requires transmembrane protease serine 2 (TMPRSS2) for S protein priming (activation by proteolysis). Other proteases, like cathepsin L (CTSL), might nevertheless contribute to SARS-CoV-2 entrance. This mostly happens in cells with low or nonexistent ACE2 expression, which calls for alternative receptors like cluster of differentiation 147 (CD147). Although co-expression of receptors and proteases is essential for successful SARS-CoV-2 infection, it is still unknown how viral receptors and accompanying proteases are distributed geographically in testicular cells. Study design, size, duration To overcome this restriction, we focused on using immunohistochemistry to map the spatial distribution of the SARS-CoV-2 receptors ACE2 and CD147, as well as their priming proteases for the viral spike (S) protein, TMPRSS2 and CTSL, which are necessary for viral fusion with host cells, in human testicular tissue. Participants/materials, setting, methods Laboratory experiments were executed under the Joint Ethics Committee of the Hospital and University (approval number 2020-094 (077-DEFI-078-CE)). Human testicular tissues were obtained from cases presenting Sertoli cell syndrome, maturation arrest, and hypospermatogenesis at the Hospital Pathology department, with five patients per pathology. Formalin-fixed paraffin-embedded tissue sections 4 μm were incubated with anti-antibodies ACE2 (orb638860, 1:100 and 1:150, Byorbit), TMPRSS2 (ab109131, 1:1000, Abcam), CD147 (MA5-29060, 1:2500, Invitrogen) and CTSL (MA5-32602, 1:100, Invitrogen). Main results and the role of chance The present study did not intend to study the virus entry proteins in different testicular pathologies. As spermatogenesis is a complex process involving many intricately linked cells, we selected these syndromes specifically to identify individual cells more clearly, Sertoli cells in Sertoli cell-only syndrome cases, spermatocytes in maturation arrest cases and spermatids in hypospermatogenesis cases. The hypospermatogenesis group consisted of cases whose azoospermia was due to duct obstruction, showing conserved spermatogenesis. Interstitial cells, including endothelium, Leydig and myoid peritubular cells, and the seminiferous epithelium (Sertoli cells, spermatogonia, spermatocytes, and spermatids), were discovered to contain ACE2 and TMPRSS2, demonstrating co-expression of both receptor and protease in all testicular cells. All cell types, with the exception of endothelium and peritubular cells, showed the presence of CD147, whereas CTSL was only found in Leydig, peritubular, and Sertoli cells. As a result, only Leydig and Sertoli cells co-expressed CD147 and CTSL. The findings of proteomic databases indicating testicular cells have SARS-CoV-2 receptors and proteases are supported by our discoveries, demonstrating that testicular cells may be directly infected and injured, halting spermatogenesis and acting as viral vectors for the SARS-CoV-2. Limitations, reasons for caution Our results support viral tropism for human testicular cells and raise the possibility of testicular manifestations, although this does not necessarily mean that the virus infects testicular cells directly and further studies are needed to discern SARS-CoV-2 infection and transmission targeting alternative receptors and to identify the underlying mechanisms. Wider implications of the findings Male reproductive system susceptibility to viral infections has long been demonstrated. To prevent infection by allowing the virus to attach to germ cells, impair sperm production, and increase the risk of meiotic errors and viruses spreading through sperm, our study emphasizes the importance of safe practices in the wider community. Trial registration number not applicable

O-307 Aneuploidy in oocytes of women of advanced maternal age: impact on embryo development and evidence for a novel mechanism of meiotic error

Abstract Study question In oocytes of advanced maternal age (AMA) women, what is the association of aneuploidy with embryo development and what are the mechanisms leading to aneuploidy? Summary answer We confirmed precocious separation of sister chromatids as the main cause of aneuploidy. Known chromosome segregation errors explained 90.4% of the observed abnormal chromosome copy numbers in polar bodies. What is known already Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years), and can affect over half of oocytes in this age group. This underlies the rationale for polar body (PB) biopsy as a form of early PGT-A, as performed in the ‘ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis’ (ESTEEM) randomised controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), meiosis II (MII) nondisjunction (ND) and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Study design, size, duration Data was sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridisation (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs, and on embryo morphology in a subset of embryos, up to day 6 post insemination (dpi), from both the intervention (PB biopsy for PGT-A) and from control (no biopsy) arms. Participants/materials, setting, methods ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (2 pronuclei) in the PB biopsy group and 1082 in the control group were compared using age-adjusted multilevel mixed-effect ordinal logistic regression for each day of embryo scoring and summarized with adjusted odds ratios (aOR) and 95% confidence intervals (CI). Main results and the role of chance Overall, 461/693 PB pairs showed abnormal segregation in 1162/10810 chromosomes. The main observed abnormal segregations were compatible with PSSC in meiosis I (MI) (n = 568/1162; 48,9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%) and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9,6%), we observed a chromosome copy number in PB1 and PB2 that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 dpi compared to the control group, indicating that polar body biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day (using the before-mentioned embryo scoring subcategories), aneuploidy was associated with a decrease in embryo quality on day 3 (aOR 0.62, 95% CI 0.43-0.90), day 4 (aOR 0.15, 95% CI 0.06-0.39) and day 5 (aOR 0.28, 95% CI 0.14-0.58). Limitations, reason for caution RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Wider implications of the findings Our finding of frequent unexplained chromosome copy numbers in polar bodies indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by missegregation of chromosomes during meiosis I and II. Study funding/competing interest(s) Data was mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. None of the authors have competing interests. This work is dedicated to the memory of Professor Joep Geraedts. Trial registration number NCT01532284 Study funding Yes Funding source Funding by national/international organization(s)

Simultaneous genome-wide haplotyping and copy number detection enabling PGT-A and PGT-M testing in equine in vitro produced embryos

The number of equine embryos being produced increases yearly (37,094 +10% in 2021; newsletter IETS December 2022). The reasons include the growing popularity of in vitro embryo production (IVP), which increases the genetic yield from high quality mares, maximizes the use of expensive straws of semen from high quality stallions and enables IVP to be performed outside the reproductive season. On the 4th October 2022, a record price of €95,000 was paid for an embryo. However, despite the embryos’ promising pedigrees, it remains unknown if transfer of such embryos will result in a healthy foal with the aspired phenotypical characteristics. This is largely because in horses, preimplantation genetic testing for aneuploidies and monogenic diseases or traits (PGT-A and PGT-M) remains underdeveloped especially when compared to humans. In addition, equine IVP has been reported to double early pregnancy losses, possibly because of an increased incidence of genetic errors. Here, we explored the possibility of concurrent genome-wide PGT-A and PGT-M in horses and assessed the incidence of chromosomal aberrations in IVP arrested or blastocyst stage embryos. We used GGP Equine SNP arrays (Illumina) with over 70,000 SNP positions to analyze whole-genome amplified DNA (Repli-G Single cell kit, Qiagen) from 14 trophectoderm biopsies and corresponding whole blastocysts and 26 individual cells from six embryos arrested at cleavage stage following IVP. Haplarithmisis (Zamani–Esteki et al. Am. J. Hum.Genet. 2015; 96:894-912) was applied to simultaneously determine the genome-wide copy number and haplotype. Five biopsies and seven single cells failed to give a result. From six arrested embryos, five had chromosomal errors in most of their cells, including (combinations of) triploidy and one or more monosomies and trisomies of meiotic and / or mitotic origin. Two biopsies showed a loss of maternal chromosome 28 and 31 respectively, which was confirmed in the corresponding blastocysts, suggesting meiotic errors. In one of those biopsies, additional complex aneuploidies not present in the blastocyst were found, suggesting a mitotic origin. Thus, aneuploidy and ploidy errors are highly prevalent in IVP arrested embryos compared to blastocysts. Like other mammals, these errors may contribute to embryonic arrest before or after clinical pregnancy detection. As a proof of principle, we determined the inherited haplotypes for four common disease-associated genes with high carrier frequency in different horse breeds (GBE1, PLOD1, B3GALNT2, MUTYH), and for one color coat-associated gene (STX17) in one biopsy-embryo combination, confirming the potential of generic genome-wide PGT-M concurrent with PGT-A testing. Similar to humans, we postulate that embryo selection will improve embryo transfer success rates in horses and enable selection for traits and against diseases thus accelerating breeding programs.

Aging and oocyte competence: A molecular cell perspective.

Follicular microenvironment is paramount in the acquisition of oocyte competence, which is dependent on two interconnected and interdependent processes: nuclear and cytoplasmic maturation. Extensive research conducted in human and model systems has provided evidence that those processes are disturbed with female aging. In fact, advanced maternal age (AMA) is associated with a lower chance of pregnancy and live birth, explained by the age-related decline in oocyte quality/competence. This decline has largely been attributed to mitochondria, essential for oocyte maturation, fertilization, and embryo development; with mitochondrial dysfunction leading to oxidative stress, responsible for nuclear and mitochondrial damage, suboptimal intracellular energy levels, calcium disturbance, and meiotic spindle alterations, that may result in oocyte aneuploidy. Nuclear-related mechanisms that justify increased oocyte aneuploidy include deoxyribonucleic acid (DNA) damage, loss of chromosomal cohesion, spindle assembly checkpoint dysfunction, meiotic recombination errors, and telomere attrition. On the other hand, age-dependent cytoplasmic maturation failure is related to mitochondrial dysfunction, altered mitochondrial biogenesis, altered mitochondrial morphology, distribution, activity, and dynamics, dysmorphic smooth endoplasmic reticulum and calcium disturbance, and alterations in the cytoskeleton. Furthermore, reproductive somatic cells also experience the effects of aging, including mitochondrial dysfunction and DNA damage, compromising the crosstalk between granulosa/cumulus cells and oocytes, also affected by a loss of gap junctions. Old oocytes seem therefore to mature in an altered microenvironment, with changes in metabolites, ribonucleic acid (RNA), proteins, and lipids. Overall, understanding the mechanisms implicated in the loss of oocyte quality will allow the establishment of emerging biomarkers and potential therapeutic anti-aging strategies. This article is categorized under: Reproductive System Diseases > Molecular and Cellular Physiology.

Genetic control of meiosis surveillance mechanisms in mammals.

Meiosis is a specialized cell division that generates haploid gametes and is critical for successful sexual reproduction. During the extended meiotic prophase I, homologous chromosomes progressively pair, synapse and desynapse. These chromosomal dynamics are tightly integrated with meiotic recombination (MR), during which programmed DNA double-strand breaks (DSBs) are formed and subsequently repaired. Consequently, parental chromosome arms reciprocally exchange, ultimately ensuring accurate homolog segregation and genetic diversity in the offspring. Surveillance mechanisms carefully monitor the MR and homologous chromosome synapsis during meiotic prophase I to avoid producing aberrant chromosomes and defective gametes. Errors in these critical processes would lead to aneuploidy and/or genetic instability. Studies of mutation in mouse models, coupled with advances in genomic technologies, lead us to more clearly understand how meiosis is controlled and how meiotic errors are linked to mammalian infertility. Here, we review the genetic regulations of these major meiotic events in mice and highlight our current understanding of their surveillance mechanisms. Furthermore, we summarize meiotic prophase genes, the mutations that activate the surveillance system leading to meiotic prophase arrest in mouse models, and their corresponding genetic variants identified in human infertile patients. Finally, we discuss their value for the diagnosis of causes of meiosis-based infertility in humans.

Genetic aetiology of Down syndrome birth: novel variants of maternal DNMT3B and RFC1 genes increase risk of meiosis II nondisjunction in the oocyte.

The aim of the present work was to explore the intriguing association of maternal folate regulator gene polymorphisms and mutations with the incidence of chromosome 21 nondisjunction and Down syndrome birth. We tested polymorphisms/mutations of DNMT3B and RFC1 genes for their association with meiotic errors in oocyte among the 1215 Down syndrome child-bearing women and 900 controls. We observed that 23 out of 31 variants of DNMT3B and RFC1 exhibited an association with meiosis II nondisjunction in maternal age-independent manner. Additionally, we have reported 17 novel mutations and 1 novel polymorphic variant that are unique to the Indian Bengali speaking cohort and increased odds in favour of meiosis II nondisjunction. We hypothesize that the risk variants and mutations of DNMT3B and RFC1 genes may cause reduction in two or more recombination events and also cause peri-centromeric single exchange that increases the risk of nondisjunction at any age of women. In silico analyses predicted the probable damages of the transcripts or proteins from the respective genes owing to the said polymorphisms. These findings from the largest population sample tested ever revealed that mutations/polymorphisms of the genes DNMT3B and RFC1 impair recombination that leads to chromosome 21 nondisjunction in the oocyte at meiosis II stage and bring us a significant step closer towards understanding the aetiology of chromosome 21 nondisjunction and birth of a child with Down syndrome to women at any age.

Genetic deconvolution of fetal and maternal cell-free DNA in maternal plasma enables next-generation non-invasive prenatal screening

Current non-invasive prenatal screening (NIPS) analyzes circulating fetal cell-free DNA (cfDNA) in maternal peripheral blood for selected aneuploidies or microdeletion/duplication syndromes. Many genetic disorders are refractory to NIPS largely because the maternal genetic material constitutes most of the total cfDNA present in the maternal plasma, which hinders the detection of fetus-specific genetic variants. Here, we developed an innovative sequencing method, termed coordinative allele-aware target enrichment sequencing (COATE-seq), followed by multidimensional genomic analyses of sequencing read depth, allelic fraction, and linked single nucleotide polymorphisms, to accurately separate the fetal genome from the maternal background. Analytical confounders including multiple gestations, maternal copy number variations, and absence of heterozygosity were successfully recognized and precluded for fetal variant analyses. In addition, fetus-specific genomic characteristics, including the cfDNA fragment length, meiotic error origins, meiotic recombination, and recombination breakpoints were identified which reinforced the fetal variant assessment. In 1129 qualified pregnancies tested, 54 fetal aneuploidies, 8 microdeletions/microduplications, and 8 monogenic variants were detected with 100% sensitivity and 99.3% specificity. Using the comprehensive cfDNA genomic analysis tools developed, we found that 60.3% of aneuploidy samples had aberrant meiotic recombination providing important insights into the mechanism underlying meiotic nondisjunctions. Altogether, we show that the genetic deconvolution of the fetal and maternal cfDNA enables thorough and accurate delineation of fetal genome which paves the way for the next-generation prenatal screening of essentially all types of human genetic disorders.

Meiosis in allopolyploid Arabidopsis suecica

SUMMARYPolyploidy is a major force shaping eukaryote evolution but poses challenges for meiotic chromosome segregation. As a result, first‐generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. How established polyploids adapt their meiotic behaviour to ensure genome stability and accurate chromosome segregation remains an active research question. We present here a cytological description of meiosis in the model allopolyploid species Arabidopsis suecica (2n = 4x = 26). In large part meiosis in A. suecica is diploid‐like, with normal synaptic progression and no evidence of synaptic partner exchanges. Some abnormalities were seen at low frequency, including univalents at metaphase I, anaphase bridges and aneuploidy at metaphase II; however, we saw no evidence of crossover formation occurring between non‐homologous chromosomes. The crossover number in A. suecica is similar to the combined number reported from its diploid parents Arabidopsis thaliana (2n = 2x = 10) and Arabidopsis arenosa (2n = 2x = 16), with an average of approximately 1.75 crossovers per chromosome pair. This contrasts with naturally evolved autotetraploid A. arenosa, where accurate chromosome segregation is achieved by restricting crossovers to approximately 1 per chromosome pair. Although an autotetraploid donor is hypothesized to have contributed the A. arenosa subgenome to A. suecica, A. suecica harbours diploid A. arenosa variants of key meiotic genes. These multiple lines of evidence suggest that meiosis in the recently evolved allopolyploid A. suecica is essentially diploid like, with meiotic adaptation following a very different trajectory to that described for autotetraploid A. arenosa.

Oocyte aneuploidy rates in river and swamp buffalo types (Bubalus bubalis) determined by Multi-color Fluorescence In Situ Hybridization (M-FISH)

Aneuploidy is one of the main causes of fetal and embryonic mortality in mammals. Nonetheless, its incidence in domestic ruminants has been investigated little. Indeed, no incidence data have ever been reported for water buffalo. To establish the incidence of aneuploidy in this species, we analysed in vitro matured metaphase II (MII) oocytes with corresponding first polar bodies (I PB) of the river (2n = 50) and swamp (2n = 48) buffaloes. For the first time, six river type probes (corresponding to chromosomes 1–5 and heterosome X), were tested on swamp buffalo metaphases using Multicolor-Fluorescent In Situ Hybridization (M-FISH) before their use on oocytes MII metaphases. Of the 120 total Cumulus Oocyte Complexes (COCs, 60 for each buffalo type) subjected to in vitro maturation, 104 reached the MII stage and were analysed by M-FISH. Haploid chromosome arrangement and visible I PB were observed in 89 of the oocytes (45 in river and 44 in swamp type). In the river type, the analysis revealed one oocyte was disomic for the chromosome X (2.22%). In the swamp type, one oocyte was found to be nullisomic for chromosome X (2.27%); another was found to be nullisomic for chromosome 5 (2.27%). We also observed one oocyte affected by a premature separation of sister chromatids (PSSC) on the chromosome X (2.27%). In both buffalo types, no abnormalities were detected in other investigated chromosomes. Based on merged data, the overall aneuploidy rate for the species was 3.37%. Oocytes with unreduced chromosomes averaged 1.92% across the two types, with 1.96% in river and 1.88% in swamp. The interspecies comparison between these data and cattle and pig published data revealed substantial difference in both total aneuploidy and diploidy rates. Reducing the negative impact of the meiotic segregation errors on the fertility is key to more sustainable breeding, an efficient embryo transfer industry and ex-situ bio-conservation. In this respect, additional M-FISH studies are needed on oocytes of domestic species using larger sets of probes and/or applying next generation sequencing technologies.

Age-dependent integrity of the meiotic spindle assembly checkpoint in females requires Aurora kinase B.

A hallmark of advanced maternal age is a significant increase in meiotic chromosome segregation errors, resulting in early miscarriages and congenital disorders. These errors most frequently occur during meiosis I (MI). The spindle assembly checkpoint (SAC) prevents chromosome segregation errors by arresting the cell cycle until proper chromosome alignment is achieved. Unlike in mitosis, the SAC in oocytes is desensitized, allowing chromosome segregation in the presence of improperly aligned chromosomes. Whether SAC integrity further deteriorates with advancing maternal age, and if this decline contributes to increased segregation errors remains a fundamental question. In somatic cells, activation of the SAC depends upon Aurora kinase B (AURKB), which functions to monitor kinetochore–microtubule attachments and recruit SAC regulator proteins. In mice, oocyte‐specific deletion of AURKB (Aurkb cKO) results in an increased production of aneuploid metaphase II‐arrested eggs and premature age‐related infertility. Here, we aimed to understand the cause of the short reproductive lifespan and hypothesized that SAC integrity was compromised. In comparing oocytes from young and sexually mature Aurkb cKO females, we found that SAC integrity becomes compromised rapidly with maternal age. We show that the increased desensitization of the SAC is driven by reduced expression of MAD2, ZW10 and Securin proteins, key contributors to the SAC response pathway. The reduced expression of these proteins is the result of altered protein homeostasis, likely caused by the accumulation of reactive oxygen species. Taken together, our results demonstrate a novel function for AURKB in preserving the female reproductive lifespan possibly by protecting oocytes from oxidative stress.

Haplotyping-based preimplantation genetic testing reveals parent-of-origin specific mechanisms of aneuploidy formation

Chromosome instability is inherent to human IVF embryos, but the full spectrum and developmental fate of chromosome anomalies remain uncharacterized. Using haplotyping-based preimplantation genetic testing for monogenic diseases (PGT-M), we mapped the parental and mechanistic origin of common and rare genomic abnormalities in 2300 cleavage stage and 361 trophectoderm biopsies. We show that while single whole chromosome aneuploidy arises due to chromosome-specific meiotic errors in the oocyte, segmental imbalances predominantly affect paternal chromosomes, implicating sperm DNA damage in segmental aneuploidy formation. We also show that postzygotic aneuploidy affects multiple chromosomes across the genome and does not discriminate between parental homologs. In addition, 6% of cleavage stage embryos demonstrated signatures of tripolar cell division with excessive chromosome loss, however hypodiploid blastomeres can be excluded from further embryo development. This observation supports the selective-pressure hypothesis in embryos. Finally, considering that ploidy violations may constitute a significant proportion of non-viable embryos, using haplotyping-based approach to map these events might further improve IVF success rate.

Age-cumulative Effect of REC8 Reduction on Meiotic Chromosome Segregation Errors in Mice

Abstract Objective: This study aimed to explore the relationship between cohesin subunit REC8 reduction and meiosis chromosome segregation errors in the ovary. Methods: Rec8 +/− mice were generated using CRIPSR/Cas9 gene editing. The association between age and REC8 expression levels in the ovary was determined by Western blotting. Chromosome segregation errors were investigated by immunofluorescence imaging of superovulated oocytes. Wild-type and Rec8 +/− female mice at 5, 8, 20, 36, and 40 weeks were used to evaluate ovarian reserve by ovarian clearing and immunolabeling. Results: Ovary REC8 expression levels gradually decreased with age, while chromosome segregation errors increased with age. Segregation errors were more common in Rec8 +/− mice, suggesting an association with REC8 expression. The ovarian reserve capacity decreased significantly with age. The ovarian reserve in Rec8 +/− mice was inferior to that of age-matched wild-type mice, indicating important roles of age and REC8 levels in the ovarian reserve. Conclusions: REC8 reduction has an age-cumulative effect on meiotic chromosome segregation errors in mouse ovaries. Rec8 haploinsufficiency poses a major challenge in generating normal and reproductive oocytes in aging mice.

ACCURATE GENOMIC PREDICTION OF MOSAICISM THROUGH CELL DIVISION ORIGIN OF ANEUPLOIDY ANALYSIS IN THE PREIMPLANTATION EMBRYO

Simple intermediate copy number thresholds have proven to provide poor predictive value for diagnosing preimplantation mosaicism (both analytically and clinically). This study aims to validate a method to determine the cell division origin of aneuploidy from a single trophectoderm biopsy, and its ability to accurately predict mosaicism in the remaining embryo. A high throughput and PGT-validated DNA array platform was used to analyze over 800,000 SNPs within trophectoderm biopsies from a subset of PGT cases where parental DNA was available. Mendelian inheritance analyses were performed on embryonic genotypes within aneuploid chromosomes to predict their parental and cell division origins. Positive controls included embryos with a male karyotype (n=279) with known maternal origin of the X chromosome, and unbalanced embryos from PGT-SR cases (n=69) with known meiotic and parental origins of aneuploidy. The method was applied to clinical biopsies with aneuploidy and compared to rebiopsies (n=80) to evaluate the accuracy of predicting mosaicism (mitotic) and uniform (meiotic) aneuploidy. All samples were analyzed while blinded to prior results. Following validation, the incidence of mitotic origin of aneuploidy was investigated in 942 embryos. Blinded predictions of the X chromosome origin in male embryos, and parental and cell division origin of aneuploidy in PGT-SR derived unbalanced embryos were 100% concordant with expected results, demonstrating the validity of predicting parental and meiotic cell division origins of aneuploidy. Blinded analysis of rebiopsies showed that all embryos with mitotic origin of aneuploidy predicted in the original biopsy were confirmed as mosaic, and all embryos with meiotic origin were confirmed as uniform aneuploid, demonstrating the validity of predicting mitotic origins of aneuploidy and mosaicism. The overall incidence of mitotic whole chromosome aneuploidy (mosaicism) in embryos was 3%. The overall embryonic aneuploidy rate was 31% (mean maternal age 34.5+4). Aneuploid chromosomes primarily originated from the maternal genome (80%) and maternal whole chromosome aneuploidy primarily originated from meiotic errors (95%), while paternal aneuploidy more often originated from mitotic errors (35%)(p<0.05). These data indicate very high specificity in predicting mosaicism from a single aneuploid trophectoderm biopsy and a low prevalence of mosaicism (3%) in the human blastocyst. This is in stark contrast to existing methods of predicting mosaicism with intermediate copy number thresholds, where rebiopsy confirmation rates are ∼40%, and prevalence is estimated to be ∼20%. Improving the specificity of predicting mosaicism (reducing false positives) from a single biopsy may provide a unique opportunity to investigate the true clinical consequence of transferring bona fide mosaic embryos for the first time.

YOUNGER PATIENTS ARE MORE LIKELY TO HAVE MOSAIC-ONLY EMBRYOS DUE TO THEIR LOWER LIKELIHOOD OF AGE-RELATED WHOLE CHROMOSOME ANEUPLOIDY

While embryo mosaicism is most likely a frequent biological occurrence, during ART treatment mosaicism frequencies had been thought to be influenced by embryo culture environment, sperm quality, insemination method, and/or biopsy protocol. 1 Notwithstanding these associations, mosaicism rates had been thought to be largely independent of female age.2,3 A recent study shown a statistical trend towards decreased rates of mosaicism in women of advanced age (>37 years) compared with younger groups 4. Based on these observations, our study aims to assess the rate of mosaicism across patient age in a large cohort of screened blastocysts from a single IVF center. Patients who underwent IVF from January 2020 - March 2021 were included. All embryos underwent PGT-A with FAST-SeqS. Embryos were assigned an interpretation of aneuploid, mosaic, or euploid; indeterminate/no call embryos were not included in the analysis. The PGT lab utilized for testing did not report mosaicism when present with a full aneuploidy; those embryos were assigned the interpretation of aneuploid. Thus, any embryos designated as mosaic are “mosaic-only.” Cohorts were segregated into 5 groups based on SART age groups: (A: <35; B: 35-37; C: 38-40; D: 41-42; E: ≥43 years, respectively). Frequencies of mosaicism were compared across age groups with a Chi squared analysis and a multivariate regression analysis was performed to adjust for confounding variables. A total of 7,940 embryo biopsies were analyzed. A 13.01% prevalence of mosaic-only embryos was found in the cohort (n=1,035). When analyzing these frequencies per age, younger age groups had a significant higher prevalence of mosaic-only embryos: Group A: 15.2% (n=524); B: 14.13% (n=274); C: 10.9% (n=177); D: 6.6% (n=45) and E: 5% (n=13) (p=<0.0001). After adjusting for day of biopsy and embryo grading, an increase in age was significantly associated with lower odds of isolated mosaicism (OR 1.06 CI95% 1.05-1.06). Day of biopsy, and embryonic quality were not associated with increased odds of isolated mosaicism. A sub-analysis analyzing the mosaicism levels detected with the NGS platform found no differences in frequencies of high and low level mosaicisms across age groups. With the introduction of NGS, reproductive specialists have expanded knowledge surrounding the complexities of early human genomics. Our analysis demonstrates that younger women were more likely to have mosaic embryos without concurrent full aneuploidies. Patients of advanced age are more prone to meiotic segregation abnormalities, resulting in an increased probability of experiencing full and/or complex aneuploidies. For younger patients who are less likely to have meiotic errors, any abnormal findings are more likely to be mitotic in origin, and therefore mosaic.

RECOMBINATION EVENTS THAT PREDISPOSE MATERNAL CHROMOSOMES TO INCREASED MISSEGREGATION ARE MORE THAN JUST A NUMBER

The gain or loss of chromosomes during oogenesis is the most significant contributor to reproductive failure and miscarriage. The number of recombination sites fixed during fetal development potentially sets up and influences the generation of an aneuploid oocyte. The aim of this study was to investigate recombination events across all 23 pairs of embryonic chromosomes to shed a light on inheritance and molecular mechanisms associated with meiotic segregation errors.