Zhiqiang Yan

Background: Oocyte cryopreservation is widely used in assisted reproductive technology, but its effects on embryonic development remain a concern. This study aimed to determine whether oocyte cryopreservation induces transcriptional alterations at the early cleavage and blastocyst stages. Methods: We performed single‐cell RNA sequencing on mouse Metaphase II oocyte and embryos at early/late 1 cell, early/late 2 cell, morula and blastocyst stages derived from cryopreserved (cryo) and fresh (fresh) oocytes. Transcriptomic profiles were compared between the two groups. Results: Global transcriptomes were highly similar between cryo and fresh blastocysts. Oocyte cryopreservation did not alter the sex ratio or the developmental progression through preimplantation development. In blastocysts, cryopreservation affected only a few individual genes. In both male and female blastocysts, although oocyte cryopreservation increased the proportion of low Xist ‐expressing cells, it did not disrupt X‐linked gene dosage. In the inner cell mass (ICM) of blastocysts, a negative correlation between X‐linked gene expression and cell cycle progression was identified, and this negative correlation was more pronounced in trophectoderm (TE) cells. Overall, this stage‐dependent coordination remained unperturbed by cryopreservation. CellChat analysis revealed that the signalling interaction strength between ICM and TE also showed no significant changes in the cryo group. Conclusions: Oocyte cryopreservation does not severely disrupt overall transcriptional integrity, developmental potency and X‐chromosome dosage compensation of embryos, but it may cause subtle molecular changes. These findings highlight the need for continued refinement of cryopreservation methods and further investigation into the sub‐acute safety of assisted reproductive technology procedures.

Background: In vitro maturation (IVM) provides a safer alternative to conventional in vitro fertilization (IVF) for women with polycystic ovary syndrome (PCOS) by mitigating the risk of ovarian hyperstimulation. However, concerns persist regarding whether IVM perturbs epigenetic reprogramming in the offspring. Current evidence is constrained by candidate-gene approaches or a lack of parental controls. This study aimed to evaluate the genome-wide DNA methylation safety of IVM compared with conventional IVF using a rigorous trio-based design. Methods: This secondary epigenetic analysis was nested within a randomized controlled trial (RCT) (ClinicalTrials.gov: NCT03463772). We included 10 nuclear families (trios), comprising five IVM-conceived and five IVF-conceived singleton offspring alongside their biological parents. Both groups utilized a uniform freeze-only single-blastocyst transfer strategy to minimize hormonal confounding. Genomic DNA from umbilical cord blood (UCB) and parental peripheral blood was analyzed using reduced representation bisulfite sequencing (RRBS). Genome-wide methylation patterns and differentially methylated regions (DMRs) were subsequently compared between the groups. Results: Clinical characteristics were comparable between the IVM and IVF groups. Genome-wide analyses demonstrated high concordance in UCB methylation patterns, revealing no significant differences in global CpG methylation levels or distributions across key genomic features (promoters, CpG islands, and gene bodies). Only three rare DMRs were identified in UCB (representing ~ 0.0001% of the genome), none of which mapped to imprinted or developmentally critical loci. Furthermore, methylation variability remained consistent between the groups. Conclusions: Our findings provide robust mechanistic evidence supporting the epigenetic safety of IVM. The remarkable stability of the neonatal methylome confirms that specific IVM conditions do not compromise early developmental programming, thereby endorsing IVM as a safe and viable alternative for women with PCOS. Trial registration: ClinicalTrials.gov registry, NCT03463772. Registered on March 13, 2018.

STUDY QUESTION: Can preimplantation genetic testing for monogenic defects (PGT-M) be achieved by performing third-generation sequencing (TGS) only on the proband for families with de novo variants or incomplete pedigrees? SUMMARY ANSWER: Whole-genome TGS facilitates a simplified PGT-M workflow by establishing reliable haplotypes solely from proband sequencing involving de novo variants or incomplete pedigrees. WHAT IS KNOWN ALREADY: PGT-M enables the accurate exclusion of embryos carrying pathogenic variants. However, its application to de novo variants or incomplete pedigrees is hindered by haplotype phasing. Moreover, direct variant detection suffers from detection failure and erroneous genotyping due to uneven coverage and allele dropout caused by whole-genome amplification. Current solutions, such as gamete or embryo analysis and targeted TGS, remain constrained by procedural complexity and lack of universality across different genes and mutation types. STUDY DESIGN, SIZE, DURATION: This prospective study enrolled 16 families requiring PGT-M with de novo variants or incomplete pedigrees at the Reproductive Medicine Center of Peking University Third Hospital from July 2023 to August 2025. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 9 families with incomplete pedigrees and 7 families with de novo variants, covering 10 distinct disease-causing genes or regions. To assess the capability of TGS for haplotype phasing, we evaluated its performance regarding genomic coverage and the retrieval of informative single-nucleotide polymorphisms (SNPs). Haplotypes were constructed using proband TGS data, and linkage analysis was performed by integrating linked heterozygous SNPs with next-generation sequencing data from the couple and embryos to determine pathogenic status. Subsequently, we developed a simplified strategy that inferred inheritance by comparing heterozygous SNPs from the proband’s haplotype directly against corresponding homozygous sites in the embryos. The diagnostic outcomes of this simplified workflow were statistically evaluated and compared with those of the standard TGS strategy to assess concordance. MAIN RESULTS AND THE ROLE OF CHANCE: Phase blocks generated by TGS achieved >75% coverage for the vast majority of OMIM genes, most of which contained more than 100 heterozygous informative SNPs located in the gene body and their 1 Mb flanking regions, indicating a wide range of applicability in a variety of gene variants. Haplotypes were successfully constructed for all 16 enrolled families using TGS data, with 14 families having completed embryo testing, while the 2 families withdrew due to personal reasons. To date, prenatal diagnosis via amniocentesis in three families has confirmed the fetuses to be free of pathogenic variants. A simplified strategy was further applied to 14 families that completed the embryo testing process. This approach achieved applicability rates of 91.9% and 80.0% in embryos from non-D4Z4 and D4Z4 families, respectively. While diagnosis was precluded in a subset of embryos due to aneuploidy or insufficient SNP retrieval, the diagnostic outcomes for all remaining embryos were fully concordant with those of the standard TGS strategy. LIMITATIONS, REASONS FOR CAUTION: The applicability of this approach is primarily contingent upon embryo chromosomal euploidy and sufficient retrieval of informative SNPs. Additionally, the relatively high cost of whole-genome TGS remains a barrier to widespread adoption. Given the limited cohort size (n = 16) of this study, the applicability of this method necessitates further validation in larger clinical populations. WIDER IMPLICATIONS OF THE FINDINGS: Direct haplotype construction via proband whole-genome TGS provides an effective clinical strategy to expand the applicability of PGT-M, particularly for families with de novo variants or incomplete pedigrees. Furthermore, the simplified TGS workflow demonstrates the potential to improve clinical efficiency and reduce costs relative to the standard TGS protocol within its applicable scope. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (82125013, 82288102, 825B2046). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Purpose Aneuploidy is the most common genetic abnormality in human embryos and is one of the leading causes of embryo transfer failure. We aimed to identify candidate genes linked to mitotic-origin aneuploidy. Methods A control group of 588 euploid embryos and a case group of 236 mosaic embryos were utilized. Sequence alignment was first conducted to identify single nucleotide polymorphism (SNP) loci. Quality control (QC) and principal component analysis (PCA) were then performed to filter the SNPs and samples. The association test was carried out to identify significant variants. Fine mapping and gene sorting were used to screen and sort the candidate genes. Results Following variant identification, 10,650,011 SNPs were detected in 824 enrolled embryos. After quality control (QC), principal component analysis (PCA), and imputation, 496,728 SNPs across 762 embryos (226 cases and 536 controls) were retained. Association analysis identified 70 SNPs reaching genome-wide significance (p < 5e⁻⁸). Following annotation, 37 variants within 27 genes were considered functional. Gene Ontology (GO) analysis revealed enrichment in innate immune and protein homeostasis pathways. Fine mapping and gene prioritization highlighted EMP2 as a candidate gene of mitotic error. We found that EMP2 may play a critical role in cell-cycle control and endometrial receptivity. Conclusion In this study, we performed genome-wide association analysis on embryonic sequencing data and identified EMP2 on chromosome 16 as a potential gene implicated in mitotic-origin aneuploidy. Further verification experiments are required to confirm the functions of candidate genes during embryogenesis.

Purpose To determine whether recombination occurring around the breakpoints of translocated chromosomes may affect the accuracy of the current linkage analysis-based diagnosis of reciprocal translocation (RT) embryos. Methods Here, we have selected 688 single sperm cells sampled from 14 RT carriers with signed informed consent before semen collection and systematically investigated their chromosomal segregation patterns and potential recombination of translocated chromosomes. Results We found that 39.96% of sperm cells showed alternative segregation patterns to produce normal and chromosomally balanced sperm cells, while 39.01% presented with an adjacent-1 segregation pattern, and 16.06% presented with an adjacent-2 segregation pattern, and 4.59% presented with a 3:1 segregation pattern. Moreover, we discovered a previously unknown segregation pattern at 0.38% occurrence. Notably, of the 887 recombination events on RT-associated chromosomes identified, 18 recombination events were located within the range of 5 Mb around breakpoints, among which 10/18 (55.6%) were located within 1–3 Mb of the breakpoint. Conclusion Our study provides evidence for recombination within the 5 Mb region flanking the breakpoints of translocated chromosomes, and these results suggest a non-negligible risk in the accurate diagnosis of chromosomal translocation in embryos through current linkage analysis-based diagnostic methods.

The spatiotemporal turnover of metabolites is essential for oocyte maturation, embryonic development, and cell lineage differentiation. Here, we analyzed the metabolic profiles of individual living mouse oocytes and studied how bisphosphoglycerate mutase (BPGM), an important maternal factor, influences metabolite regulation during oocyte maturation. We found that BPGM is expressed in mouse follicles, oocytes, and embryos, as well as in human embryos. Notably, deletion of Bpgm significantly reduced the rate of oocyte maturation and reduced mouse fertility, which was observed as reduced pups per litter. Also, the expression levels for meiosis-related genes and genes related to glucose metabolic pathways (glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway) were altered in BPGM-deficient mouse oocytes. We used a highly sensitive, live-cell sampling approach to carry out metabolite assays using induced nanoelectrospray-ionization mass spectrometry technology on 1 picolitre of aspirated cytoplasm from oocytes. BPGM gene disruption impaired glucose metabolism pathways, tyrosine metabolism, and amino acid biosynthesis. Together, our findings indicate that Bpgm participates in oocyte and embryo development, and we demonstrate the feasibility of studying metabolite composition and other phenotypic features of single oocytes.

Pre-implantation genetic testing for aneuploidy (PGT-A) is used in approximately half of in vitro fertilization cycles. Given the limited understanding of the genetics of human embryos, the current use of PGT-A is based on biologically uncertain assumptions and unvalidated guidelines, leading to the possibility of disposing of embryos with pregnancy potential. We isolated and sequenced all single cells (1133) from in vitro cultured 20 human blastocysts. We found that all blastocysts exhibited mosaicism with mitotic-induced aneuploid cells and showed an ~25% aneuploidy rate per embryo. Moreover, 70% (14/20) of blastocysts contained ‘chromosome-complementary’ cells, suggesting genetic mosaicism is underestimated in routine PGT-A. Additionally, the analysis of 20,945 single cells from day 8–14 embryos (in vitro cultured) and embryonic/fetal organs showed that 97% of the analyzed embryos/organs were mosaic. Over 96% of their aneuploid cells harbored ≤ 2 chromosome errors. Our findings have revealed a high prevalence of mosaicism in human embryos.