STUDY QUESTION:What are the risk factors associated with meiotic errors in blastocysts with mosaic biopsy results? SUMMARY ANSWER:Meiotic errors were identified in 3.9% of blastocysts with mosaic biopsy results and were correlated with high-level mosaicism, maternal origin, and advanced maternal age. WHAT IS KNOWN ALREADY:Chromosomal mosaicism mainly arises from postzygotic mitotic errors, except for rare events from rescued meiotic errors. Preimplantation genetic testing for aneuploidy origin (PGT-AO) can distinguish meiotic errors from mitotic errors; however, the clinical value of PGT-AO remains to be explored. STUDY DESIGN SIZE DURATION:A retrospective cohort of 391 blastocysts with mosaic biopsy results from a university-based fertility centre in China between January 2020 and December 2024 was analysed by PGT-AO. Risk factors associated with meiotic errors in blastocysts with mosaic biopsy results were explored. Pregnancy outcomes following 96 mosaic embryo transfers (METs) of embryos with mitotic errors were compared with those following transfer of 288 matched euploid embryos. Additionally, 7 donated blastocysts were separated for single-cell DNA sequencing. PARTICIPANTS/MATERIALS SETTING METHODS:The parental origin and cell-division origin were analysed in 391 blastocysts classified as euploid-aneuploid mosaic from a cohort of 8932 blastocysts detected using the single-nucleotide polymorphism (SNP) array. In the prospective study, pregnancy outcomes following METs of embryos with mitotic errors were compared with those following transfer of matched euploid embryos at a 1:3 ratio using propensity score matching. The primary outcome was the live birth/ongoing pregnancy rate (LB/OPR) of METs involving mitotic errors. Multivariate logistic regression analysis was used to evaluate risk factors for pregnancy outcomes. Prenatal and placental samples were analysed by SNP array and/or FISH for genetic verification. Single-cell DNA sequencing of 314 cells separated from 7 donated blastocysts (4 with high-level mosaicism, 2 with meiotic aneuploidy, and 1 with low-level mosaicism) was conducted to assess actual mosaicism. The chromosomal constitution of the blastocysts was comprehensively evaluated at the single-cell level, and concordance with the initial PGT-A results was assessed. MAIN RESULTS AND THE ROLE OF CHANCE:A SNP-based mosaicism quantification platform was established and validated using mixtures of single cells of varying ploidy to mimic clinical mosaic samples. In the retrospective cohort, the error origin was successfully determined for 384 of the 391 blastocysts with euploid-aneuploid mosaicism. The overall meiotic error rate was only 3.9% (15/384). Meiotic error was identified in 9.6% of blastocysts with mosaicism from women of advanced maternal age, which was significantly greater than the 3.0% observed in blastocysts with mosaicism from young women (OR = 3.43, 95% CI 1.12-10.46; P = 0.039). In addition, meiotic error was identified in 7.2% (10/139) of blastocysts with high-level mosaicism but 2.0% (5/245) of blastocysts with low-level mosaicism (OR = 3.72, 95% CI 1.25-11.12; P = 0.012). Meiotic error was also significantly greater in blastocysts with maternal-origin mosaicism than in those with paternal-origin mosaicism (8.9% vs 0.4%, OR = 21.66; P < 0.001). Clinical outcomes were comparable between METs and euploid embryo transfers, with no significant difference in LB/OPR (47.9% vs 52.8%, P = 0.409). Single-cell sequencing of donated blastocysts with high-level mosaicisms of mitotic errors demonstrated highly variable mosaic levels, ranging from a reproducibility rate of 8.7-100% for initial mosaic abnormalities at the single-cell level. LARGE SCALE DATA:Due to the individual privacy of the patients, the data are not publicly available. LIMITATIONS REASONS FOR CAUTION:Intrinsic technical noise cannot be completely distinguished from genuine mosaicism in the PGT-AO platform. The sample size of the MET cohort was limited, with few cases undergoing prenatal/postnatal genetic validation. In the single-cell study, the number of verified embryos was relatively small, and the threshold of copy number variation (CNV) detection was 10 Mb, which may have led to the underestimation of CNVs under 10 Mb. WIDER IMPLICATIONS OF THE FINDINGS:PGT-AO analysis demonstrated a low prevalence of meiotic errors in human blastocysts with mosaic biopsy results. PGT-AO is recommended for embryos from patients of advanced maternal age, embryos with high-level mosaicism, and embryos with maternal-origin mosaicism in PGT cycles. FUNDING:This study was supported by grants from the National Key Research and Development Program of China (No. 2023YFC2705503), National Natural Science Foundation of China (No. 82071716), Natural Science Foundation of Guangdong Province (No. 2025A1515010982), Key Clinical Technique of Guangzhou (No. 2023P-ZD19), and Medical Scientific Research Foundation of Guangdong Province (No. A2025203). DISCLOSURES:All authors declare no conflicts of interest.

Over the past decade, the field of human embryo editing has witnessed remarkable advancements and triggered significant ethical debates. The groundbreaking tool, CRISPR/Cas9, has revolutionized the landscape of genetic engineering by enabling modifications at the genomic level in germ cells. Since the first case of human embryo gene editing in 2015, the field has rapidly progressed, presenting promising avenues for therapeutic interventions. However, it still grapples with safety concerns, including off-target effects, mosaicism, and the long-term impacts of genetic alterations, as well as ongoing ethical controversies. In this review, we will systematically overview the significant research in this field and provide insights into the potential applications of basic research in early embryonic development and the treatment of genetic diseases.

Dear Editor, By taking advantage of parallel sequencing of genome and transcriptome (G&T-seq),1 we demonstrated the distinct transcriptome profiles of human preimplantation blastocysts in perspectives of embryo digital karyotype, developmental speed and implantation competence. Our study provided valuable information for further research in the physiology behind human embryo development and laid the foundation for embryo selection from the view of the transcriptome. Preimplantation genetic test for aneuploidy (PGT-A) serves as an important invasive method to select euploid embryos. However, even PGT-A cannot guarantee a successful pregnancy,2 for almost 50% of euploid blastocysts could not result in a live birth. It means that there is still a big room to improve the capability of embryo selection besides aneuploidy screening. RNA sequencing might have the potential for assessing embryo competence.3, 4 Here we investigated the distinct transcriptome profiles in human pre-implantation blastocysts with the application of G&T-seq (Figure 1A). We have verified this method in biopsied samples from 41 donated blastocysts in terms of the transcriptome consistency of samples from the same blastocyst, the prediction value of aneuploidies by transcriptome (Figure S1), as well as the lineage characteristic of inner cell mass (ICM) and trophectoderm (TE) (Figure S2), indicating the clinical safety and reproducibility of this method. G&T-seq is a unique technology for studying the transcriptome of chromosomal mosaicism, taking advantage of separate genome sequencing and RNA sequencing. In comparisons of transcriptomes of 28 TE few-cell samples from eight mosaic embryos with 17 TE few-cell samples from five euploidies (Figure 1B), we identified 79 genes upregulated and 37 genes downregulated (Figure 1C). Notably, ectoderm and primitive endoderm genes, including KLF4, TGFBR1, ITGB5 and GATA6, were significantly upregulated in TE of mosaic blastocysts (Figure 1D). Furthermore, upregulated genes were mainly enriched in embryonic development, stem cell proliferation, endoderm development and other pathways (Table S1), implying that there might be a lineage separation disorder in TE cells with chromosomal mosaicism, and the inadequately developed trophoblast may contribute to the adverse pregnancy outcomes of mosaic embryos. Human blastocysts have different developmental speeds. It may take 5–7 days for an embryo to develop to the grade 4 stage according to the Gardner grading system, which is the stage allowing TE biopsy. Clinically, blastocysts biopsied on day 6 or day 7 (named D6 or D7 blastocyst) are defined as growth-retarded blastocysts with lower implantation potential compared with day 5 blastocysts. The reason for retarded development speed remains to be clarified. To investigate the transcriptome related to blastocyst developmental speed and implantation potential, we collected TE few-cell samples prospectively in 105 couples who underwent PGT in our reproductive centre (Figure 2A and Table S2). Totally, 143 blastocysts (D5 n = 82, D6 n = 54 and D7 n = 7) were confirmed to be euploidies by G&T-seq, of which the detection efficiency of chromosomal screening was similar to that of conventional NGS in the same period (Table S3). Transcriptionally, these samples were obviously clustered according to the biopsied day (Figure 2B). It seemed that TE cells differentiated more maturely in the growth-retarded blastocysts than that of the D5 blastocysts, for the number of expressed genes and the average levels of TE marker genes5 (Table S4) increased in day 6/7 TE samples (Figure 2C,D). To further look at the transcriptional changes of normally developed D5 blastocysts from the 5th day to the 7th day after fertilization, nine donated D5 euploid blastocysts were cultured one or two more days, and then sequentially biopsied and treated by G&T-seq in the form of TE few-cells samples, which were named as D5_D6 or D5_D7 samples. The transcriptome characteristics of these TE samples were totally different from D5 blastocysts, but similar to the growth-retarded D6 or D7 blastocysts (Figure 2E). The pseudotime trajectory in D6 or D7 samples was nearly coincident with that in D5_D6 or D5_D7 samples, respectively, and roughly arranged according to the day after fertilization (Figure 2F,G). Genes with higher expression levels on day 5 after fertilization were mainly enriched in the regulation of mitochondrial membrane potential, autophagy, and stem cell population maintenance, while genes with higher expression levels in the later development stage (D6/7) were mainly enriched in amino acid metabolic and protein biogenesis process, stem cell differentiation, canonical Wnt signalling pathway, placenta development, steroid biosynthetic process and cadherin binding related to cell-cell adhesion (Figure 2H and Figure S3). Similar transcriptome characteristics of the growth-retarded blastocysts with D5_D6 or D5_D7 samples indicate that trophectoderm may differentiate autonomously as a clock tick after fertilization, but not depending on the morphology of blastocysts. This novel finding may at least partly explain the low development potential of growth-retarded blastocysts from the point of cross-talk between an embryo and endometrium, since they were transferred to the uterus at the same window as the D5 blastocysts.6, 7 On the other hand, we compared differentially expressed genes (DEGs) between grow-retarded D6/7 TE samples and normally developed D5_D6/D5_D7 samples. The up-regulated DEGs were enriched in GO terms with regard to the establishment or maintenance of cell polarity (Figure S4), which is one of the most important events during early embryonic divisions.8 Our findings might indicate that delayed-growing blastocysts have dysfunction related to cell polarity, which might be consistent with the publication from Wang et al., suggesting that the growth-retarded blastocysts and arrest embryos may share some common mechanism.9 The factors affecting the speed of embryo development might have originated from earlier embryological events, and analysis from time-lapse imaging may shed light on the related issues. Finally, we compared the transcriptome profiles between pregnant and non-pregnant blastocysts. The baseline characteristics were comparable between the two groups, except for the ratio of D6/7 blastocysts and corresponding ICM grade (Table S5). No significant clusters were found, indicating that the overall transcriptomes in TE samples from preimplantation blastocysts with different pregnancy outcomes were quite similar (Figure 3A). There were eight DEGs, including significantly upregulated genes SOX4, TMSB4X, IFNAR1, C3orf14 and CISD2, and downregulated genes LRRC4, HTT and HES4 in the non-pregnant group (adjusted p < .05, Figure 3B,C). Moreover, we established a logistic regression model for predicting euploid blastocyst pregnancy outcomes by combining the transcriptome markers and clinical characteristics. With the use of binary logistic analysis, we integrated patients' age, endometrial preparation protocol for embryo transfer cycle, embryo morphological grade, day after fertilization of embryo transferred, and highlighted DEGs mentioned above. Results revealed that genes LRRC4, IFNAR1, HES4 and HTT, as well as the ICM grade, were significantly correlated with pregnancy outcomes in these young females (Table S6). We could not verify the transcriptional profiles reported by Wang et al.,3 which might be due to the larger sample size and good prognosis of young patients in our study. In conclusion, we evaluated the transcriptome-wide approach G&T-seq for assessing embryo competence and found aberrant expression of genes related to implantation competence and mosaicism, which might provide valuable information for embryo selection from the view of the transcriptome. Yanwen Xu and Canquan Zhou contributed to the design of the work. Song Li, Bing Cai, Chenhui Ding and Muhua Lai performed the experiments. Song Li, Jialiu Liu and Yan Xu conducted the data analysis. Song Li and Jialiu Liu wrote the first draft, and Yanwen Xu revised the manuscript. All authors contributed to editing and reviewing the final version of the manuscript. This study was supported by the National Key Research and Development Program of China (No. 2023YFC2705503 and 2018YFC1003102), the National Natural Science Foundation of China (No. 82071716 and 81771588) and the Natural Science Foundation of Guangdong Province (No. 201804020087). The authors declare no conflict of interest. This study was approved by the Clinical Research and Laboratory Animal Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (No. [2020]110), and all patients provided informed consent. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Zona pellucida glycoprotein-1 (ZP1) is essential for maintaining oocyte structural integrity and facilitating fertilization. Mutations in ZP1 are strongly associated with primary infertility disorders such as fertilization failure and empty follicle syndrome; however, the absence of accurate experimental models has hindered mechanistic understanding and obscured the etiological basis of ZP1-related infertility. In this study, CRISPR/Cas9-mediated genome editing was employed to generate two ZP1-edited cynomolgus macaques ( Macaca fascicularis), designated #ZP1-1 (male) and #ZP1-2 (female). Following sexual maturation, oocytes retrieved from #ZP1-2 through superovulation exhibited a marked increase in zona pellucida-deficient oocytes and a significant reduction in maturation rates compared to controls. Integrated analyses, including immunofluorescence staining, transmission electron microscopy, transcriptomic profiling of oocytes, and histopathological examination of ovarian tissue, revealed disrupted folliculogenesis and oocyte anomalies consistent with phenotypes observed in human empty follicle syndrome. These findings establish the ZP1-knockout cynomolgus macaque as the first non-human primate model of ZP1-related infertility, providing a valuable platform for elucidating disease mechanisms and informing the development of targeted interventions for infertility arising from ZP gene mutations.

N6-methyladenosine (m6A), the most abundant mRNA modification in eukaryotes, plays an essential role in regulating gene expression. Our prior research, alongside that of others, demonstrated that conditional uterine knockout of methyltransferase-like 3 (METTL3), the enzyme responsible for m6A modification, led to complete failure of embryo implantation and decidualization. Intriguingly, METTL3 expression is downregulated rather than upregulated in human endometrial stromal cells (HESCs) during in vitro decidualization and in mouse decidual tissues during pregnancy. We hypothesized that this decline in METTL3 expression is indispensable for successful decidualization. To test this hypothesis, we overexpressed METTL3 in HESCs and observed impaired decidualization in vitro. Additionally, we generated genetically engineered mice with uterine-specific METTL3 overexpression using Pgr-Cre, which exhibited subfertility mainly due to impaired decidualization. Further investigation revealed a marked decrease in HAND2, a well-established regulator of decidualization, following METTL3 overexpression. Mechanistically, we uncovered that METTL3 overexpression destabilizes HAND2 mRNA via m6A modification at the 5'-UTR. In summary, our study underscores the critical role of programmed METTL3 downregulation in decidualization by sustaining HAND2 expression.

Mouse studies have established the crucial role for uterine m6A modification in embryo implantation and decidualization. Nevertheless, the importance of this epigenetic modification in the analogous biological process in humans remains incompletely understood. Here, we show that methyltransferase-like 3 [METTL3; N(6)-adenosine-methyltransferase catalytic subunit METTL3], the core component of the m6A writer complex, was significantly decreased in the endometrium of women with recurrent implantation failure during the window of implantation. Furthermore, we demonstrated that small interfering RNA (siRNA)-mediated knockdown of METTL3 in cultured human endometrial stromal cells (HESCs) resulted in impaired decidualization, which was primarily attributed to the downregulation of WNT4, a crucial factor for decidualization. Mechanistically, we discovered that METTL3 positively regulates the expression of the progesterone receptor (PGR) protein through m6A modification at the 5' untranslated region (5'-UTR) of PGR mRNA. In turn, WNT4 functions downstream of PGR, serving as a secondary target of METTL3. In conclusion, this study provides evidence that the METTL3-PGR-WNT4 pathway is essential for human decidualization. Our findings offer novel insights into the molecular mechanisms underlying human decidualization, potentially paving the way for future therapeutic strategies in reproductive medicine.

Background Antral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined. Methods Whole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9(Q308X/S415T) mouse model was generated based on the variant found in one of the patients. Results Two probands with bi-allelic GDF9 variants (GDF9(His209GlnfsTer6/S428T), GDF9(Q321X/S428T)) and eight GDF9(S428T) heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9(Q308X/S415T) mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9(Q308X/S415T) group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement. Conclusions GDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

Despite significant advancements in assisted reproductive technology, there is still high demand for improvements in the clinical pregnancy rate and reductions in the time needed to achieve a live birth. One bottleneck issue is the effective selection of oocytes and/or embryos of better quality in the laboratory. The quality of oocytes and embryos cannot be accurately assessed with routine clinical methods. Thus, assessment methods that are more objective, accurate, quantifiable, non-invasive, rapid, and repeatable are needed in the clinic. This perspective highlights the latest developments in non-invasive assessments of oocyte and embryo quality and presents new trends and insights into the biomedical engineering technologies used to evaluate oocyte and embryo quality.

Mutations in the Rhodopsin(RHO)gene are the main cause of autosomal dominant retinitis pigmentosa(adRP),84％ of which are pathogenic gain-of-function point mutations.Treatment strategies for adRP typically involve silencing or ablating the pathogenic allele,while normal RHO protein replacement has no meaningful therapeutic benefit.Here,we present an adenine base editor(ABE)-mediated therapeutic approach for adRP caused by RHO point mutations in vivo.The correctable pathogenic mutations are screened and verified,including T17M,Q344ter,and P347L.Two adRP animal models are created carrying the class 1(Q344ter)and class 2(T17M)mutations,and dual AAV-delivered ABE can effectively repair both mutations in vivo.The early intervention of ABE8e efficiently corrects the Q344ter mutation that causes a severe form of adRP,delays photoreceptor death,and restores retinal function and visual behavior.These results suggest that ABE is a promising alternative to treat RHO mutation-associated adRP.Our work provides an effective spacer-mediated point mutation correction therapy for dominantly inherited ocular disorders.

STUDY QUESTION Can blastocyst aneuploidy be predicted for patients with previous aneuploid pregnancy loss (PAPL) and receiving preimplantation genetic testing for aneuploidy (PGT-A)? SUMMARY ANSWER Multivariable logistic regression models were established to predict high risk of blastocyst aneuploidy using four identified factors, presenting good predictive performance. WHAT IS KNOWN ALREADY Aneuploidy is the most common embryonic chromosomal abnormality leading to pregnancy loss. Several studies have demonstrated a higher embryo aneuploidy rate in patients with PAPL, which has suggested that PGT-A should have benefits in PAPL patients intending to improve their pregnancy outcomes. However, recent studies have failed to demonstrate the efficacy of PGT-A for PAPL patients. One possible way to improve the efficacy is to predict the risk of blastocyst aneuploidy risk in order to identify the specific PAPL population who may benefit from PGT-A. STUDY DESIGN, SIZE, DURATION We conducted a multicenter retrospective cohort study based on data analysis of 1119 patients receiving PGT-A in three reproductive medical centers of university affiliated teaching hospitals during January 2014 to June 2020. Acohort of 550 patients who had one to three PAPL(s) were included in the PAPL group. In addition, 569 patients with monogenic diseaseswithout pregnancy loss were taken as the non-PAPL group. PARTICIPANTS/MATERIALS, SETTING, METHODS PGT-A was conducted using single nucleotide polymorphism microarrays and next-generation sequencing. Aneuploidy rates in Day 5 blastocysts of each patient were calculated and high-risk aneuploidy was definedas a rate of ≥50%. Candidate risk factors for high-risk aneuploidy were selected using the Akaike information criterion andwere subsequently included in multivariable logistic regression models. Overall predictive accuracy was assessed using the confusionmatrix, discrimination by area under the receiver operating characteristic curve (AUC), and calibration by plotting the predictedprobabilities versus the observed probabilities. Statistical significance was set at P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE Blastocyst aneuploidy rates were 30 ± 25% and 21 ± 19% for PAPL and non-PAPL groups, respectively. Maternal age (odds ratio (OR) = 1.31, 95% CI 1.24-1.39, P < 0.001), number of PAPLs (OR = 1.40, 95% CI 1.05-1.86, P = 0.02), estradiol level on the ovulation trigger day (OR = 0.47, 95% CI 0.30-0.73, P < 0.001), and blastocyst formation rate (OR = 0.13, 95% CI 0.03-0.50, P = 0.003) were associated with high-risk of blastocyst aneuploidy. The predictive model based on the above four variables yielded AUCs of 0.80 using the training dataset and 0.83 using the test dataset, with average and maximal discrepancies of 2.89% and 12.76% for the training dataset, and 0.98% and 5.49% for the test dataset, respectively. LIMITATIONS, REASONS FOR CAUTION Our conclusions might not be compatible with those having fewer than four biopsied blastocysts and diminished ovarian reserves, since all of the included patients had four or more biopsied blastocysts and had exhibited good ovarian reserves. WIDER IMPLICATIONS OF THE FINDINGS The developed predictive model is critical for counseling PAPL patients before PGT-A byconsidering maternal age, number of PAPLs, estradiol levels on the ovulation trigger day, and the blastocyst formation rate. This prediction model achieves good risk stratification and so may be useful for identifying PAPL patients who may have higher risk of blastocyst aneuploidy and can therefore acquire better pregnancy outcomes by PGT-A. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the National Natural Science Foundation of China under Grant (81871159). No competing interest existed in the study. TRIAL REGISTRATION NUMBER N/A.

Embryo implantation, a crucial step in human reproduction, is tightly controlled by estrogen and progesterone (P4) via estrogen receptor alpha and progesterone receptor (PGR), respectively. Here, we report that N6-methyladenosine (m6A), the most abun-dant mRNA modification in eukaryotes, plays an essential role in embryo implantation through the maintenance of P4 signaling. Conditional deletion of methyltransferase-like 3 (Mettl3), encoding the m6A writer METTL3, in the female reproductive tract using a Cre mouse line with Pgr promoter (Pgr-Cre) resulted in complete implantation failure due to pre-implantation embryo loss and defective uterine receptivity. Moreover, the uterus of Mettl3 null mice failed to respond to artificial decidualization. We further found that Mettl3 deletion was accompanied by a marked decrease in PGR protein expression. Mechanistically, we found that Pgr mRNA is a direct target for METTL3-mediated m6A modification. A luciferase assay revealed that the m6A modification in the 5 ' untranslated region (5 '-UTR) of Pgr mRNA enhances PGR protein translation efficiency in a YTHDF1-dependent manner. Finally, we demonstrated that METTL3 is required for human endometrial stromal cell decidualization in vitro and that the METTL3-PGR axis is conserved between mice and humans. In summary, this study provides evidence that METTL3 is essential for normal P4 signaling during embryo implantation via m6A-mediated translation control of Pgr mRNA.

Background: The goal of the assisted reproductive treatment is to transfer one euploid blastocyst and to help infertile women giving birth one healthy neonate. Some algorithms have been used to assess the ploidy status of embryos derived from couples with normal chromosome, who subjected to preimplantation genetic testing for aneuploidy (PGT-A) treatment. However, it is currently unknown whether artificial intelligence model can be used to assess the euploidy status of blastocyst derived from populations with chromosomal rearrangement. Methods: From February 2020 to May 2021, we collected the whole raw time-lapse videos at multiple focal planes from in vitro cultured embryos, the clinical information of couples, and the comprehensive chromosome screening results of those blastocysts that had received PGT treatment. Initially, we developed a novel deep learning model called the Attentive Multi-Focus Selection Network (AMSNet) to analyze time-lapse videos in real time and predict blastocyst formation. Building upon AMSNet, we integrated additional clinically predictive variables and created a second deep learning model, the Attentive Multi-Focus Video and Clinical Information Fusion Network (AMCFNet), to assess the euploidy status of embryos. The efficacy of the AMCFNet was further tested in embryos with parental chromosomal rearrangements. The receiver operating characteristic curve (ROC) was used to evaluate the superiority of the model. Results: A total of 4112 embryos with complete time-lapse videos were enrolled for the blastocyst formation prediction task, and 1422 qualified blastocysts received PGT-A ( n = 589) or PGT for chromosomal structural rearrangement (PGT-SR, n = 833) were enrolled for the euploidy assessment task in this study. The AMSNet model using seven focal raw time-lapse videos has the best real-time accuracy. The real-time accuracy for AMSNet to predict blastocyst formation reached above 70% on the day 2 of embryo culture, and then increased to 80% on the day 4 of embryo culture. Combing with 4 clinical features of couples, the AUC of AMCFNet with 7 focal points increased to 0.729 in blastocysts derived from couples with chromosomal rearrangement. Conclusion: Integrating seven focal raw time-lapse images of embryos and parental clinical information, AMCFNet model have the capability of assessing euploidy status in blastocysts derived from couples with chromosomal rearrangement.

Recombination is essential for physical attachments and genetic diversity. The Han Chinese population is the largest ethnic group worldwide, therefore, the construction of a genetic map regarding recombination for the population is essential. In this study, 164 and 240 couples who underwent preimplantation genetic testing for monogenic diseases or segmental rearrangement were included in the analysis. Blastocysts and probands from couples who underwent preimplantation genetic testing for monogenic diseases by single nucleotide polymorphism array were included for recombination analysis. The location of recombination was determined from haplotype phase transitions in parent-offspring pairs at loci where the parents were heterozygous. The genetic map for Chinese in vitro fertilization embryos was constructed by the expectation-maximization algorithm with chip-level data. Our results confirmed that homologous recombination occurred more often in maternal chromosomes, and the age effect was more significant in maternal homologous recombination. A total of 6,494 homologous recombination hotspots (32.3%) were identified in genes of Online Mendelian Inheritance in Man. A uniform association between homologous recombination and aneuploidy was not established. In addition, carriers with identified breakpoints of reciprocal translocations were analyzed, and locations of breakpoints were found partly overlapped with homologous recombination hotspots, implying a possible similar mechanism behind both events. This study highlights the significance of constructing a recombination map, which may improve the accuracy of haplotype analysis for preimplantation genetic testing for monogenic diseases. Overlapping locations of translocation and recombination are worthy of further investigation.

Purpose This study evaluated the relationship between cytoplasmic granulation patterns and the developmental potential of mature sibling oocytes. Methods Data from 54 cycles of preimplantation genetic tests for structural rearrangement from July 2019 to June 2022 were analyzed. In total, 564 embryos were cultured using a time-lapse system. Sibling oocytes were divided into four groups based on cytoplasmic granulation patterns: fine granulation (FG) group (n = 177), central granulation (CG) group (n = 183), dispersed granulation (DG) group (n = 161), and uneven granulation (UG) group (n = 43). The CG group was further divided into three groups (grades I, II, and III) based on the tertile of the ratio of central granular distribution area to oocyte area. Fertilization rate, embryo morphokinetics, chromosomal ploidy, and clinical outcomes of the groups were compared. Results No significant differences were observed in morphokinetic parameters, fertilization rate, embryo production, blastocyst formation, and aneuploidy rates among the different cytoplasmic-granulation pattern groups. However, embryos derived from CG oocytes showed significantly higher aneuploidy rates in grade III compared to grade I (86.21% vs 61.54%, P = 0.036) or grade II (86.21% vs 56.00%, P = 0.013). Thirty embryos were transferred to the uteri of female patients and the clinical pregnancy and live birth rates did not significantly differ among groups. Conclusions Cytoplasmic granulation patterns may not affect embryo fertilization, development speed, and aneuploidy rates. However, a higher grade of CG may be associated with increased aneuploidy rates. Larger sample sizes are required to explore the impact of oocyte cytoplasmic granulation patterns on embryo implantation potential.

Research question: Does blastocyst storage time have an impact on pregnancy and neonatal outcomes following the first single vitrified/warmed high-quality blastocyst transfer cycle for young women?Design: Retrospective cohort study in a university-affiliated reproductive medical centre.Results: A total of 2938 patients undergoing their first frozen embryo transfer (FET) cycle with a single high-quality blastocyst (Day 5: 3BB and above; Day 6: 4BB and above) transferred were divided into five groups: Group A with storage time <3 months (n = 1621), Group B with storage time of 4-6 months (n = 657), Group C with storage time of 7-12 months (n = 225), Group D with storage time of 13-24 months (n = 104), and Group E with storage time of 25-98 months (n = 331). After adjusting for confounding factors by multivariate logistic regression, there were no significant differences in live birth rate [Group A as reference; Group B: adjusted odds ratio (aOR) 0.954 (95% CI 0.791- 1.151); Group C: aOR 0.905 (95% CI 0.674-1.214); Group D: aOR 0.727 (95% CI 0.474-1.114); Group E: aOR 1.185 (955 CI 0.873-1.608)], b-human-chorionic-gonadotropin-positive rate, clinical pregnancy rate and miscarriage rate between Group A and the other groups. Among all singletons born after FET, there were no significant differences with regards to gestational age, preterm birth, birthweight, low birthweight, high birthweight and macrosomia.Conclusion: Long-term cryostorage of human vitrified high-quality blastocysts does not affect pregnancy or neonatal outcomes.

The results from different studies are inconsistent regarding whether development potential correlated with embryo development speed after single euploid blastocyst transfer. The age-associated reproductive decline is not only because of the difference in aneuploidy rates but also because of metabolic and epigenetic changes of the embryos. Therefore, we aimed to assess the independent effect of embryo development speed on implantation potential in young women. A total of 326 young women who underwent preimplantation genetic testing for monogenic diseases with aneuploidy screening were analyzed. Day-5 and day-6 euploid blastocysts yielded similar implantation rates (65.20 vs. 61.22%). The odds ratio (OR) remained non-significant after adjusting for confounders (adjusted OR = 0.84, 95% confidence interval 0.52-1.36). There was a trend that day-6 euploid blastocysts had a higher miscarriage rate (13.33 vs. 9.20%). However, the live birth delivery rate of day-5 blastocysts was similar to that of day-6 blastocysts (59.20 vs. 53.06%). In the stratified analysis, live birth delivery rates were similar between day-5 and day-6 similarly graded euploid blastocysts (excellent and good, 62.04 vs. 64.71%; average, 58.73 vs. 53.70%; poor, 43.75 vs. 44.44%). Embryo development speed has no obvious impact on implantation competence in young women's vitrified/warmed euploid embryo transfer cycles.

Preimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism. PGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45–50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss. All nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families. NGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.

Background: Preimplantation genetic test for monogenic disorders (PGT-M) has been used to select genetic disease-free embryos for implantation during in vitro fertilization (IVF) treatment. However, embryos tested by PGT-M have risks of harboring chromosomal aneuploidy. Hence, a universal method to detect monogenic diseases and genomic imbalances is required.Methods: Here, we report a novel PGT-A/M procedure allowing simultaneous detection of monogenic diseases and genomic imbalances in one experiment. Library was prepared in a special way that multiplex polymerase chain reaction (PCR) was integrated into the process of whole genome amplification. The resulting library was used for one-step low-pass whole genome sequencing (WGS) and high-depth target enrichment sequencing (TES).Results: The TAGs-seq PGT-A/M was first validated with genomic DNA (gDNA) and the multiple displacement amplification (MDA) products of a cell line. Over 90% of sequencing reads covered the whole-genome region with around 0.3–0.4 × depth, while around 5.4%–7.3% of reads covered target genes with >10000 × depth. Then, for clinical validation, 54 embryos from 8 women receiving PGT-M of β-thalassemia were tested by the TAGs-seq PGT-A/M. In each embryo, an average of 20.0 million reads with 0.3 × depth of the whole-genome region was analyzed for genomic imbalance, while an average of 0.9 million reads with 11260.0 × depth of the target gene HBB were analyzed for β-thalassemia. Eventually, 18 embryos were identified with genomic imbalance with 81.1% consistency to karyomapping results. 10 embryos contained β-thalassemia with 100% consistency to conventional PGT-M method.Conclusion: TAGs-seq PGT-A/M simultaneously detected genomic imbalance and monogenic disease in embryos without dramatic increase of sequencing data output.