GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement.

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 Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients. RESULTS: Two probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T 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. Gdf9Q308X/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 Gdf9Q308X/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.

Organic-inorganic hybrid ZIF-8/MXene/cellulose-based textiles with improved antibacterial and electromagnetic interference shielding performance.

Despite the tremendous efforts on developing antibacterial wearable textile materials containing Ti3C2Tx MXene, the singular antimicrobial mechanism, poor antibacterial durability, and oxidation susceptibility of MXene limits their applications. In this context, flexible multifunctional cellulosic textiles were prepared via layer-by-layer assembly of MXene and the in-situ synthesis of zeolitic imidazolate framework-8 (ZIF-8). Specifically, the introduction of highly conductive MXene enhanced the interface interactions between the ZIF-8 layer and cellulose fibers, endowing the green-based materials with outstanding synergistic photothermal/photodynamic therapy (PTT/PDT) activity and adjustable electromagnetic interference (EMI) shielding performance. In-situ polymerization formed a MXene/ZIF-8 bilayer structure, promoting the generation of reactive oxygen species (ROS) while protecting MXene from oxidation. The as-prepared smart textile exhibited excellent bactericidal efficacy of >99.99 % against both Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) after 5 min of NIR (300 mW cm-2) irradiation which is below the maximum permissible exposure (MPE) limit. The sustained released Zn2+ from the ZIF-8 layer achieved a bactericidal efficiency of over 99.99 % within 48 h without NIR light. Furthermore, this smart textile also demonstrated remarkable EMI shielding efficiency (47.7 dB). Clearly, this study provides an elaborate strategy for designing and constructing multifunctional cellulose-based materials for a variety of applications.

Knowledge-embedded spatio-temporal analysis for euploidy embryos identification in couples with chromosomal rearrangements.

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.

Predicting risk of blastocyst aneuploidy among women with previous aneuploid pregnancy loss: a multicenter-data-based multivariable model.

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. A cohort of 550 patients who had one to three PAPL(s) were included in the PAPL group. In addition, 569 patients with monogenic diseases without 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 defined as a rate of ≥50%. Candidate risk factors for high-risk aneuploidy were selected using the Akaike information criterion and were subsequently included in multivariable logistic regression models. Overall predictive accuracy was assessed using the confusion matrix, discrimination by area under the receiver operating characteristic curve (AUC), and calibration by plotting the predicted probabilities 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 by considering 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.

Effects of different oocyte cytoplasmic granulation patterns on embryo development and euploidy: a sibling oocyte control study.

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.

Storage time does not influence pregnancy and neonatal outcomes for first single vitrified high-quality blastocyst transfer cycle.

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)], ß-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.

Clinical application of next generation sequencing-based haplotype linkage analysis in the preimplantation genetic testing for germline mosaicisms.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mapping of meiotic recombination in human preimplantation blastocysts.

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.

METTL3 is essential for normal progesterone signaling during embryo implantation via m6A-mediated translation control of progesterone receptor.

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 abundant 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.

Simultaneous detection of genomic imbalance in patients receiving preimplantation genetic testing for monogenic diseases (PGT-M).

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 ß-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 ß-thalassemia. Eventually, 18 embryos were identified with genomic imbalance with 81.1% consistency to karyomapping results. 10 embryos contained ß-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.

Digital Economy, Environmental Regulation, and Ecological Well-Being Performance: A Provincial Panel Data Analysis from China.

China is currently in a strategic opportunity period for green and high-quality development, and developing the digital economy is an important choice to achieve environmental pollution control, improve regional ecological efficiency, and enhance social welfare. In this context, the impact of the digital economy on ecological well-being performance and the role of environmental regulation need to be examined. In this study, the super-efficiency SBM-DEA model was used to measure the level of ecological well-being performance in 30 provinces of China from 2011 to 2019. On this basis, the mediating effect model and spatial Durbin model were adopted to explore the transmission mechanism and regional heterogeneity of the impact of the digital economy on ecological well-being performance. The empirical results show that the digital economy significantly contributes to regional ecological well-being performance in China, and there is significant spatial spillover as well. Moreover, the findings still hold under robustness tests. The results also show that environmental regulation is an important transmission path for the digital economy to enhance regional ecological well-being performance, and the impact of environmental regulation on ecological well-being performance varies by region; specifically, the impact in eastern China is positive but not significant. However, the digital economy plays a significant positive role in promoting ecological well-being performance in the central and western regions, and is more obvious in the central region. Finally, suggestions are put forward to enhance the role of the digital economy in regional ecological well-being performance, which is of great significance for promoting green economic growth and high-quality development.

Risk factors related to chromosomal mosaicism in human blastocysts.

RESEARCH QUESTION: Do factors relating to patients, ART, or both, affect the incidence of chromosomal mosaicism in human blastocysts? DESIGN: Blastocysts (n = 5718) from 1198 PGT-A cycles were biopsied between 2015 and 2021. All samples were amplified with multiple displacement amplification, and MiSeq system was used for next-generation sequencing. Mosaicism prevalence, and ART and patient factors potentially affecting mosaicism, were analysed. RESULTS: Among 5718 blastocysts detected, 1245 were mosaic (21.8%). Day-6 biopsied blastocysts yielded a statistically significantly higher mosaicism rate than day-5 blastocysts (24.5% versus 19.1%; OR 1.174; 95% CI 1.017 to 1.355, P = 0.028). Mosaicism rate increased as morphological score declined (excellent quality [13.2%], good quality [19.0%], average quality [22.0%] and poor quality [26.4%], P < 0.001). Compared with excellent-quality embryos, the OR of mosaicism was 1.490 (95% CI 1.069 to 2.078, P = 0.019) for good-quality, 1.751 (95% CI 1.274 to 2.407, P = 0.001) for average-quality, and 2.113 (95% CI 1.512 to 2.952, P < 0.001) for poor-quality embryos. Biopsy technicians were related to the incidence of mosaicism. One of the four technicians had a significantly higher mosaicism rate than the others (27.9%; 20.9%; 20.5%; 20.5%, respectively; P < 0.001). Parental ages, female BMI, history of infertility, sperm quality, antral follicular counts, ovarian stimulation protocols, stimulation length, total gonadotrophin dosage and number of retrieved oocytes, were not related to the incidence of mosaicism. CONCLUSION: Slow developing, poor-quality blastocysts are at higher risk of mosaicism. Biopsy technician may contribute to artificial mosaicism as an extrinsic factor.

Developmental mRNA m5C landscape and regulatory innovations of massive m5C modification of maternal mRNAs in animals.

m5C is one of the longest-known RNA modifications, however, its developmental dynamics, functions, and evolution in mRNAs remain largely unknown. Here, we generate quantitative mRNA m5C maps at different stages of development in 6 vertebrate and invertebrate species and find convergent and unexpected massive methylation of maternal mRNAs mediated by NSUN2 and NSUN6. Using Drosophila as a model, we reveal that embryos lacking maternal mRNA m5C undergo cell cycle delays and fail to timely initiate maternal-to-zygotic transition, implying the functional importance of maternal mRNA m5C. From invertebrates to the lineage leading to humans, two waves of m5C regulatory innovations are observed: higher animals gain cis-directed NSUN2-mediated m5C sites at the 5' end of the mRNAs, accompanied by the emergence of more structured 5'UTR regions; humans gain thousands of trans-directed NSUN6-mediated m5C sites enriched in genes regulating the mitotic cell cycle. Collectively, our studies highlight the existence and regulatory innovations of a mechanism of early embryonic development and provide key resources for elucidating the role of mRNA m5C in biology and disease.

Evaluating the application value of NGS-based PGT-A by screening cryopreserved MDA products of embryos from PGT-M cycles with known transfer outcomes.

PURPOSE: To determine the application value of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidies (PGT-A). METHODS: We conducted a retrospective case-control study on a cohort of frozen-thawed embryo transfer (FET) cycles following preimplantation genetic testing for monogenic disorders (PGT-M) between 2014 and 2017. Cycles that produced live births or early miscarriages were divided into live birth group (n = 76) or miscarriage group (n = 19), respectively. The NGS-based aneuploidy screening was performed on the multiple displacement amplification (MDA) products of the embryonic trophectoderm biopsy samples that were cryopreserved following PGT-M. RESULTS: In the live birth group, 75% (57/76) embryos were euploid and 14.5% (11/76) were aneuploid. The remaining 10.5% (8/76) embryos were NGS-classified mosaic with the high- (≥ 50%) and low-level (< 50%) mosaicism rates at 7.9% (6/76) and 2.6% (2/76), respectively. In the miscarriage group, only 23.5% (4/17) embryos were aneuploid, while 58.8% (10/17) were euploid and 17.6% (3/17) were NGS-classified mosaic with the high- and low-level mosaicism rates at 11.8% (2/17) and 5.9% (1/17), respectively. For live birth and miscarriage groups, the transferable rate was 82.9% (63/76) and 70.6% (12/17), respectively, whereas the untransferable rate was 17.1% (13/76) and 29.4% (5/17), respectively. CONCLUSION: The application of NGS-based PGT-A remains questionable, as it may cause at least one in six embryos with reproductive potential to be discarded and prevent miscarriage in less than one in three embryos in single-gene disease carriers.

The inconsistency between two major aneuploidy-screening platforms-single-nucleotide polymorphism array and next-generation sequencing-in the detection of embryo mosaicism.

BACKGROUND: In preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos. RESULTS: We retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively. CONCLUSIONS: The consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos.

Similar implantation competence in euploid blastocysts developed on day 5 or day 6 in young women: a retrospective cohort study.

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.

Comparison of two mainstream endometrial preparation regimens in vitrified-warmed embryo transfers after PGT.

RESEARCH QUESTION: Which of the two mainstream endometrial preparation regimens, assisted natural cycle (NC) or hormone replacement treatment cycle (HRT), help frozen-thawed embryo transfer (FET) cycles after preimplantation genetic testing (PGT) achieve better clinical outcomes? DESIGN: This retrospective analysis included 3400 vitrified-warmed single blastocyst transfer cycles after PGT from January 2011 to November 2020, and involved 2332 patients with regular menstrual cycles. The decision to proceed with an assisted NC (n = 827) or HRT (n = 2573) before FET was reached based on a combination of patient preference and physician guidance. Clinical pregnancy rate, live birth rate, early miscarriage rate and obstetric outcomes were compared. RESULTS: No significant difference was observed between the assisted NC and HRT groups in terms of clinical pregnancy rate (51.6% versus 50.7%, P = 0.634), live birth rate (44.0% versus 43.4%, P = 0.746) or early miscarriage rate (12.6% versus 12.0%, P = 0.707). Multivariate analysis indicated that the endometrial preparation protocol was not an independent factor for a clinical pregnancy or live birth. In the HRT group, the Caesarean section rate (64.7% versus 51.9%, P < 0.001) and pregnancy complication rate (20.2% versus 13.8%, P = 0.003) were significantly higher. The two groups were not statistically different with respect to gestational age, early preterm birth rate, fetal weight or fetal birth defect rate. CONCLUSIONS: For patients undergoing a PGT-FET cycle involving a single blastocyst transfer, using assisted NC and HRT for the endometrial preparation could lead to comparable rates of clinical pregnancy and live birth. Additionally, NC is safer than HRT in terms of avoiding pregnancy complications and adverse obstetric outcomes.

Effect of carriers' sex on meiotic segregation patterns and chromosome stability of reciprocal translocations.

RESEARCH QUESTION: Does the sex of reciprocal translocation carriers affect meiotic segregation patterns and stability of non-translocated chromosomes during meiosis? DESIGN: A total of 790 couples who underwent preimplantation genetic testing for reciprocal translocations by using the single nucleotide polymorphism (SNP) array platform between October 2016 and December 2019 were included. Among them, 294 couples had their euploid embryos distinguished between normal euploidies and balanced translocation carriers. RESULTS: Female translocation carriers had a significantly lower incidence of alternate segregation pattern than male carriers (43.26% versus 47.98%, P = 0.001), but a higher incidence of 3:1 segregation pattern (6.70% versus 4.29%, P < 0.001). Stratified analysis showed only female translocation carriers with acrocentric chromosome (Acr-ch) involved had a lower incidence of alternate segregation pattern and a higher incidence of 3:1 segregation pattern compared with male carriers (41.63% versus 47.73%, P = 0.012; 9.32% versus 5.03%, P = 0.001). In 2233 embryos of 294 couples with identification of normal and balanced embryos, no significant differences were found in the paternal-origin aneuploidy rate (5.61% versus 5.82%, P = 0.861) and the maternal-origin aneuploidy rate (12.82% versus 12.08%, P = 0.673) in both male and female carriers. After excluding segmental aneuploidies, no differences were found between male and female carriers in both paternal-origin aneuploidy rate (2.14% versus 1.75%, P = 0.594) and maternal-origin aneuploidy rate (11.75% versus 11.06%, P = 0.683). CONCLUSION: The sex of the translocation carriers affected meiotic segregation patterns with no effect on the stability of non-translocated chromosomes during meiosis.

Oocyte Degeneration After ICSI Is Not an Indicator of Live Birth in Young Women.

Introduction: Intracytoplasmic sperm injection (ICSI) was introduced in 1990s as one of the most dramatic breakthroughs in assisted reproductive technology. Even with advances in ICSI technology, this mechanical micromanipulation carries a 5 to 19% risk of oocyte degeneration. Whether the presence of oocyte degeneration reflects the sibling oocyte quality and predicts the sibling embryo development potential and clinical pregnancy outcomes remains controversial. There is no study showing the competence of the sibling embryos from the prospective of cumulative live birth rate. Whether oocyte degeneration is associated with poor quality of the remainder of the cohort remains further to be elucidated. Method: This retrospective observational study included a total of 488 OPU cycles underwent ICSI with fresh cleavage stage embryo transfer and successive frozen/thawed embryo transfer (FET) cycles from January 2018 to December 2019. All female patients were under the age of 35 years, and underwent ICSI with or without oocyte degeneration (OD). Cycles with at least one oocyte degenerated were defined as oocyte degeneration group (OD group), and cycles with no oocyte degenerated were defined as non-OD group. The OD group was further divided to three subgroups according to different oocyte degeneration rate (<10%, 10-20%, and >20%). Results: There were no significant differences with regards to implantation rate (38.5% vs 35.1%, P=0.302), clinical pregnancy rate (54.9% vs 50.3%, P=0.340), and LBR per OPU cycle (47.0% vs 42.9%, P=0.395) between OD and non-OD groups. Initial gonadotropin dosage, E2 level on hCG day and the number of matured oocytes appeared to be independent risk factors for OD. The adjusted odds ratio of live birth rate per OPU cycle were similar in different oocyte degeneration rate subgroups. The ongoing pregnancy/LBR per transfer in FET cycles was not significantly different between OD group and non-OD groups (38.8% vs 43.9%, P=0.439). The cumulative LBR per OPU cycle was also comparable between OD and non-OD group (63.4% vs 64.8%, P=0.760). Conclusion: The results provide cycle-based evidence that the presence of oocyte degeneration after ICSI is not an indicator for predicting the cumulative live birth rate per OPU cycle in young women.

Successful four-factor preimplantation genetic testing: α- and ß-thalassemia, human leukocyte antigen typing, and aneuploidy screening.

Our study established an effective next-generation sequencing (NGS) protocol for four-factor preimplantation genetic testing (PGT) using  α- and ß-thalassemia, human leukocyte antigen (HLA) typing, and aneuploidy screening. Three couples, in whom both partners were α- and ß-double thalassemia carriers, underwent PGT between 2016 and 2018. These individuals sought an opportunity for hematopoietic stem cell transplantation to save their children from ß-thalassemia major. A total of 35 biopsied trophectoderm samples underwent multiple displacement amplification (MDA). PGT for α- and ß-thalassemia and HLA typing were performed on MDA products using NGS-based single-nucleotide polymorphism (SNP) haplotyping. Although two samples failed MDA, 94.3% (33/35) of samples were successfully amplified, achieving conclusive PGT results. Furthermore, 51.5% (17/33) of the embryos were diagnosed as unaffected non-carriers or carriers. Of the 17 unaffected embryos, nine (52.9%) were tested further  and identified as euploid via NGS-based aneuploid screening, in which five had HLA types matching affected children. One family did not achieve any unaffected euploid embryos. The two other families transferred HLA-matched and unaffected euploid embryos, resulting in two healthy 'savior babies.' NGS-PGT results were confirmed in prenatal diagnosis. Therefore, NGS-SNP was effective in performing PGT for multipurpose detection within a single PGT cycle.