Exploring the efficacy and beneficial population of preimplantation genetic testing for aneuploidy start from the oocyte retrieval cycle: a real-world study.

BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) is widely used as an embryo selection technique in in vitro fertilization (IVF), but its effectiveness and potential beneficiary populations are unclear. METHODS: This retrospective cohort study included patients who underwent their first oocyte retrieval cycles at CITIC-Xiangya between January 2016 and November 2019, and the associated fresh and thawed embryo transfer cycles up to November 30, 2020. PGT-A (PGT-A group) and intracytoplasmic sperm injection (ICSI)/IVF (non-PGT-A group) cycles were included. The numbers of oocytes and embryos obtained were unrestricted. In total, 60,580 patients were enrolled, and baseline data were matched between groups using 1:3 propensity score matching. Sensitivity analyses, including propensity score stratification and traditional multivariate logistic regression, were performed on the original unmatched cohort to check the robustness of the overall results. Analyses were stratified by age, body mass index, ovarian reserve/responsiveness, and potential indications to explore benefits in subgroups. The primary outcome was cumulative live birth rate (CLBR). The other outcomes included live birth rate (LBR), pregnancy loss rate, clinical pregnancy rate, pregnancy complications, low birth weight rate, and neonatal malformation rate. RESULTS: In total, 4195 PGT-A users were matched with 10,140 non-PGT-A users. A significant reduction in CLBR was observed in women using PGT-A (27.5% vs. 31.1%; odds ratio (OR) = 0.84, 95% confidence interval (CI) 0.78-0.91; P < 0.001). However, women using PGT-A had higher first-transfer pregnancy (63.9% vs. 46.9%; OR = 2.01, 95% CI 1.81-2.23; P < 0.001) and LBR (52.6% vs. 34.2%, OR = 2.13, 95% CI 1.92-2.36; P < 0.001) rates and lower rates of early miscarriage (12.8% vs. 20.2%; OR = 0.58, 95% CI 0.48-0.70; P < 0.001), preterm birth (8.6% vs 17.3%; P < 0.001), and low birth weight (4.9% vs. 19.3%; P < 0.001). Moreover, subgroup analyses revealed that women aged ≥ 38 years, diagnosed with recurrent pregnancy loss or intrauterine adhesions benefited from PGT-A, with a significant increase in first-transfer LBR without a decrease in CLBR. CONCLUSION: PGT-A does not increase and decrease CLBR per oocyte retrieval cycle; nonetheless, it is effective in infertile populations with specific indications. PGT-A reduces complications associated with multiple gestations.

The effect of carrier characteristics and female age on preimplantation genetic testing results of blastocysts from Robertsonian translocation carriers.

PURPOSE: To analyze factors affecting segregation and ploidy results from Robertsonian carriers, and determine chromosomes involved impact chromosome stability during meiosis and mitosis. METHODS: This retrospective study include 928 oocyte retrieval cycles from 763 couples with Robertsonian translocations undergoing preimplantation genetic testing for structural rearrangements (PGT-SR) using next-generation sequencing (NGS) between December 2012 and June 2020.The segregation patterns of the trivalent of 3423 blastocysts were analyzed according to the carrier's sex and age. A total of 1492 couples who received preimplantation genetic testing for aneuploidy (PGT-A) were included as the control group and matched according to maternal age and testing time stage. RESULTS: A total of 1728 (50.5%) normal/balanced embryos were identified from 3423 embryos diagnosed. The rate of alternate segregation in male Robertsonian translocation carriers was significantly higher than that in female carriers (82.3% vs. 60.0%, P < 0.001). However, the segregation ratio exhibited no difference between young and older carriers. Further, increasing maternal age decreased the proportion of transferable embryo cycle in both female and male carriers. And the ratio of chromosome mosaic from the Robertsonian translocation carrier group was significantly higher than that in the PGT-A control group (1.2% vs. 0.5%, P < 0.01). CONCLUSIONS: The meiotic segregation modes were affected by the carrier sex and were independent of the carrier's age. Advanced maternal age decreased the probability of obtaining a normal/balanced embryo. In additional, the Robertsonian translocation chromosome could increase the possibility of chromosome mosaicism during mitosis in blastocysts.

Conventional IVF is feasible in preimplantation genetic testing for aneuploidy.

PURPOSE: To investigate the feasibility of the application of conventional in vitro fertilization (cIVF) for couples undergoing preimplantation genetic testing for aneuploidies (PGT-A) with non-male factor infertility. METHODS: To evaluate the efficiency of sperm whole-genome amplification (WGA), spermatozoa were subjected to three WGA protocols: Picoplex, ChromInst, and multiple displacement amplification (MDA). In the clinical studies, 641 couples who underwent PGT-A treatment for frozen embryos between January 2016 and December 2021 were included to retrospectively compare the chromosomal and clinical outcomes of cIVF and intracytoplasmic sperm injection (ICSI). Twenty-six couples were prospectively recruited for cIVF and PGT-A treatment between April 2021 and April 2022; parental contamination was analyzed in biopsied samples; and 12 aneuploid embryos were donated to validate the PGT-A results. RESULTS: Sperm DNA failed to amplify under Picoplex and ChromInst conditions but could be amplified using MDA. In frozen PGT-A cycles, no significant differences in the average rates of euploid, mosaic, and aneuploid embryos per cycle between the cIVF-PGT-A and ICSI-PGT-A groups were observed. The results of the prospective study that recruited couples for cIVF-PGT-A treatment showed no paternal contamination and one case of maternal contamination in 150 biopsied trophectoderm samples. Among the 12 donated embryos with whole-chromosome aneuploidy, 11 (91.7%) presented uniform chromosomal aberrations, which were in agreement with the original biopsy results. CONCLUSIONS: Under the Picoplex and ChromInst WGA protocols, the risk of parental contamination in the cIVF-PGT-A cycles was low. Therefore, applying cIVF to couples with non-male factor infertility who are undergoing PGT-A is feasible.

Non-invasive preimplantation genetic testing for conventional IVF blastocysts.

BACKGROUND: Previous studies suggested that non-invasive preimplantation genetic testing (niPGT) for intracytoplasmic sperm injection (ICSI) blastocysts can be used to identify chromosomal ploidy and chromosomal abnormalities. Here, we report the feasibility and performance of niPGT for conventional in vitro fertilization (IVF) blastocysts. METHODS: This was a prospective observational study. In the preclinical stage, whole genome amplification and NGS were performed using the sperm spent culture medium (SCM). Then, trophectoderm (TE) biopsies and corresponding SCM derived from 27 conventional IVF monopronuclear embryos were collected. In the clinical stage, samples from 25 conventional IVF cycles and 37 ICSI cycles from April 2020-August 2021 were collected for performance evaluation. RESULTS: Preclinically, we confirmed failed sperm DNA amplification under the current amplification system. Subsequent niPGT from the 27 monopronuclear blastocysts showed 69.2% concordance with PGT results of corresponding TE biopsies. In the clinical stage, no paternal contamination was observed in any of the 161 SCM samples from conventional IVF. While maternal contamination was observed in 29.8% (48/161) SCM samples, only 2.5% (4/161) samples had a contamination ratio ≥ 50%. Compared with that of TE biopsy, the performances of NiPGT from 161 conventional IVF embryos and 122 ICSI embryos were not significantly different (P > 0.05), with ploidy concordance rates of 75% and 74.6% for IVF and ICSI methods, respectively. Finally, evaluation of the euploid probability of embryos with different types of niPGT results showed prediction probabilities of 82.8%, 77.8%, 62.5%, 50.0%, 40.9% and 18.4% for euploidy, sex-chromosome mosaics only, low-level mosaics, multiple abnormal chromosomes, high-level mosaics and aneuploidy, respectively. CONCLUSIONS: Our research results preliminarily confirm that the niPGT approach using SCM from conventional IVF has comparable performance with ICSI and might broadening the application scope of niPGT.

Segmental aneuploidies with 1 Mb resolution in human preimplantation blastocysts.

PURPOSE: This study aimed to investigate the spectrum and characteristics of segmental aneuploidies (SAs) of <10 megabase (Mb) length in human preimplantation blastocysts. METHODS: Preimplantation genetic testing for aneuploidy was performed in 15,411 blastocysts from 5171 patients using a validated 1 Mb resolution platform. The characteristics and spectrum of SAs, including the incidence, sizes, type, inheritance pattern, clinical significance, and embryo distribution, were studied. RESULTS: In total, 6.4% of the 15,411 blastocysts carried SAs of >10 Mb, 4.9% of embryos had SAs ranging between 1 to 10 Mb, and 84.3% of 1 to 10 Mb SAs were <5 Mb in size. Inheritance pattern analysis indicated that approximately 63.8% of 1 to 10 Mb SAs were inherited and were predominantly 1 to 3 Mb in size. Furthermore, 18.4% of inherited SAs and 51.9% de novo 1 to 10 Mb SAs were pathogenic or likely pathogenic (P/LP). Different from whole-chromosome aneuploidies, reanalysis indicated that 50% of the de novo 1 to 10 Mb SAs and 70% of the >10 Mb SAs arose from mitotic errors. CONCLUSION: Based on the established platform, 1 to 10 Mb SAs are common in blastocysts and include a subset of P/LP SAs. Inheritance pattern analysis and clinical interpretation based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines contributed to determine the P/LP SAs.

A novel multifunctional haplotyping-based preimplantation genetic testing for different genetic conditions.

STUDY QUESTION: Is there an efficient and cost-effective detection platform for different genetic conditions about embryos? SUMMARY ANSWER: A multifunctional haplotyping-based preimplantation genetic testing platform was provided for detecting different genetic conditions. WHAT IS KNOWN ALREADY: Genetic disease and chromosomal rearrangement have been known to significantly impact fertility and development. Therefore, preimplantation genetic testing for aneuploidy (PGT-A), monogenic disorders (PGT-M) and structural rearrangements (PGT-SR), a part of ART, has been presented together to minimize the fetal genetic risk and increase pregnancy rate. For patients or their families who are suffering from chromosome abnormality, monogenic disease, unexplained repeated spontaneous abortion or implantation failure, after accepting genetic counseling, they may be suggested to accept detection from more than one PGT platforms about the embryos to avoid some genetic diseases. However, PGT platforms work through different workflows. The high costliness, lack of material and long-time operation of combined PGT platforms limit their application. STUDY DESIGN, SIZE, DURATION: All 188 embryonic samples from 43 families were tested with HaploPGT platform, and most of their genetic abnormalities had been determined by different conventional PGT methods beforehand. Among them, there were 12 families only carrying structural rearrangements (115 embryos) in which 9 families accepted implantation and 5 families had normal labor ART outcomes, 7 families only carrying monogenic diseases (26 embryos) and 3 families carrying both structural rearrangements and monogenic diseases (26 embryos). Twelve monopronucleated zygotes (1PN) samples and 9 suspected triploid samples were collected from 21 families. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Here, we raised a comprehensive PGT method called HaploPGT, combining reduced representation genome sequencing, read-count analysis, B allele frequency and haplotyping analysis, to simultaneously detect different genetic disorders in one single test. MAIN RESULTS AND THE ROLE OF CHANCE: With 80 million reads (80M) genomic data, the proportion of windows (1 million base pairs (Mb)) containing two or more informative single nucleotide polymorphism (SNP) sites was 97.81%, meanwhile the genotyping error rate stabilized at a low level (2.19%). Furthermore, the informative SNPs were equally distributed across the genome, and whole-genomic haplotyping was established. Therefore, 80M was chosen to balance the cost and accuracy in HaploPGT. HaploPGT was able to identify abnormal embryos with triploid, global and partial loss of heterozygosity, and even to distinguish parental origin of copy number variation in mosaic and non-mosaic embryos. Besides, by retrospectively analyzing 188 embryonic samples from 43 families, HaploPGT revealed 100% concordance with the available results obtained from reference methods, including PGT-A, PGT-M, PGT-SR and PGT-HLA. LIMITATIONS, REASON FOR CAUTION: Despite the numerous benefits HaploPGT could bring, it still required additional family members to deduce the parental haplotype for identifying balanced translocation and monogenic mutation in tested embryos. In terms of PGT-SR, the additional family member could be a reference embryo with unbalanced translocation. For PGT-M, a proband was normally required. In both cases, genomic information from grandparents or parental siblings might help for haplotyping theoretically. Another restriction was that haploid, and diploid resulting from the duplication of a haploid, could not be told apart by HaploPGT, but it was able to recognize partial loss of heterozygosity in the embryonic genome. In addition, it should be noted that the location of rearrangement breakpoints and the situation of mutation sites were complicated, which meant that partial genetic disorders might not be completely detected. WIDER IMPLICATIONS OF THE FINDINGS: HaploPGT is an efficient and cost-effective detection platform with high clinical value for detecting genetic status. This platform could promote the application of PGT in ART, to increase pregnancy rate and decrease the birth of children with genetic diseases. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Natural Science Foundation of China (81873478, to L.H.), National Key R&D Program of China (2018YFC1003100, to L.H.), the Natural Science Foundation of Hunan Province (Grant 2022JJ30414, to P.X.), Hunan Provincial Grant for Innovative Province Construction (2019SK4012) and the Scientific Research Foundation of Reproductive and Genetic Hospital of China International Trust & Investment Corporation (CITIC)-Xiangya (YNXM-201910). Haplotyping analysis has been licensed to Basecare Co., Ltd. L.K., Y.M., K.K., D.Z., N.L., J.Z. and R.D. are Basecare Co., Ltd employees. The other authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Comprehensive preimplantation genetic testing by massively parallel sequencing.

STUDY QUESTION: Can whole genome sequencing (WGS) offer a relatively cost-effective approach for embryonic genome-wide haplotyping and preimplantation genetic testing (PGT) for monogenic disorders (PGT-M), aneuploidy (PGT-A) and structural rearrangements (PGT-SR)? SUMMARY ANSWER: Reliable genome-wide haplotyping, PGT-M, PGT-A and PGT-SR could be performed by WGS with 10× depth of parental and 4× depth of embryonic sequencing data. WHAT IS KNOWN ALREADY: Reduced representation genome sequencing with a genome-wide next-generation sequencing haplarithmisis-based solution has been verified as a generic approach for automated haplotyping and comprehensive PGT. Several low-depth massively parallel sequencing (MPS)-based methods for haplotyping and comprehensive PGT have been developed. However, an additional family member, such as a sibling, or a proband, is required for PGT-M haplotyping using low-depth MPS methods. STUDY DESIGN, SIZE, DURATION: In this study, 10 families that had undergone traditional IVF-PGT and 53 embryos, including 13 embryos from two PGT-SR families and 40 embryos from eight PGT-M families, were included to evaluate a WGS-based method. There were 24 blastomeres and 29 blastocysts in total. All embryos were used for PGT-A. Karyomapping validated the WGS results. Clinical outcomes of the 10 families were evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: A blastomere or a few trophectoderm cells from the blastocyst were biopsied, and multiple displacement amplification (MDA) was performed. MDA DNA and bulk DNA of family members were used for library construction. Libraries were sequenced, and data analysis, including haplotype inheritance deduction for PGT-M and PGT-SR and read-count analysis for PGT-A, was performed using an in-house pipeline. Haplotyping with a proband and parent-only haplotyping without additional family members were performed to assess the WGS methodology. Concordance analysis between the WGS results and traditional PGT methods was performed. MAIN RESULTS AND THE ROLE OF CHANCE: For the 40 PGT-M and 53 PGT-A embryos, 100% concordance between the WGS and single-nucleotide polymorphism (SNP)-array results was observed, regardless of whether additional family members or a proband was included for PGT-M haplotyping. For the 13 embryos from the two PGT-SR families, the embryonic balanced translocation was detected and 100% concordance between WGS and MicroSeq with PCR-seq was demonstrated. LIMITATIONS, REASONS FOR CAUTION: The number of samples in this study was limited. In some cases, the reference embryo for PGT-M or PGT-SR parent-only haplotyping was not available owing to failed direct genotyping. WIDER IMPLICATIONS OF THE FINDINGS: WGS-based PGT-A, PGT-M and PGT-SR offered a comprehensive PGT approach for haplotyping without the requirement for additional family members. It provided an improved complementary method to PGT methodologies, such as low-depth MPS- and SNP array-based methods. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the research grant from the National Key R&D Program of China (2018YFC0910201 and 2018YFC1004900), the Guangdong province science and technology project of China (2019B020226001), the Shenzhen Birth Defect Screening Project Lab (JZF No. [2016] 750) and the Shenzhen Municipal Government of China (JCYJ20170412152854656). This work was also supported by the National Natural Science Foundation of China (81771638, 81901495 and 81971344), the National Key R&D Program of China (2018YFC1004901 and 2016YFC0905103), the Shanghai Sailing Program (18YF1424800), the Shanghai Municipal Commission of Science and Technology Program (15411964000) and the Shanghai 'Rising Stars of Medical Talent' Youth Development Program Clinical Laboratory Practitioners Program (201972). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Complex mosaic blastocysts after preimplantation genetic testing: prevalence and outcomes after re-biopsy and re-vitrification.

RESEARCH QUESTION: What is the incidence of complex mosaic in preimplantation genetic testing (PGT) blastocysts and can it be managed in clinical practice? DESIGN: A retrospective study of PGT cycles conducted between January 2018 and October 2019 at a single centre. Biopsies of blastocysts were collected and analysed by next-generation sequencing (NGS). Complex mosaic blastocysts were defined as those with three or more mosaic chromosomes. The cryopreserved complex mosaic blastocysts underwent a second round of biopsy, NGS analysis and vitrification. The euploid blastocysts identified by the re-biopsy were warmed again for embryo transfer. The main outcomes included the prevalence of the complex mosaic and the ongoing pregnancy rate. RESULTS: The prevalence of the complex mosaic was 2.4% (437/17,979). The prevalence of the complex mosaic was not associated with maternal age and morphological quality. A total of 89 complex mosaic blastocysts underwent re-biopsy and 96.6% (86/89) survived the first warming. For the re-biopsy samples, 61.6% (53/86) were euploid. The poor-quality blastocysts had higher rates of aneuploidy compared with good-quality blastocysts. The survival rate for blastocysts undergoing the second warming was 100% (18/18) and resulted in an ongoing pregnancy rate of 38.9% (7/18) as well as the birth of six healthy infants. CONCLUSION: Re-biopsy may rescue blastocysts with development potential for transfer and improve the cumulative pregnancy rate per stimulation cycle in patients containing complex mosaic blastocysts.

Reproductive risks and preimplantation genetic testing intervention for X-autosome translocation carriers.

RESEARCH QUESTION: What is the genetic cause of multiple congenital disabilities in a girl with a maternal balanced X-autosome translocation [t(X-A)]? Is preimplantation genetic testing (PGT), to distinguish non-carrier from euploid/balanced embryos and prioritize transfer, an effective and applicable strategy for couples with t(X-A)? DESIGN: Karyotype analysis, whole-exome sequencing and X inactivation analysis were performed for a girl with congenital cardiac anomalies, language impairment and mild neurodevelopmental delay. PGT based on next-generation sequencing after microdissecting junction region (MicroSeq) to distinguish non-carrier and carrier embryos was used in three couples with a female t(X-A) carrier (cases 1-3). RESULTS: The girl carried a maternal balanced translocation 46,X,t(X;1)(q28;p31.1). Whole-exome sequencing revealed no monogenic mutation related to her phenotype, but she carried a rare skewed inactivation of the translocated X chromosome that spread to the adjacent interstitial 1p segment, contrary to her mother. All translocation breakpoints in cases 1-3 were successfully identified and each couple underwent one PGT cycle. Thirty oocytes were retrieved, and 13 blastocysts were eligible for biopsy, of which six embryos had a balanced translocation and only four were non-carriers. Three cryopreserved embryo transfers with non-carrier status embryos resulted in the birth of two healthy children (one girl and one boy), who were subsequently confirmed to have normal karyotypes. CONCLUSIONS: This study reported a girl with multiple congenital disabilities associated with a maternal balanced t(X-A) and verified that the distinction between non-carrier and carrier embryos is an effective and applicable strategy to avoid transferring genetic and reproductive risks to the offspring of t(X-A) carriers.

Reproductive outcomes after preimplantation genetic testing in mosaic Turner syndrome: a retrospective cohort study of 100 cycles.

PURPOSE: The purpose of this study is to explore the reproductive outcomes of women with Turner syndrome (TS) in preimplantation genetic testing (PGT) cycles. METHODS: A retrospective study of 100 controlled ovarian stimulating cycles, 68 TS (sixty-four mosaic Turner syndrome (MTS) and four pure Turner syndrome (PTS)) women underwent PGT was conducted from 2013 to 2018. RESULTS: Embryo X chromosome abnormal rates of TS women were significantly higher than women with normal karyotype (7.04 vs 1.61%, P<0.01). Cumulative live birth rates (CLBR) after PGT-NGS treatment were lower in TS than control (31.15 vs 45.59%, P<0.05). Clinical pregnancy rates per transfer (CPR), miscarriage rates (MR) and live birth rates per transfer (LBR) remained comparable between TS and control group. Reproductive outcomes (X chromosome abnormal rates, CPR, MR, LBR and CLBR) among low (<10%), medium (10-50%) and high (>50%) level 45,X mosaicism groups were not statistically different. CONCLUSIONS: To avoid high risk of embryo X chromosome abnormalities, prenatal or preimplantation genetic testing should be recommended to mosaic or pure TS patients.

Clinical outcomes following preimplantation genetic testing and microdissecting junction region in couples with balanced chromosome rearrangement.

PURPOSE: The purpose of this study is to summarize the clinical outcomes of apparently balanced chromosome rearrangement (ABCR) carriers in preimplantation genetic testing (PGT) cycles by next-generation sequencing following microdissecting junction region (MicroSeq) to distinguish non-carrier embryos from balanced carriers. METHODS: A retrospective study of 762 ABCR carrier couples who requested PGT for structural rearrangements combined with MicroSeq at the Reproductive and Genetic Hospital of CITIC-Xiangya was conducted between October 2014 and October 2019. RESULTS: Trophectoderm biopsy was performed in 4122 blastocysts derived from 917 PGT-SR cycles and 3781 blastocysts were detected. Among the 3781 blastocysts diagnosed, 1433 (37.9%, 1433/3781) were balanced, of which 739 blastocysts were carriers (51.57%, 739/1433) and 694 blastocysts were normal (48.43%, 694/1433). Approximately 26.39% of cycles had both carrier and normal embryo transfer, and the average number of biopsied blastocysts was 6.7. In the cumulative 223 biopsied cycles with normal embryo transfer, all couples chose to transfer the normal embryos. In the 225 cycles with only carrier embryos, the couples chose to transfer the carrier embryos in 169/225 (75.11%) cycles. A total of 732 frozen embryo transfer cycles were performed, resulting in 502 clinical pregnancies. Cumulatively, 326 babies were born; all of these babies were healthy and free of any developmental issues. CONCLUSION: Our study provides the first evaluation of the clinical outcomes of a large sample with ABCR carrier couples undergoing the MicroSeq-PGT technique and reveals its powerful ability to distinguish between carrier and non-carrier balanced embryos.

X-chromosome inactivation pattern of amniocytes predicts the risk of dystrophinopathy in fetal carriers of DMD mutations.

OBJECTIVE: To predict the risk of dystrophinopathy in fetal carriers of dystrophin gene (DMD) mutations. METHODS: Twenty-three pregnant women, with a total of 25 female fetuses carrying DMD mutations, were recruited. Among them, 13 pregnant women who participated in this study were only used to analyse the incidence of induced abortion after fetuses were diagnosed as dystrophinopathy carriers. Eleven fetal carriers from 10 pregnant women were tested to analyse X-chromosome inactivation (XCI) using amniocytes to assess the risk of dystrophinopathy. Follow-ups were conducted on all cases. RESULTS: Approximately one-third of fetuses were aborted before assessing the risk of dystrophinopathy. XCI analysis of amniocytes showed that 10 fetuses had random XCI patterns, and one fetus exhibited a highly skewed XCI pattern (100:0) with primary expression of the maternal X chromosome that carried the mutant allele. These 11 fetal carriers were born, and follow-up showed that the girl who showed the skewed XCI pattern as a fetus was diagnosed with Duchenne muscular dystrophy (DMD) at the age of four. The others did not present with dystrophinopathy-associated symptoms. CONCLUSIONS: XCI was significantly implicated in symptomatic female carriers of dystrophinopathies, and XCI pattern analysis of amniocytes may be useful in predicting the risk of dystrophinopathy in fetal carriers.

Prevalence and authenticity of de-novo segmental aneuploidy (>16 Mb) in human blastocysts as detected by next-generation sequencing.

RESEARCH QUESTION: What is the prevalence and authenticity of de-novo segmental aneuploidies (>16 Mb) detected by next-generation sequencing (NGS) in human preimplantation blastocysts? DESIGN: Between April 2013 and June 2016, 5735 blastocysts from 1854 couples (average age 33.11 ± 5.65 years) underwent preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) or for aneuploidy (PGT-A) using NGS on trophectoderm (TE) biopsy samples. The prevalence of de-novo segmental aneuploidy was calculated from these results. Forty blastocysts with de-novo segmental aneuploidy detected by NGS, which had been donated for research, were warmed for further fluorescence in-situ hybridization (FISH) analysis to confirm their authenticity. RESULTS: The frequency of de-novo segmental aneuploidies in blastocysts was 10.13% (581/5735); the phenomenon was not related to maternal age and occurred on all chromosomes. Of the 40 donated blastocysts, 39 were successfully warmed and fixed for FISH analysis at the single-cell level. The de-novo segmental aneuploidies identified by NGS were confirmed by FISH in all 39 blastocysts. However, the de-novo segmental aneuploidies in these blastocysts were not all pure patterns, with 66.67% (26/39) of blastocysts exhibiting mosaic patterns varying from 8.30% to 92.86% of cells with de-novo segmental aneuploidy. The concordance rate between NGS and FISH in TE and inner cell mass (ICM) samples was 47.69% (31/65). CONCLUSIONS: De-novo segmental aneuploidy above 16 Mb occurred in blastocysts and could be detected by NGS, while some aneuploidies existed as mosaics in both TE and ICM.

Polar body transfer restores the developmental potential of oocytes to blastocyst stage in a case of repeated embryo fragmentation.

PURPOSE: We aimed to determine the developmental potential of human reconstructed oocytes after polar body genome transfer (PBT) and to report the case of a woman with multiple cycles of severe embryo fragmentation. METHODS: Fresh and cryopreserved first polar bodies (PB1s) were transferred to enucleated metaphase II oocytes (PB1T), while fresh PB2s were removed from fertilized oocytes and used instead of the female pronucleus in donor zygotes. Reconstructed oocytes underwent intracytoplasmic sperm injection (ICSI) and were cultured to blastocyst. Biopsied trophectoderm cells of PBT-derived blastocysts were screened for chromosomes by next-generation sequencing (NGS). Then, cryopreserved PB1T was carried out in one woman with a history of several cycles of extensive embryo fragmentation, and the blastocysts derived from PB1T were screened for aneuploidy but not transferred to the patient. RESULTS: There were no significant differences in the rates of normal fertilization and blastocyst formation between fresh and cryopreserved PB1T and control oocytes. Of the three fresh and three cryopreserved PB1T-derived blastocysts, two and one blastocysts exhibited normal diploidy respectively. In contrast, 17 PB2 transfers yielded 16 two pronuclei (2PN) zygotes with one normal and one small-sized pronucleus each and no blastocyst formation. In the female patient, 18 oocytes were inseminated by ICSI in the fourth cycle and the PB1s were biopsied. Although the embryos developed from the patient's own oocytes showed severe fragmentation, the oocytes reconstructed after PB1T produced three chromosomally normal blastocysts. CONCLUSIONS: Normal blastocysts can develop from human reconstructed oocytes after PB1T. The application of the first PB transfers may be beneficial to patients with a history of poor embryo development and excessive fragmentation.

Telomerase-mediated telomere elongation from human blastocysts to embryonic stem cells.

High telomerase activity is a characteristic of human embryonic stem cells (hESCs), however, the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion, but stabilized at later passages. We report that the core unit of telomerase, hTERT, was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore, this was regulated in a Wnt-ß-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation, demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation, suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines, we were able to establish that telomere length remained relatively stable at 12.02 ± 1.01 kb during later passages (15-95). In contrast, telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary, we propose that correct, stable telomere length may serve as a potential biomarker for genetically stable hESCs.

Physiological oxygen prevents frequent silencing of the DLK1-DIO3 cluster during human embryonic stem cells culture.

Genetic and epigenetic alterations are observed in long-term culture (>30 passages) of human embryonic stem cells (hESCs); however, little information is available in early cultures. Through a large-scale gene expression analysis between initial-passage hESCs (ihESCs, <10 passages) and early-passage hESCs (ehESCs, 20-30 passages) of 12 hESC lines, we found that the DLK1-DIO3 gene cluster was normally expressed and showed normal methylation pattern in ihESC, but was frequently silenced after 20 passages. Both the DLK1-DIO3 active status in ihESCs and the inactive status in ehESCs were inheritable during differentiation. Silencing of the DLK1-DIO3 cluster did not seem to compromise the multilineage differentiation ability of hESCs, but was associated with reduced DNA damage-induced apoptosis in ehESCs and their differentiated hepatocyte-like cell derivatives, possibly through attenuation of the expression and phosphorylation of p53. Furthermore, we demonstrated that 5% oxygen, instead of the commonly used 20% oxygen, is required for preserving the expression of the DLK1-DIO3 cluster. Overall, the data suggest that active expression of the DLK1-DIO3 cluster represents a new biomarker for epigenetic stability of hESCs and indicates the importance of using a proper physiological oxygen level during the derivation and culture of hESCs.

Trace levels of mitomycin C disrupt genomic integrity and lead to DNA damage response defect in long-term-cultured human embryonic stem cells.

How to maintain the genetic integrity of cultured human embryonic stem (hES) cells is raising crucial concerns for future clinical use in regenerative medicine. Mitomycin C(MMC), a DNA damage agent, is widely used for preparation of feeder cells in many laboratories. However, to what extent MMC affects the karyotypic stability of hES cells is not clear. Here, we measured residual MMC using High Performance Liquid Chromatography-Mass Spectrometry/Mass Spectrometry following each step of feeder preparation and found that 2.26 ± 0.77 and 3.50 ± 0.92 ng/ml remained in mouse feeder cells and human feeder cells, respectively. In addition, different amounts of MMC caused different chromosomal aberrations in hES cells. In particular, one abnormality, dup(1)(p32p36), was the same identical to one we previously reported in another hES cell line. Using Affymetrix SNP 6.0 arrays, the copy number variation changes of the hES cells maintained on MMC-inactivated feeders (MMC-feeder) were significantly more than those cultured on γ-inactivated feeder (IR-feeder) cells. Furthermore, DNA damage response (DDR) genes were down-regulated during long-term culture in the MMC-containing system, leading to DDR defect and shortened telomeres of hES cells, a sign of genomic instability. Therefore, MMC-feeder and MMC-induced genomic variation present an important safety problem that would limit such hES from being applied for future clinic use and drug screening.

Clinical outcomes in carriers of insertional translocation: a retrospective analysis of comprehensive chromosome screening results.

Objective: To evaluate the clinical outcomes in the carriers of insertional translocation (IT). Design: Retrospective case series. Setting: University-affiliated reproductive medical center. Patients: Twenty-three couples with ITs. Intervention: No direct interventions were involved; however, this study included patients who underwent preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR). Main Outcome Measure: Outcome of preimplantation genetic testing for structural chromosomal rearrangements and percentage of blastocysts available for transfer. Results: Among 23 IT carriers, 15 were simple interchromosome ITs (type A), 3 were intrachromosome IT carriers (type B), and 5 were interchromosome IT carriers combined with other translocations (type C). A total of 190 blastocysts from 30 cycles were biopsied, 187 embryos were tested successfully, and only 57 blastocysts (30.5%) from 21 patients were available for transfer (normal or balanced). The unbalanced rearrangement rate of type C was 79.2% (42/53), and the proportion of type A was 50.0% (57/114), which was significantly higher than that of type B (5%, 1/20). In type A, the probability of embryos harboring unbalanced rearrangement in female carriers was 56.0% (51/91), which was higher than that in male carriers (26.1%, 6/23). Furthermore, the haploid autosomal length value of the inserted fragment was correlated linearly with the incidence of abnormal embryos. In type A gametes, most gametes produced by 2:2 separation without crossover, and no 3:1 separation gamete was observed. Conclusions: The chance of identifying normal or balanced and mosaic blastocysts per mature oocytes in patients with ITs are 16.6% (67/404). Greater IT complexity results in fewer transferable embryos. For simple interchromosome ITs, female carriers and those with higher haploid autosomal length values have a higher risk of producing embryos with unbalanced rearrangement.

A single-cell transcriptome atlas of human euploid and aneuploid blastocysts.

Aneuploidy is frequently detected in early human embryos as a major cause of early pregnancy failure. However, how aneuploidy affects cellular function remains elusive. Here, we profiled the transcriptomes of 14,908 single cells from 203 human euploid and aneuploid blastocysts involving autosomal and sex chromosomes. Nearly all of the blastocysts contained four lineages. In aneuploid chromosomes, 19.5% ± 1.2% of the expressed genes showed a dosage effect, and 90 dosage-sensitive domains were identified. Aneuploidy leads to prevalent genome-wide transcriptome alterations. Common effects, including apoptosis, were identified, especially in monosomies, partially explaining the lower cell numbers in autosomal monosomies. We further identified lineage-specific effects causing unstable epiblast development in aneuploidies, which was accompanied by the downregulation of TGF-ß and FGF signaling, which resulted in insufficient trophectoderm maturation. Our work provides crucial insights into the molecular basis of human aneuploid blastocysts and may shed light on the cellular interaction during blastocyst development.

Ultra-low-coverage genome-wide association study-insights into gestational age using 17,844 embryo samples with preimplantation genetic testing.

BACKGROUND: Very low-coverage (0.1 to 1×) whole genome sequencing (WGS) has become a promising and affordable approach to discover genomic variants of human populations for genome-wide association study (GWAS). To support genetic screening using preimplantation genetic testing (PGT) in a large population, the sequencing coverage goes below 0.1× to an ultra-low level. However, the feasibility and effectiveness of ultra-low-coverage WGS (ulcWGS) for GWAS remains undetermined. METHODS: We built a pipeline to carry out analysis of ulcWGS data for GWAS. To examine its effectiveness, we benchmarked the accuracy of genotype imputation at the combination of different coverages below 0.1× and sample sizes from 2000 to 16,000, using 17,844 embryo PGT samples with approximately 0.04× average coverage and the standard Chinese sample HG005 with known genotypes. We then applied the imputed genotypes of 1744 transferred embryos who have gestational ages and complete follow-up records to GWAS. RESULTS: The accuracy of genotype imputation under ultra-low coverage can be improved by increasing the sample size and applying a set of filters. From 1744 born embryos, we identified 11 genomic risk loci associated with gestational ages and 166 genes mapped to these loci according to positional, expression quantitative trait locus, and chromatin interaction strategies. Among these mapped genes, CRHBP, ICAM1, and OXTR were more frequently reported as preterm birth related. By joint analysis of gene expression data from previous studies, we constructed interrelationships of mainly CRHBP, ICAM1, PLAGL1, DNMT1, CNTLN, DKK1, and EGR2 with preterm birth, infant disease, and breast cancer. CONCLUSIONS: This study not only demonstrates that ulcWGS could achieve relatively high accuracy of adequate genotype imputation and is capable of GWAS, but also provides insights into the associations between gestational age and genetic variations of the fetal embryos from Chinese population.