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

RESEARCH QUESTION:What are the clinical outcomes for carriers of complex chromosomal rearrangements (CCR) and couples with both partners harbouring chromosomal rearrangements who undergo preimplantation genetic testing (PGT)? DESIGN:This case series included eight couples with atypical chromosomal rearrangements (five CCR carriers and three couples with both partners harbouring chromosomal rearrangements) who underwent blastocyst trophectoderm biopsy as well as PGT. The clinical outcomes of the transfers were recorded. RESULTS:PGT was performed on eight carriers of atypical chromosomal rearrangements. A total of 223 oocytes (157 from CCR carriers and 66 from couples where both partners harboured chromosomal rearrangements) were retrieved from the eight participants. Thirty-three (14.8%) blastocysts were obtained, of which 5 (15.1%) were euploid, 2 (6.1%) were mosaic and the remaining 26 (78.8%) were aneuploid. For CCR carriers, there were 3, 0 and 21 euploid, mosaic and aneuploid blastocysts, respectively, and for couples where both partners harboured chromosomal rearrangements there were 2, 2 and 5. Four euploid embryos and one mosaic embryo were successfully transferred, but none of them produced a pregnancy. CONCLUSIONS:Carriers of atypical chromosomal rearrangements had a high probability of creating aneuploid embryos, and therefore PGT showed a low usefulness for CCR carriers. Thus, spermatozoa/egg donation IVF was advocated. Couples with both partners harbouring chromosomal rearrangements had a reasonable rate of transferable embryos and may benefit from PGT.

A male full-term neonate was admitted at 30 minutes of life with pallor and 10 minutes of respiratory distress. Physical examination revealed pallor, increased intercanthal distance, low-set ears, a palpable cystic mass in the neck, hepatomegaly, a pedunculated, globular appendage attached to the right thumb, and an ectopic toenail on the right second toe. Laboratory testing showed severe anemia with hemoglobin of 44 g/L. Bone marrow examination demonstrated hypoplasia. Whole-exome sequencing identified a heterozygous pathogenic variant in the RPS19 gene, c.175T>C (p.Ser59Pro), establishing the diagnosis of Diamond-Blackfan anemia. On follow-up to 2 years and 2 months of age, both hemoglobin and reticulocyte counts remained within normal ranges. This case illustrates early-onset severe anemia in a neonate with genetically confirmed Diamond-Blackfan anemia and expands the phenotypic spectrum, informing clinical recognition and management.

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

Background: Charcot-Marie-Tooth type 1A (CMT1A), the most frequent type of Charcot-MarieTooth disease, is mainly caused by a 1.4-Mb duplication containing the PMP22 gene. There is no effective treatment other than general supportive care and symptomatic treatment. Preimplantation genetic testing for monogenic defects (PGT-M) is an alternative approach for obtaining healthy babies. Methods: A new technology and analysis method based on next-generation sequencing (NGS) was developed to detect duplication mutations directly. Simultaneously, aneuploidy and linkage analyses were performed to achieve a comprehensive and accurate embryo diagnosis.Results: Eight couples were recruited in this study; PMP22 duplication was validated in seven couples, and PMP22 splicing mutation was found in one. Forty-five embryos from 12 PGT cycles were successfully detected using this novel method. The direct detection results for all embryos were consistent with the linkage analyses, suggesting a 100 % accuracy rate, and the aneuploidy rate of the biopsied blastocysts was 33.3 %. Eventually, 18 of the 45 diagnosed embryos were deemed suitable for transfer. Four healthy babies from three families were delivered and their genetic status confirmed by amniocentesis. Additionally, there were no adverse effects of anesthesia or increased pregnancy complications during PGT-M in female patients with CMT1A.Conclusions: This study provided a simple, reliable, and efficient method that can directly detect PMP22 mutations based on NGS data and does not require positive family members. A clinical

Purpose While efforts have been made to establish blastocyst grading systems in the past decades, little research has examined the quality of biopsy specimens. This study is the first to correlate the morphology of biopsied trophectoderm (TE) cells to their quality and subsequent genetic testing results of preimplantation genetic testing (PGT), through an innovative Morphological Analysis and Genetic Integrality Criterion (MAGIC) system. Methods Biopsied TE cells were first evaluated according to the MAGIC procedure, followed by whole-genome amplification (WGA) and library construction, and then sequenced using the Illumina X Ten Platform. Copy number variation (CNV) and allele drop-out (ADO) rates as well as test failure rates were compared and analyzed. Results Our data explores the relationship between TE cell morphology and its quality and final genetic testing outcome, which is established based on the MAGIC system. MAGIC guarantees that only high- or good-quality TE cells are used for genetic testing to generate excellent data uniformity and lower ADO rates. Low-quality cells containing biopsied TE cell mass are responsible for the “background noise” of CNV analysis. Conclusion The MAGIC application has effectively decreased the false-positive mosaicism, hence to ensure the stability and veracity of detection results, to avoid misdiagnoses, and to improve accuracy, as well as to avoid re-biopsy procedures. The study also contributes to understand how the IVF laboratory and the molecular biology laboratory depend on each other to achieve good-quality PGT results, which are clinically relevant for the patients.

Hereditary tumor syndromes have garnered substantial attention due to their adverse effects on both the physical and psychological health of patients, as well as the elevated risk of transmission to subsequent generations. This has prompted a growing interest in exploring preimplantation genetic testing (PGT) as a treatment option to mitigate and eliminate these impacts. Several studies have demonstrated that de novo variants have become a great cause of many hereditary tumor syndromes, which introduce certain difficulties to PGT. In the absence of adequate genetic linkage information (parents and offspring), haplotype construction seems unrealizable. In the study, researchers used single sperm or affected embryos as proband to perform single-nucleotide polymorphism linkage analysis for cases with de novo variants. For complicated variants, the strategy that sperm combined with embryo detection will increase accuracy while avoiding the limitations and potential failures of using a single detection material. The study recruited 11 couples with male de novo carriers, including 3 tumor types and 4 genes. To date, 4 couples have been clinically confirmed as pregnant and three healthy babies have been born. The results of amniocentesis or umbilical cord blood verification were consistent with the results of PGT-M. The study aims to introduce the application of the PGT-M strategy in hereditary tumor syndromes.

Abstract Pre-implantation Genetic Testing for Aneuploidy (PGT-A) is widely used in half of in vitro fertilization (IVF) cycles, leading to the unwarranted disposal of embryos with pregnancy potential. Due to the limited understanding of genetics of human embryo, current PGT-A is built on biologically uncertain assumptions and on unvalidated guidelines1. Here, we sequenced all single cells (1,072) from 20 human blastocysts and analyzed 13,897 single cells from post-implantation embryos as well as fetal organs. Unexpectedly, all blastocysts contained mitotic aneuploid cells and showed about 25% aneuploidy rate per embryo. Among the 20 blastocysts, 70% (14/20) contained chromosome ‘complementary’ cells, suggesting the possible underestimation of mosaicism in traditional PGT-A. All the post-implantation embryos and fetal organs were mosaic, and over 80% of their aneuploid cells harbored ≤ 2 chromosome errors. Our findings showed all human embryos are naturally aneuploid-mosaic and this may provide guidance to the implementation of PGT-A in clinical practice.

Research question: Can preimplantation genetic testing for structural rearrangement (PGT-SR) based on low-coverage next -generation sequencing (NGS) accurately discriminate between normal and carrier embryos of reciprocal translocation (RecT) and Robertsonian translocation (RobT)?Design: A total of 109 couples with RecT or RobT were included in this study. The ages, bad obsteric histories (BOH), blood karyotype and IVF cycle information, including the number of cumulus-oocyte complexes, metaphase II oocytes, two pronuclei oocytes and blastocysts were recorded. 0.1 x whole genome sequencing (WGS) of embryos followed by copy number variation (identifying unbalanced/balanced) and 2 x WGS of parents and embryos followed by haplotype analysis (discriminating between normal and carrier) were carried out in PGT-SR cycles. The embryos without translocation were transferred and clinical outcomes evaluated. Results: Among all the couples in this study, 67 patients had RecT and 42 had RobT. After unbalanced and balanced detection, 103 balanced embryos underwent a further normal and carrier discrimination procedure, and 53 normal embryos were identified. Finally, 32 normal embryos were transferred, with an ongoing pregnancy rate of 46.88% (15/32). All ongoing pregnancies underwent amniocentesis, and the amninocentesis karyotyping results showed 100% concordance with PGT-SR diagnosis.Conclusions: Our low-coverage NGS-based PGT-SR method can accurately discriminate between normal and carrier status of balanced embryos. The method is cost-effective and has broad clinical applicability.

Congenital contractural arachnodactyly （CCA,OMIM:121050）,also known as Beals syndrome,belongs to a group of rare autosomal dominant （AD） diseases of connective tissue （Maslen et al.,1997）.People with CCA share many distinguishing features,such as arachnodactyly,camp tod actyly,multiple joint contractures（especially finger,elbow,and knee joints）,crumpled ears,scoliosis,pectus deformities,and muscular hypoplasia （Jurko et al.,2013）.It exhibits no specific geographic or ethnic predilection （Frederic et al.,2009）.

BACKGROUND:Haplotyping reveals chromosome blocks inherited from parents to in vitro fertilized (IVF) embryos in preimplantation genetic diagnosis (PGD), enabling the observation of the transmission of disease alleles between generations. However, the methods of haplotyping that are suitable for single cells are limited because a whole genome amplification (WGA) process is performed before sequencing or genotyping in PGD, and true haplotype profiles of embryos need to be constructed based on genotypes that can contain many WGA artifacts.RESULTS:Here, we offer scHaplotyper as a genetic diagnosis tool that reconstructs and visualizes the haplotype profiles of single cells based on the Hidden Markov Model (HMM). scHaplotyper can trace the origin of each haplotype block in the embryo, enabling the detection of carrier status of disease alleles in each embryo. We applied this method in PGD in two families affected with genetic disorders, and the result was the healthy live births of two children in the two families, demonstrating the clinical application of this method.CONCLUSION:Next generation sequencing (NGS) of preimplantation embryos enable genetic screening for families with genetic disorders, avoiding the birth of affected babies. With the validation and successful clinical application, we showed that scHaplotyper is a convenient and accurate method to screen out embryos. More patients with genetic disorder will benefit from the genetic diagnosis of embryos. The source code of scHaplotyper is available at GitHub repository: https://github.com/yzqheart/scHaplotyper.

目的 在致病突变明确的家系中通过产前遗传诊断来阻断肥厚型心肌病(hypertrophy cardiomyopathy,HCM)的代际传递,减少HCM患者数量.方法 对分别携带MYH7基因Arg663Ser和Arg453His致病突变的2例HCM患者进行家系分析,通过Sanger测序检测致病突变位点,通过毛细管电泳进行短串联重复序列(short tandem repeats,STR)分型及及连锁分析.在17-20周行羊穿术采集羊水,羊水细胞提取基因组DNA,进行遗传检测.结果 MYH7基因Arg663Ser和Arg453His突变在各自家系中与HCM家系共分离,为所在家系的致病突变.我们选择了杂合度高的6个位于MYH7基因附近的STR(D14S50、D14S283、D14S990、D14S972、D14S64和D14S264)进行分型检测,确认在第一个家系中D14S50的173bp长度等位基因和D14S990的151bp长度等位基因与Arg663Ser突变连锁.在第二个家系中,D14S50的169bp长度等位基因和D14S283的145bp长度等位基因与Arg453His突变连锁.羊水DNA的Sanger测序和STR分型均显示2例胚胎均未携带MYH7基因致病突变.新生儿脐带血的复检结果与产前诊断结果一致.结论 产前遗传诊断能够在孕早期明确诊断胎儿是否携带家族HCM致病突变,为咨询者夫妇提供合理的遗传咨询依据,对阻断HCM在家系中遗传具有重要意义.

Preimplantation genetic diagnosis (PGD) of genetic diseases, combined with human leukocyte antigen (HLA) typing (PGD-HLA), is a useful technique to have healthy offspring that are compatible with a sibling for hematopoietic stem cells transplantation (HSCT) to treat their genetic diseases. Here, we report a new strategy using single nucleotide polymorphism (SNP) linkage analysis for monogenic disease PGD combined with HLA typing, to simultaneously obtain the information of chromosomal aneuploidy, target mutations and HLA typing through a single low-depth next generation sequencing (NGS) procedure. In this study, five couples with probands underwent SNP linkage analysis for PGD-HLA typing were recruited. Within these five couples, two couples fortunately harvested four unaffected and HLA matched embryos with their siblings. After embryo transfer, two healthy neonates were born successfully. Subsequently, cord blood hematopoietic stem cells obtained from these two neonates were collected and frozen for treating their sick siblings. This novel strategy could provide abundant and specific SNPs for each family, therefore linkage information adjacent and even within HLA clusters were apparent. This study offers a highly flexible and precise method which could eliminate misdiagnosis caused by chromosomal recombination of the HLA gene, thus potentially benefit the success rate of HSCT.

PURPOSE:Preimplantation genetic diagnosis (PGD) analysis can be challenging for couples who carry more than one genetic condition. In this study, we describe a new PGD strategy to select which embryo(s) to transfer for two clinically challenging cases. Both cases lack essential family members for linkage analysis including de novo mutation combined with reciprocal translocation.METHODS:Diverging from conventional method, we performed direct point mutation detection, quantitative analysis of gene copy number, combined with linkage analysis assisted by SNP information from single sperm (or polar bodies), thus establishing an all-in-one protocol for single embryonic cell preimplantation diagnosis for two co-existing genetic conditions (monogenic disease and chromosomal abnormality) on the NGS-based platform.RESULTS:Using this newly developed method, 15 embryos from two cases were screened, and two embryos were determined as free of the monogenic disease and specific chromosomal abnormalities created by the prospective father's reciprocal translocations.CONCLUSION:This novel PGD strategy could effectively select unaffected embryo(s) for couples affected with or carrying a monogenetic disease and a reciprocal chromosome translocation concurrently.

目的 胚胎植入前遗传学诊断技术(PGD)是减少出生缺陷儿的有效方法.遗传咨询和预实验是PGD流程关键环节,影响着PGD最终结局. 方法 根据遗传病的遗传方式不同,设计个性化预实验策略,包括家系突变位点验证,单细胞基因组扩增测序,连锁分析. 结果 遗传咨询结合预实验中突变位点在家系成员中的验证结果,可以判断常染色体显性和X-连锁隐性遗传病致病基因及基因突变位点的可靠性,而对于X-连锁隐性遗传病致病基因的寻找,需从家族中的先症者入手而非携带者.与此同时,预实验还能发现家系中存在的一些特殊问题,比如染色体平衡易位. 结论 遗传咨询和预实验,需成为单基因遗传病胚胎诊断流程的第一步,可以帮助我们判断基因及基因突变位点的致病性,制定个性化单基因胚胎检测策略,提高诊断的准确性,将因基因或者突变位点判断错误而导致的患儿出生风险降到最小.

Birth defects are caused by multiple factors, such as chromosome abnormality, environmental factors, and maternal factors. In this study, we focused on exploring the genetic causes of a non-consanguineous couple who suffered from four times of unsuccessful pregnancy due to unexplained recurrent fetal malformations with similar symptoms and normal chromosome copy number variations. Using trio-whole exome sequencing(trio-WES) for this couple and one of the affected fetuses, we found a mutation, c.1996 delC on the maternal imprinted gene MAGEL2 that was carried by the affected fetus and husband, leading to Schaaf-Yang syndrome. To screen this mutation, we further performed preimplantation genetic diagnosis(PGD) strategy followed by a gene pedigree validation and pathogenicity analysis. After the transfer of a PGD-screened embryo, a normal newborn without previous abnormal symptoms was born(February 15, 2019). We present the first data that identified a pathogenic gene(MAGEL2 c.1996 delC) in a fetus with Schaaf-Yang syndrome in the EAS(East Asian) database and overcame this genetic defect by using processed PGD for this couple based on the WES results.

目的 以小鼠月经样模型为基础,探索缺血再灌注在月经发生中的作用. 方法 假孕NIH小鼠子宫诱导蜕膜化,完成蜕膜化后将小鼠分为对照组(n=8)、假手术组(n=8)与缺血再灌注组(n=10).对照组小鼠无操作,假手术组仅实行开腹手术,而缺血再灌注组小鼠开腹后对单侧子宫角进行血管夹持手术阻断血流,30 min后恢复血流,造成单侧子宫角内发生缺血再灌注,另一侧子宫角无操作.手术后观察阴道出血状况,且术后6h与24h取材进行大体观察与组织形态学观察,统计子宫内膜崩解的小鼠数量. 结果 处理后24h,对照组小鼠子宫内膜全部保持完好,无一例(0/8)发生内膜崩解;假手术组仅一例(1/8)发生内膜崩解;缺血再灌注组大部分小鼠(8/10)双侧子宫角的子宫内膜均发生崩解并伴随阴道出血现象. 结论 缺血再灌注能够直接导致绝大部分小鼠蜕膜化子宫内膜发生完全崩解.

Knowledge on the influence of specific genotypes on the phenotypic expression of hypertrophic cardiomyopathy (HCM) is emerging. The objective of this study was to evaluate the genotype-phenotype relation in HCM patients and to construct a score to predict the genetic yield based to improve counseling. Unrelated HCM patients who underwent genetic testing were included in the analysis. Multivariate logistic regression was performed to identify variables that predict a positive genetic test. A weighted score was constructed based on the odds ratios. In total, 378 HCM patients were included of whom 141 carried a mutation (global yield 37%), 181 were mutation negative and 56 only carried a variant of unknown significance. We identified age at diagnosis <45 years, familial HCM, familial sudden death, arrhythmic syncope, maximal wall thickness ≥20 mm, asymmetrical hypertrophy and the absence of negative T waves in the lateral ECG leads as significant predictors of a positive genetic test. When we included these values in a risk score we found very high correlation between the score and the observed genetic yield (Pearson r = 0.98). MYBPC3 mutation carriers more frequently suffered sudden cardiac death compared to troponin complex mutations carriers (p = 0.01) and a similar trend was observed compared to MYH7 mutation carriers (p = 0.08) and mutation negative patients (p = 0.11). To conclude, a simple score system based on clinical variables can predict the genetic yield in HCM index patients, aiding in counseling HCM patients. MYBPC3 mutation carriers had a worse outcome regarding sudden cardiac death.

Whole-exome sequencing (WES) is widely used to detect genetic mutations that cause Mendelian diseases, and has been successfully applied in combination with preimplantation genetic diagnosis (PGD) to avoid the transmission of genetic defects. We investigated 40 nonconsanguineous families with unexplained, recurrent fetal malformations (two or more malformed fetuses) from May 2016 to December 2018. Using Trio-WES, we identified 32 disease-associated variants in 40 families (80% positive rate), which were subsequently verified. Known Mendelian diseases were identified in 12 families (30%), highly suspected Mendelian diseases in 12 families (30%), variants with uncertain significance in 8 families (20%), and no noticeable variants for 8 families (20%). Further analysis showed variants in 22 genes may cause fetal malformations. Four gene variants were detected in fetuses for the first time, which expanded the spectrum of the disease phenotype. Two novel candidate genes may be related to fetal malformations. Of 26 couples receiving PGD on disease-associated genes, 3 healthy newborns were delivered, and 4 couples are undergoing pregnancies. We reported the fetal data and developed an optimized genetic testing strategy. Our finding strongly suggests the presence of single gene Mendelian disorders in 60% of those families, and PGD services for couples to have healthy babies.

Reciprocal translocation is a chromosomal structural abnormality that arises when two non-homologous chromosomes rearrange and attach with each other, an incidence that occurs in about 1/500to 1/625 newborns （Mackie and Scriven, 2002）. This event typically does not lead to any significant loss of genetic material, thus reciprocal translocation carriers do not exhibit any severe abnormal