Current non-invasive prenatal screening (NIPS) analyzes circulating fetal cell-free DNA (cfDNA) in maternal peripheral blood for selected aneuploidies or microdeletion/duplication syndromes. Many genetic disorders are refractory to NIPS largely because the maternal genetic material constitutes most of the total cfDNA present in the maternal plasma, which hinders the detection of fetus-specific genetic variants. Here, we developed an innovative sequencing method, termed coordinative allele-aware target enrichment sequencing (COATE-seq), followed by multidimensional genomic analyses of sequencing read depth, allelic fraction, and linked single nucleotide polymorphisms, to accurately separate the fetal genome from the maternal background. Analytical confounders including multiple gestations, maternal copy number variations, and absence of heterozygosity were successfully recognized and precluded for fetal variant analyses. In addition, fetus-specific genomic characteristics, including the cfDNA fragment length, meiotic error origins, meiotic recombination, and recombination breakpoints were identified which reinforced the fetal variant assessment. In 1129 qualified pregnancies tested, 54 fetal aneuploidies, 8 microdeletions/microduplications, and 8 monogenic variants were detected with 100% sensitivity and 99.3% specificity. Using the comprehensive cfDNA genomic analysis tools developed, we found that 60.3% of aneuploidy samples had aberrant meiotic recombination providing important insights into the mechanism underlying meiotic nondisjunctions. Altogether, we show that the genetic deconvolution of the fetal and maternal cfDNA enables thorough and accurate delineation of fetal genome which paves the way for the next-generation prenatal screening of essentially all types of human genetic disorders.

Background: The revised guidelines for the management of medullary thyroid carcinoma recommend that genetic counseling regarding reproductive options, including preimplantation genetic diagnosis (PGD), be considered for all RET mutation carriers of reproductive age to avoid the transmission of multiple endocrine neoplasia type 2 (MEN2). However, the high complexity and cost of PGD have hindered its widespread use. Thus, it is necessary to establish a simple and relatively inexpensive method to facilitate the PGD of MEN2. Patients and Methods: A customized Nimblegen EZ sequence capture array was designed to capture the targeted regions, including the RET gene, and 1 Mb range on each side of the RET gene. Targeted, capture-based next generation sequencing of three members of one family with MEN2A (the couple and the paternal father) was conducted to identify the informative markers. The diagnosis of the embryos was achieved through haplotype analysis based on informative markers and causative mutation. Results: Based on the sequencing results, 173 informative markers were detected, which were sufficient for the subsequent use for PGD. Seven informative markers and the causative mutation (RET C634Y) were selected and subjected to Sanger sequencing. Through haplotype analysis, 4 embryos without inheritance of the mutation haplotype of the RET gene were diagnosed as unaffected. One unaffected embryo was transferred, with one healthy baby born at 38 gestational weeks. Conclusion: Targeted, capture-based next generation sequencing for identification of informative markers together with Sanger sequencing is an easy and efficient method for the PGD of monogenic diseases, such as MEN2.

Recent studies have suggested that sperm mitochondrial DNA copy number (mtDNA-CN), DNA fragmentation index (DFI), and reactive oxygen species (ROS) content are crucial to sperm function. However, the associations between these measurements and embryo development and pregnancy outcomes in assisted reproductive technology (ART) remain unclear. Semen samples were collected from 401 participants, and seminal quality, parameters of sperm concentration, motility, and morphology were analyzed by a computer-assisted sperm analysis system. DFI, mtDNA-CN, and ROS levels were measured using sperm chromatin structure assay, real-time quantitative polymerase chain reaction, and ROS assay, respectively. Among the participants, 126 couples underwent ART treatments, including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and 79 of the couples had embryos transferred. In 401 semen samples, elevated mtDNA-CN and DFI were associated with poor seminal quality. In 126 ART couples, only mtDNA-CN was negatively correlated with the fertilization rate, but this correlation was not significant after adjusting for male age, female age, seminal quality, ART strategy, number of retrieved oocytes, controlled stimulation protocols, and cycle rank. Regarding pregnancy outcomes, sperm mtDNA-CN, ROS, and DFI were not associated with the clinical pregnancy rate or live birth rate in 79 transferred cases. In conclusion, increased mtDNA-CN and DFI in sperm jointly contributed to poor seminal quality, but sperm mtDNA-CN, ROS, and DFI were not associated with clinical outcomes in ART.