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• Novel variants of ZP1, ZP2 and ZP3 are associated with female infertility. • In-vitro functional studies of ZP protein variants indicate pathogenesis. • The ZP3 variant was overcome with ICSI treatment. • Targeted genetic diagnosis of ZP genes may guide fertilization methods. Which genetic variants might explain the causes of empty follicle syndrome (EFS) and abnormal zona pellucida (ZP) and affect the success of treatment with assisted reproductive technologies (ART)? Whole-exome sequencing was performed in probands with EFS and abnormal ZP. Sanger sequencing was used for variant validation. Using HEK-293T cells, the effects of ZP1 and ZP2 variants on protein expression were explored by western blotting, and the effect of the ZP1 variant on protein location was investigated via immunofluorescence. The protein structure was also analysed to investigate the pathogenicity of variants. A homozygous nonsense variant in ZP1 (c.874C>T, p.Gln292*) was detected in a patient with EFS. A novel homozygous frameshift variant in ZP2 (c.836_837delAG, p.Glu279Valfs*6) and a novel heterozygous missense variant in ZP3 (c.1159G>A, p.Val387Met) were identified in two patients with ZP morphological abnormalities, respectively. Western blotting and immunofluorescence analysis showed that the ZP1 variant results in a premature stop codon, leading to the truncated ZP1 protein. The ZP2 variant, which is situated in the N-terminus, triggers the degradation of a premature termination protein. Additionally, the patient with the ZP3 variant achieved clinical pregnancy following intracytoplasmic sperm injection treatment. These findings expand the mutational spectrum of ZP1, ZP2 and ZP3 , and provide new evidence for genetic diagnosis of female infertility. The targeted genetic diagnosis of ZP genes is recommended to choose appropriate fertilization methods and improve success rates of treatment with ART. [ABSTRACT FROM AUTHOR]

Meckel syndrome (MKS), also known as the Meckel–Gruber syndrome, is a severe pleiotropic autosomal recessive developmental disorder caused by dysfunction of the primary cilia during early embryogenesis. The diagnostic criteria are based on clinical variability and genetic heterogeneity. Mutations in the MKS1 gene constitute approximately 7% of all MKS cases. Herein, we present a non-consanguineous couple with three abnormal pregnancies as the fetuses showed MKS-related phenotypes of the central nervous system malformation and postaxial polydactyly. Whole-exome sequencing identified two novel heterozygous mutations of MKS1 : c.350C>A and c.1408-14A>G. The nonsense mutation c.350C>A produced a premature stop codon and induced the truncation of the MKS1 protein (p.S117*). Reverse-transcription polymerase chain reaction (RT-PCR) showed that c.1408-14A>G skipped exon 16 and encoded the mutant MKS1 p.E471Lfs*92. Functional studies showed that these two mutations disrupted the B9–C2 domain of the MKS1 protein and attenuated the interactions with B9D2, the essential component of the ciliary transition zone. The couple finally got a healthy baby through preimplantation genetic testing for monogenic disorder (PGT-M) with haplotype linkage analysis. Thus, this study expanded the mutation spectrum of MKS1 and elucidated the genetic heterogeneity of MKS1 in clinical cases. [ABSTRACT FROM AUTHOR]

The characteristics of the human pronuclei (PNs), which exist 16-22 h after fertilization, appear to serve as good indicators to evaluate the quality of human oocyte and embryo, and may reflect the status of female and male chromosome composition. Here, a quantitative PN measurement method that is generated by applying expert experience combined with deep learning from large annotated datasets is reported. After mathematic reconstruction of PNs, significant differences are obtained in chromosome-normal rate and chromosomal small errors such as copy number variants by comparing the size of the reconstructive female PN. After integrating the whole procedure of PN dynamics and adjusting for errors that occur during PN identification, the results are robust. Notably, all positive prediction results are obtained from the female propositus population. Thus, the size of female PNs may mirror the internal quality of the chromosomal integrity of the oocyte. Embryos that develop from zygotes with larger female PNs may have a reduced risk of copy number variations., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Advanced Genetics published by Wiley Periodicals LLC.)

Background: This study aims to describe clinical and diagnostic phenotype and identify pathogenic variants of a female with unknown causes of infertility. Methods: Clinical assessment was performed for the phenotype diagnosis. Whole-exome sequencing (WES) and the followed cDNA-PCR sequencing were applied to identify the pathogenic variant and investigate the potentially aberrant mRNA splicing event. The pathogenicity of the variant was analysed using multiple in silico prediction tools, including the 3D protein remodelling. Quantitative RT-PCR (qRT-PCR) was performed to measure PATL2 mRNA expression in the peripheral blood leukocytes of the proband and controls. Results: The proband was diagnosed with the female infertility due to oocyte germinal vesicle (GV) arrest. A novel homozygous splice site variant of PATL2 (NM_001145112.2, c.871-1G>A), inherited from her asymptomatic heterozygous parents, was detected by WES. Sequencing of cDNA amplification products demonstrated that this variant resulted in the exon 10 skipping and in-frame loss of 54 nucleotides in the PATL2 transcript. Quantitative RT-PCR suggested that the mutant transcript escape the mRNA degradation. Conclusion: We identified a novel pathogenic homozygous splice site of PATL2 (c.871-1G>A) underlying the oocyte GV arrest phenotype and elucidated its molecular mechanism. This study expands the variant spectrum of PATL2 and benefits our understanding of its genotype-phenotype correlations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sun, Tong, Liu, Tian, Yang, Zhou, Liu, Huang and Li.)

Purpose: Expanded carrier screening (ECS) is an effective method to identify at-risk couples (ARCs) and avoid birth defects. This study aimed to reveal the carrier spectrum in the Chinese population and to delineate an expanded carrier gene panel suitable in China. Methods: Medical exome sequencing (MES), including 4,158 disease-causing genes, was offered to couples at two reproductive centers. It was initially used as a diagnostic yield for potential patients and then used for ECS. Clinical information and ECS results were retrospectively collected. Results: A total of 2,234 couples, representing 4,468 individuals, underwent MES. In total, 254 individuals showed genetic disease symptoms, and 56 of them were diagnosed with genetic diseases by MES. Overall, 94.5% of them were carriers of at least one disease-causing variant. The most prevalent genes were GJB2 for autosomal recessive disorders and G6PD for X-linked diseases. The ARC rate was 9.80%, and couples were inclined to undergo preimplantation genetic testing when diseases were classified as "profound" or "severe." Conclusion: This study provided insight to establish a suitable ECS gene panel for the Chinese population. Disease severity significantly influenced reproductive decision-making. The results highlighted the importance of conducting ECS for couples before undergoing assisted reproductive technology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tong, He, He, Li, Hu, Hu, Lu, Lin, Dong, Zhang, Du and Liu.)

Piwi-interacting RNAs (piRNAs), a new class of small RNAs discovered from mammalian testes, are involved in transcriptional silencing of retrotransposons and other genetic elements in germ line cells. In order to identify a full transcriptome set of piRNAs expressed in the sexually mature porcine testes, small RNA fractions were extracted and were subjected to a Solexa deep sequencing. We cloned 6,913,561 clean reads of Sus Scrofa small RNAs (18-30 nt) and performed functional characterization. Sus Scrofa small RNAs showed a bimodal length distribution with two peaks at 21 nt and 29 nt. Then from 938,328 deep-sequenced small RNAs (26-30 nt), 375,195 piRNAs were identified by a k-mer scheme and 326 piRNAs were identified by homology searches. All piRNAs predicted by the k-mer scheme were then mapped to swine genome by Short Oligonucleotide Analysis Package (SOAP), and 81.61% of all uniquely mapping piRNAs (197,673) were located to 1124 defined genomic regions (5.85 Mb). Within these regions, 536 and 501 piRNA clusters generally distributed across only minus or plus genomic strand, 48 piRNA clusters distributed on two strands but in a divergent manner, and 39 piRNA clusters distributed on two strands in an overlapping manner. Furthermore, expression pattern of 7 piRNAs identified by homology searches showed 5 piRNAs displayed a ubiquitous expression pattern, although 2 piRNAs were specifically expressed in the testes. Overall, our results provide new information of porcine piRNAs and their specific expression pattern in porcine testes suggests that piRNAs have a role in regulating spermatogenesis.