Associations between exposure to pesticides mixture and semen quality among the non-occupationally exposed males: Four statistical models.

Most epidemiological studies on the associations between pesticides exposure and semen quality have been based on a single pesticide, with inconsistent major results. In contrast, there was limited human evidence on the potential effect of pesticides mixture on semen quality. Our study aimed to investigate the relationship of pesticide profiles with semen quality parameters among 299 non-occupationally exposed males aged 25-50 without any clinical abnormalities. Serum concentrations of 21 pesticides were quantified by gas chromatography-tandem mass spectrometry (GC-MS/MS). Semen quality parameters were abstracted from medical records. Generalized linear regression models (GLMs) and three mixture approaches, including weighted quantile sum regression (WQS), elastic net regression (ENR) and Bayesian kernel machine regression (BKMR), were applied to explore the single and mixed effects of pesticide exposure on semen quality. In GLMs, as the serum levels of Bendiocarb, ß-BHC, Clomazone, Dicrotophos, Dimethenamid, Paclobutrazole, Pentachloroaniline and Pyrimethanil increased, the straight-line velocity (VSL), linearity (LIN) and straightness (STR) decreased. This negative association also occurred between the concentration of ß-BHC, Pentachloroaniline, Pyrimethanil and progressive motility, total motility. In the WQS models, pesticides mixture was negatively associated with total motility and several sperm motility parameters (ß: -3.07∼-1.02 per decile, FDR-P<0.05). After screening the important pesticides derived from the mixture by ENR model, the BKMR models showed that the decreased qualities for VSL, LIN, and STR were also observed when pesticide mixtures were at ≥ 70th percentiles. Clomazone, Dimethenamid, and Pyrimethanil (Posterior inclusion probability, PIP: 0.2850-0.8900) were identified as relatively important contributors. The study provides evidence that exposure to single or mixed pesticide was associated with impaired semen quality.

An Effective and Universal Long-Read Sequencing-Based Approach for SMN1 2 + 0 Carrier Screening through Family Trio Analysis.

BACKGROUND: Population-wide carrier screening for spinal muscular atrophy (SMA) is recommended by professional organizations to facilitate informed reproductive options. However, genetic screening for SMN1 2 + 0 carriers, accounting for 3%-8% of all SMA carriers, has been challenging due to the large gene size and long distance between the 2 SMN genes. METHODS: Here we repurposed a previously developed long-read sequencing-based approach, termed comprehensive analysis of SMA (CASMA), to identify SMN1 2 + 0 carriers through haplotype analysis in family trios (CASMA-trio). Bioinformatics pipelines were developed for accurate haplotype analysis and SMN1 2 + 0 deduction. Seventy-nine subjects from 24 families composed of, at the minimum, 3 were enrolled, and CASMA-trio was employed to determine whether an index subject with 2 SMN1 copies was a 2 + 0 carrier in these families. For the proof-of-principle, SMN2 2 + 0 was also analyzed. RESULTS: Among the 16 subjects with 2 SMN1 copies, CASMA-trio identified 5 subjects from 4 families as SMN1 2 + 0 carriers, which was consistent with pedigree analysis involving an affected proband. CASMA-trio also identified SMN2 2 + 0 in six out of 43 subjects with 2 SMN2 copies. Additionally, CASMA-trio successfully determined the distribution pattern of SMN1 and SMN2 genes on 2 alleles in all 79 subjects. CONCLUSIONS: CASMA-trio represents an effective and universal approach for SMN1 2 + 0 carriers screening, as it does not reply on the presence of an affected proband, certain single-nucleotide polymorphisms, ethnicity-specific haplotypes, or complicated single-nucleotide polymorphism analysis across 3 generations. Incorporating CASMA-trio into existing SMA carrier screening programs will greatly reduce residual risk ratio.

Comprehensive Analysis of Spinal Muscular Atrophy: SMN1 Copy Number, Intragenic Mutation, and 2 + 0 Carrier Analysis by Third-Generation Sequencing.

Population-wide carrier screening for spinal muscular atrophy (SMA) is recommended by the American College of Medical Genetics and Genomics. However, the methods used currently mainly focus on SMN1 copy number and fail to identify carriers with pathogenic intragenic mutations and silent (2 + 0) carriers. We developed a method termed comprehensive analysis of SMA (CASMA) based on long-range PCR and third-generation sequencing of full-length and downstream regions of SMN1/2. The sensitivity and specificity of CASMA to detect SMA carriers with one copy of SMN1 were 100% (n = 101) and 99.2% (n = 236), respectively. CASMA confirmed three SMN1 intragenic mutations and pinpointed an inframe mutation c.661_666del to SMN2, which was misreported to SMN1 by allele-specific long-range nested PCR plus Sanger sequencing. CASMA also correctly predicted 8 of 16 samples (50%) with SMN1 duplication alleles. CASMA was expected to increase the detection rate of SMA carriers from 91% to 98% and decrease the residual risk ratio from 1:415 to 1:1868 after negative results of two SMN1 copies in the Chinese population. CASMA presents a comprehensive approach for identifying SMN1 and SMN2 copy number, intragenic mutations, and potential silent carriers that significantly reduces the residual risk ratio in SMA carrier screening and has great clinical utility.

Comprehensive copy number analysis of Y chromosome-linked loci for detection of structural variations and diagnosis of male infertility.

Infertility affects about 15% of heterosexual couples and male factors account for ~45-50% of clinical cases. Genetic factors play an important role in male infertility and thus we try to develop a cost-effective method for screening the genetic factors in male infertility. In our retrospective proof-of-concept study, we employed the high-throughput ligation-dependent probe amplification (HLPA) to examine the copy number by 115 genomic loci covering the Y chromosome, and 5 loci covering the X chromosome-specific region. We identified 8 sex chromosome aneuploid people from the low sperm concentration (LSC) group, and Y chromosome-specific microdeletion/duplications in 211 samples from the LSC group, and in 212 samples from the control group. 35 samples showed complete loss of AZFc (BPY2 to CDY1B deletion), which was not observed in controls. Nevertheless, a partial loss of AZFc (BPY2 to BPY2B deletion) was detected at comparable frequencies in both groups (68/211 vs. 108/212, respectively). And we further found structural variations in 28.6 and 26.9% samples from infertility and fertility groups. Moreover, we found that there were lower copy numbers for heterochromatic sequences in men with LSC. Especially, we reported that ultra-low relative copy number (RCN) (<0.5) type and low RCN (0.5 to <0.75) type in Yq12 were more often in the LSC group for the first time. Our results not only shed light on the potential role of low RCN in Yq12 in male infertility but also showed that HLPA can be a powerful and cost-effective tool for clinical screening in male infertility.

Expanding the Scope of Non-invasive Prenatal Testing to Detect Fetal Chromosomal Copy Number Variations.

Non-invasive prenatal testing (NIPT) for common fetal trisomies is effective. However, the usefulness of cell-free DNA testing to detect other chromosomal abnormalities is poorly understood. We analyzed the positive rate at different read depths in next-generation sequencing (NGS) and identified a strategy for fetal copy number variant (CNV) detection in NIPT. Pregnant women who underwent NIPT by NGS at read depths of 4-6 M and fetuses with suspected CNVs were analyzed by amniocentesis and chromosomal microarray analysis (CMA). These fetus samples were re-sequenced at a read depth of 25 M and the positive detection rate was determined. With the increase in read depth, the positive CNV detection rate increased. The positive CNV detection rates at 25 M with small fragments were higher by NGS than by karyotype analysis. Increasing read depth in NGS improves the positive CNV detection rate while lowering the false positive detection rate. NIPT by NGS may be an accurate method of fetal chromosome analysis and reduce the rate of birth defects.

[Prenatal diagnosis of spinal muscular atrophy using multiple ligation-dependent probe amplification].

OBJECTIVE: To carry out prenatal diagnosis for families with high risk for spinal muscular atrophy (SMA) by using multiplex ligation-dependent probe amplification (MLPA). METHODS: Twenty-one families were enrolled. MLPA was used to detect copy numbers of SMN1 and SMN2 genes. Maternal contamination was excluded by using a short tandem repeat method. RESULTS: For 23 fetuses from the 21 families, 14 were identified as carriers, 1 as SMA patient, and 8 as normal. By linkage analysis of parental samples, three individuals were determined as silent (2+0) carriers. CONCLUSION: MLPA can determine the carrier status of SMA. The identification of three silent (2+0) carriers among the 44 parental samples indicated a risk for such families, for which genetic counseling and reproduction guidance should be provided.

Preimplantation Genetic Testing for a Chinese Family With X-Linked Lymphoproliferative Syndrome Type 1.

BACKGROUND: X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency disorder. We performed experiments based on two strategies of preimplantation genetic testing (PGT) for a family with XLP caused by a mutation in SH2D1A (c.191G > A). METHODS: First, a single-cell polymerase chain reaction (PCR) protocol was established using single lymphocytes. A nested PCR experiment was performed with direct sequencing after whole genome amplification of single cells to assess the accuracy of the genetic diagnosis. Embryos obtained after intracytoplasmic sperm injection were biopsied on day 3 and detected using the established single-cell PCR protocol. In the second PGT cycle, targeted next generation sequencing (NGS) was performed and the single nucleotide polymorphism (SNP) markers flanking SH2D1A were selected to determine the disease-carrying haplotype phase in each embryo. RESULT: In the first PGT cycle, six embryos were biopsied. Discounting an embryo from a single failed PCR experiment, five embryos were identified, including three unaffected and two hemizygous. After PCR, one normal embryo was transferred when it was developing into an early blastocyst. Although the ultrasound images indicated a viable singleton pregnancy, the implantation was on the cesarean scar. Therefore, an artificial abortion was performed. In the haplotyping cycle, six embryos were identified to have inherited a haplotype without pathogenic mutations. After the embryo implantation process failed twice, a successful singleton pregnancy was established, and subsequently, a healthy female child was born. CONCLUSION: Targeted NGS with haplotyping analysis circumvents the laborious process of multiplex PCR and is more likely to ensure diagnostic accuracy. However, when a genetic recombination occurs close to the site of mutation, confirmed identification using selected SNP markers can be challenging.

Correction: Perfluorooctane Sulfonate Disturbs Nanog Expression through miR-490-3p in Mouse Embryonic Stem Cells.

[This corrects the article DOI: 10.1371/journal.pone.0074968.].

Identification of significant biomarkers and pathways associated with gastric carcinogenesis by whole genome-wide expression profiling analysis.

The incidence of gastric cancer (GC) is extremely high in East Asia. GC is also one of the most common and lethal forms of cancer from a global perspective. However, to date, we have not been able to determine one or several genes as biomarkers in the diagnosis of GC and have also been unable to identify the genes which are important in the therapy of GC. In this study, we analyzed all genome-wide expression profiling arrays uploaded onto the Gene Expression Omnibus (GEO) database to filtrate the differentially expressed genes (DEGs) between normal stomach tissues and GC tissues. GSE13911, GSE19826 and GSE79973 were based on the GPL570 platform, and GSE29272 was based on the GPL96 platform. We screened out the DEGs from the two platforms and by selecting the intersection of these two platforms, we identified the common DEGs in the sequencing data from different laboratories. Finally, we obtained 3 upregulated and 34 downregulated DEGs in GC from 384 samples. As the number of downregulated DEGs was greater than that of the upregulated DEGs, functional analysis and pathway enrichment analysis were performed on the downregulated DEGs. Through our analysis, we identified the most significant genes associated with GC, such as secreted phosphoprotein 1 (SPP1), sulfatase 1 (SULF1), thrombospondin 2 (THBS2), ATPase H+/K+ transporting beta subunit (ATP4B), gastric intrinsic factor (GIF) and gastrokine 1 (GKN1). The prognostic power of these genes was corroborated in the Oncomine database and by Kaplan-Meier plotter (KM-plotter) analysis. Moreover, gastric acid secretion, collecting duct acid secretion, nitrogen metabolism and drug metabolism were significantly related to GC. Thus, these genes and pathways may be potential targets for improving the diagnosis and clinical effects in patients with GC.

Current pesticide profiles in blood serum of adults in Jiangsu Province of China and a comparison with other countries.

Although various pesticides were used globally, the pesticides profiles in human blood serum remain largely unknown. We determined pesticide exposure profiles using solid-phase extraction and gas chromatography tandem with triple quadrupole mass spectrometry in 200 human blood serum samples from the adult population in Jiangsu Province, China. A systematic and comprehensive literature review was carried out to identify the articles investigating pesticide exposure and compare exposure data. Of the 88 pesticides, 76 were found in the blood serum of the population in Jiangsu Province. To the best of our knowledge, 58 pesticides were reported in human blood serum for the first time, and among these pesticides, parathion-methyl, pyrimethanil, fluacrypyrim, simazine, cloquintocet-mexyl and barban were debatable in more than half of the samples. By statistical comparison of the blood serum levels of pesticides between this study and other countries, we found the levels of several organochlorine pesticides were significantly higher in the female population of Jiangsu Province. Health risks related to the pesticide profiling were then revealed, which identified higher carcinogenic toxicity and teratogenic toxicity risk in the female adults of Jiangsu Province caused by organochlorine pesticide exposure. This study not only provides a high-throughput pesticide screening method for future studies of the exposome, but also presents the first human data on exposure to a number of pesticides. It may provide a knowledge database for the risk assessment and management of the pesticides.

Targeted sequencing identifies a novel SH2D1A pathogenic variant in a Chinese family: Carrier screening and prenatal genetic testing.

X-linked lymphoproliferative disease type 1 (XLP1) is a rare primary immunodeficiency characterized by a clinical triad consisting of severe EBV-induced hemophagocytic lymphohistiocytosis, B-cell lymphoma, and dysgammaglobulinemia. Mutations in SH2D1A gene have been revealed as the cause of XLP1. In this study, a pregnant woman with recurrence history of birthing immunodeficiency was screened for pathogenic variant because the proband sample was unavailable. We aimed to clarify the genetic diagnosis and provide prenatal testing for the family. Next-generation sequencing (NGS)-based multigene panel was used in carrier screening of the pregnant woman. Variants of immunodeficiency related genes were analyzed and prioritized. Candidate variant was verified by using Sanger sequencing. The possible influence of the identified variant was evaluated through RNA assay. Amniocentesis, karyotyping, and Sanger sequencing were performed for prenatal testing. We identified a novel de novo frameshift SH2D1A pathogenic variant (c.251_255delTTTCA) in the pregnant carrier. Peripheral blood RNA assay indicated that the mutant transcript could escape nonsense-mediated mRNA decay (NMD) and might encode a C-terminal truncated protein. Information of the variant led to success prenatal diagnosis of the fetus. In conclusion, our study clarified the genetic diagnosis and altered disease prevention for a pregnant carrier of XLP1.

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish.

Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 µg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development (mbp and syn2a) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

Perfluorooctane sulfonate disturbs Nanog expression through miR-490-3p in mouse embryonic stem cells.

Perfluorooctane sulfonate (PFOS) poses potential risks to reproduction and development. Mouse embryonic stem cells (mESCs) are ideal models for developmental toxicity testing of environmental contaminants in vitro. However, the mechanism by which PFOS affects early embryonic development is still unclear. In this study, mESCs were exposed to PFOS for 24 h, and then general cytotoxicity and pluripotency were evaluated. MTT assay showed that neither PFOS (0.2 µM, 2 µM, 20 µM, and 200 µM) nor control medium (0.1% DMSO) treatments affected cell viability. Furthermore, there were no significant differences in cell cycle and apoptosis between the PFOS treatment and control groups. However, we found that the mRNA and protein levels of pluripotency markers (Sox2, Nanog) in mESCs were significantly decreased following exposure to PFOS for 24 h, while there were no significant changes in the mRNA and protein levels of Oct4. Accordingly, the expression levels of miR-145 and miR-490-3p, which can regulate Sox2 and Nanog expressions were significantly increased. Chrm2, the host gene of miR-490-3p, was positively associated with miR-490-3p expression after PFOS exposure. Dual luciferase reporter assay suggests that miR-490-3p directly targets Nanog. These results suggest that PFOS can disturb the expression of pluripotency factors in mESCs, while miR-145 and miR-490-3p play key roles in modulating this effect.