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Non-Invasive Prenatal Screening (NIPS) is a useful screening method for common aneuploidies that can occur in pregnancies. It yields high sensitivities and specificities for the targeted conditions it tests for. Most commonly, these include Trisomies in chromosomes 21, 18, and 13, as well as aneuploidies in chromosomes X and Y. It does not, however, replace diagnostic testing. We review four cases seen by our institutions of patients who had NIPS performed with low-risk results and subsequently had fetuses affected with trisomy 18.All fetal samples were evaluated by level II anatomic ultrasound and tested on amniocytes or products of conception through karyotype or chromosomal microarray following low-risk NIPS.None of the fetuses showed evidence of mosaicism and had features (both on ultrasound and postnatally) consistent with Trisomy 18. Postnatal fluorescence in situ hybridization performed on Formalin-Fixed Paraffin-Embedded tissue from 3 of the affected pregnancies' placentas identified mosaicism of trisomy 18.We discuss the possible explanations for the discrepancy between NIPS results and fetal karyotype, including, but not limited to placental mosaicism, placental size, and limitations of NIPS as a screening test.

Plasma cell neoplasm (PCN) is associated with characteristic chromosomal aberrations of diagnostic and prognostic significance. The presence of a small percentage of neoplastic cells is a drawback in the application of karyotyping and fluorescence in situ hybridization for the evaluation of bone marrow aspirate. The analysis of samples enriched for CD138

The complex structure of the chromosome 2q12.3-q13 region provides a high chance of recombination events between various low copy repeats (LCRs). Copy number variants (CNV) in this region are present in both healthy populations and individuals affected with developmental delay, autism and congenital anomalies. Variable expressivity, reduced penetrance and limited characterization of the affected genes have complicated the classification of the CNVs clinical significance.Chromosomal microarray analysis data were reviewed for 10 298 patients with neurodevelopmental disorders referred to the UPMC Medical Genetics and Genomics Laboratories. A genotype-phenotype correlation was performed among the patients harboring the 2q12.3-q13 CNVs with overlapping genomic intervals.We identified 17 (1 in ~600) individuals with rare CNVs in the 2q12.3-q13 region, including nine patients with deletions, seven individuals with duplications and one patient who had both a deletion and a duplication. Likely pathogenic CNVs with the breakpoints between LCRs encompassing the potential dosage-sensitive genes BCL2L11, BUB1, FBLN7 and TMEM87B were the most common. CNVs were also observed between LCRs surrounding the RANBP2 and LIMS1 genes.Our study provides evidence for pathogenic CNV hotspots within the chromosome 2q12.3-q13 region. We suggest CNV classification based on the affected interval and the involvement of potential dosage-sensitive genes in these patients.

An equitable approach by the American College of Medical Genetics and Genomics (ACMG) has recently recommended carrier screening for genes associated with moderate to severe autosomal recessive conditions with a carrier frequency of ≥1/200 in the Genome Aggregation Database exomes (gnomADv2.0.2). We analyzed carrier frequencies in gnomADv3.1.1 genomes representing diverse populations. ClinVar data on 35 996 pathogenic/likely pathogenic variants in 419 genes were used to estimate the gnomAD frequency of heterozygous carriers. We found that ninety-two genes had a carrier frequency of ≥1/200, of which 63 were shared between v3.1.1 and v2.0.2 and 29 were new in v3.1.1. Addition of new populations (Amish, Finnish and Middle Eastern) increased the number of new genes with a carrier frequency of ≥1/200 to 71. Changes in carrier frequencies were attributed to new gnomAD populations, different sample sizes, new ClinVar data, and technical differences between exomes and genomes. This study highlights the dynamic changes in carrier frequencies due to new datasets from diverse populations and provides updated carrier frequencies based on the combined data from 184 352 genomes and exomes in gnomAD. We recommend a periodic review for inclusion of new population data to update carrier screening panels in the future.

Carrier screening began 50 years ago with screening for conditions that have a high prevalence in defined racial/ethnic groups (e.g., Tay–Sachs disease in the Ashkenazi Jewish population; sickle cell disease in Black individuals). Cystic fibrosis was the first medical condition for which panethnic screening was recommended, followed by spinal muscular atrophy. Next-generation sequencing allows low cost and high throughput identification of sequence variants across many genes simultaneously. Since the phrase “expanded carrier screening” is nonspecific, there is a need to define carrier screening processes in a way that will allow equitable opportunity for patients to learn their reproductive risks using next-generation sequencing technology. An improved understanding of this risk allows patients to make informed reproductive decisions. Reproductive decision making is the established metric for clinical utility of population-based carrier screening. Furthermore, standardization of the screening approach will facilitate testing consistency. This practice resource reviews the current status of carrier screening, provides answers to some of the emerging questions, and recommends a consistent and equitable approach for offering carrier screening to all individuals during pregnancy or preconception.

Proper function of GABAergic synapses depends upon the postsynaptic compartment anchoring of neurotransmitter receptors to the membrane by gephyrin and collybistin (Cb). In humans, Cb is encoded by ARHGEF9 on Xq11.1. ARHGEF9 alterations, some inherited from unaffected mothers, have been reported in males with autism, seizures and severe neurodevelopmental abnormalities. In females, a spectrum of mild to moderate phenotype has been detected. We report two unrelated females with autism and mild intellectual disability. High resolution X-chromosome microarray analysis revealed de novo intragenic deletions in ARHGEF9 of 24 kb and 56 kb involving exons 5–8 and exons 3–8 and leading to truncated forms of collybistin. Peripheral blood samples revealed random X-chromosome inactivation in both patients. To explain phenotypic variability in female patients, we propose a model for disruption of collybistin and various irregular interactions in post-synaptic neurons based on X inactivation patterns. Our findings highlight the importance of ARHGEF9 integrity and suggest further research on its correlation with autism and neurobehavioral problems.

The paternal environment has been linked to infertility and negative outcomes. Such effects may be transmitted via sperm through histone modifications. To date, in-depth profiling of the sperm chromatin in men has been limited. Here, we use deep sequencing to characterize the sperm profiles of histone H3 lysine 4 tri-methylation (H3K4me3) and DNA methylation in a representative reference population of 37 men. Our analysis reveals that H3K4me3 is localized throughout the genome and at genes for fertility and development. Remarkably, enrichment is also found at regions that escape epigenetic reprogramming in primordial germ cells, embryonic enhancers, and short-interspersed nuclear elements (SINEs). There is significant overlap in H3K4me3 and DNA methylation throughout the genome, suggesting a potential interplay between these marks previously reported to be mutually exclusive in sperm. Comparisons made between H3K4me3 marked regions in sperm and the embryonic transcriptome suggest an influence of paternal chromatin on embryonic gene expression.

In clinical exome/genome sequencing, the American College of Medical Genetics and Genomics (ACMG) recommends reporting of secondary findings unrelated to a patient's phenotype when pathogenic single-nucleotide variants (SNVs) are observed in one of 59 genes associated with a life-threatening, medically actionable condition. Little is known about the incidence and sensitivity of chromosomal microarray analysis (CMA) for detection of pathogenic copy number variants (CNVs) comprising medically-actionable genes. Clinical CMA has been performed on 8865 individuals referred for molecular cytogenetic testing. We retrospectively reviewed the CMA results to identify patients with CNVs comprising genes included in the 59-ACMG list of secondary findings. We evaluated the clinical significance of these CNVs in respect to pathogenicity, phenotypic manifestations, and heritability. We identified 23 patients (0.26%) with relevant CNV either deletions comprising the entire gene or intragenic alterations involving one or more secondary findings genes. A number of patients and/or their family members with pathogenic CNVs manifest or expected to develop an anticipated clinical phenotype and would benefit from preventive management similar to the patients with pathogenic SNVs. To improve patients' care standardization should apply to reporting of both sequencing and CNVs obtained via clinical genome-wide analysis, including chromosomal microarray and exome/genome sequencing.

Whole exome sequencing (WES) is an emerging technique in prenatal diagnosis. In this retrospective study, we examined diagnostic utility and limitations of WES in prenatal cases with structural birth defects. DNA from 20 trios (fetal and parental), with normal karyotype and microarray findings, underwent WES and variant interpretation at a reference laboratory. The WES results were later re-evaluated in our academic center utilizing prenatal and postnatal phenotyping. Initial analysis using only prenatal ultrasound findings revealed no pathogenic or likely pathogenic variants in 20 pregnancies with structural birth defects. Re-analysis of WES variants and combination of prenatal and postnatal phenotyping yielded pathogenic variants in at least 20% of cases including PORCN gene in a fetus with split-hand/foot malformation, as well as variants of uncertain significance in NEB and NOTCH1 in fetuses with postnatal muscle weakness and Adams-Oliver syndrome, respectively. Furthermore, Sanger sequencing in a patient with holoprosencephaly, elucidated by postnatal MRI, revealed a pathogenic 47-base pairs deletion in ZIC2 which was missed by prenatal WES. This study suggests that incomplete prenatal phenotyping and lack of prenatal ultrasound-genotype databases are the limiting factors for current interpretation of WES data in prenatal diagnosis. Development of prenatal phenotype-genotype databases would significantly help WES interpretation in this setting. Patients who underwent prenatal clinical WES may benefit from the re-analysis based on detailed postnatal findings.

STUDY QUESTION Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes? SUMMARY ANSWER Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation. WHAT IS KNOWN ALREADY There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease. The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required. In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities. STUDY DESIGN, SIZE, DURATION Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 0.3 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets. F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases. PARTICIPANTS/MATERIALS, SETTINGS, METHODS F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development. F1 males were subsequently mated with chow-fed female mice. Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)18.5 and one delivered and followed postnatally. DNA methylation at a global level and at the differentially methylated regions of imprinted genes (H19, Imprinted Maternally Expressed Transcript (Non-Protein Coding)-H19, Small Nuclear Ribonucleoprotein Polypeptide N-Snrpn, KCNQ1 Opposite Strand/Antisense Transcript 1 (Non-Protein Coding)-Kcnq1ot1, Paternally Expressed Gene 1-Peg1 and Paternally Expressed Gene 3-Peg3) was assessed by luminometric methylation analysis and bisulfite pyrosequencing, respectively, in F1 sperm, F2 E18.5 placenta and F2 E18.5 brain cortex. MAIN RESULTS AND THE ROLE OF CHANCE F1 males exhibited lower sperm counts following lifetime exposure to both folic acid deficiency and the highest dose of folic acid supplementation (20FS), (both P < 0.05). Post-implantation losses were increased amongst F2 E18.5 day litters from 20FS exposed F1 males (P < 0.05). F2 litters derived from both 7FD and 20FS exposed F1 males had significantly higher postnatal-preweaning pup death (both P < 0.05). Sperm from 10FS exposed males had increased variance in methylation across imprinted gene H19, P < 0.05; increased variance at a few sites within H19 was also found for the 7FD and 20FS groups (P < 0.05). While the 20FS diet resulted in inter-individual alterations in methylation across the imprinted genes Snrpn and Peg3 in F2 E18.5 placenta, ≥50% of individual sites tested in Peg1 and/or Peg3 were affected in the 7FD and 10FS groups. Inter-individual alterations in Peg1 methylation were found in F2 E18.5 day 10FS group brain cortex (P < 0.05). LARGE SCALE DATA Not applicable. LIMITATIONS REASONS FOR CAUTION The cause of the increase in postnatal-preweaning mortality was not investigated post-mortem. Further studies are required to understand the mechanisms underlying the adverse effects of folic acid deficiency and supplementation on developing male germ cells. Genome-wide DNA and histone methylome studies as well as gene expression studies are required to better understand the links between folic acid exposures, an altered germ cell epigenome and offspring outcomes. WIDER IMPLICATIONS OF THE FINDINGS The findings of this study provide further support for paternally transmitted environmental effects. The results indicate that both folic acid deficiency and high dose supplementation can be detrimental to germ cell development and reproductive fitness, in part by altering DNA methylation in sperm. STUDY FUNDING AND COMPETING INTERESTS This study was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR #89944). The authors declare they have no conflicts of interest.

Background: The sperm DNA methylation landscape is unique and critical for offspring health. If gamete-derived DNA methylation escapes reprograming in early embryos, epigenetic defects in sperm may be transmitted to the next generation. Current techniques to assess sperm DNA methylation show bias toward CpG-dense regions and do not target areas of dynamic methylation, those predicted to be environmentally sensitive and tunable regulatory elements. Objectives: Our goal was to assess variation in human sperm DNA methylation and design a targeted capture panel to interrogate the human sperm methylome. Methods: To characterize variation in sperm DNA methylation, we performed whole genome bisulfite sequencing (WGBS) on an equimolar pool of sperm DNA from a wide cross section of 30 men varying in age, fertility status, methylenetetrahydrofolate reductase (MTHFR) genotype, and exposures. With our targeted capture panel, in individual samples, we examined the effect of MTHFR genotype (n=13 677CC, n=8 677TT), as well as high-dose folic acid supplementation (n=6, per genotype, before and after supplementation). Results: Through WGBS we discovered nearly 1 million CpGs possessing intermediate methylation levels (20–80%), termed dynamic sperm CpGs. These dynamic CpGs, along with 2 million commonly assessed CpGs, were used to customize a capture panel for targeted interrogation of the human sperm methylome and test its ability to detect effects of altered folate metabolism. As compared with MTHFR 677CC men, those with the 677TT genotype (50% decreased MTHFR activity) had both hyper- and hypomethylation in their sperm. High-dose folic acid supplement treatment exacerbated hypomethylation in MTHFR 677TT men compared with 677CC. In both cases, >80% of altered methylation was found in dynamic sperm CpGs, uniquely measured by our assay. Discussion: Our sperm panel allowed the discovery of differential methylation following conditions affecting folate metabolism in novel dynamic sperm CpGs. Improved ability to examine variation in sperm DNA methylation can facilitate comprehensive studies of environment–epigenome interactions. https://doi.org/10.1289/EHP4812

Study question What is the prevalence of somatic chromosomal instability among women with idiopathic primary ovarian insufficiency (POI)? Summary answer A subset of women with idiopathic POI may have functional impairment in DNA repair leading to chromosomal instability in their soma. What is known already The formation and repair of DNA double-strand breaks during meiotic recombination are fundamental processes of gametogenesis. Oocytes with compromised DNA integrity are susceptible to apoptosis which could trigger premature ovarian aging and accelerated wastage of the human follicle reserve. Genomewide association studies, as well as whole exome sequencing, have implicated multiple genes involved in DNA damage repair. However, the prevalence of defective DNA damage repair in the soma of women with POI is unknown. Study design, size, duration In total, 46 women with POI and 15 family members were evaluated for excessive mitomycin-C (MMC)-induced chromosome breakage. Healthy fertile females (n = 20) and two lymphoblastoid cell lines served as negative and as positive controls, respectively. Participants/materials, setting, methods We performed a pilot functional study utilizing MMC to assess chromosomal instability in the peripheral blood of participants. A high-resolution array comparative genomic hybridization (aCGH) was performed on 16 POI patients to identify copy number variations (CNVs) for a set of 341 targeted genes implicated in DNA repair. Main results and the role of chance Array CGH revealed three POI patients (3/16, 18.8%) with pathogenic CNVs. Excessive chromosomal breakage suggestive of a constitutional deficiency in DNA repair was detected in one POI patient with the 16p12.3 duplication. In two patients with negative chromosome breakage analysis, aCGH detected a Xq28 deletion comprising the Centrin EF-hand Protein 2 (CETN2) and HAUS Augmin Like Complex Subunit 7 (HAUS7) genes essential for meiotic DNA repair, and a duplication in the 3p22.2 region comprising a part of the ATPase domain of the MutL Homolog 1 (MLH1) gene. Limitations reasons for caution Peripheral lymphocytes, used as a surrogate tissue to quantify induced chromosome damage, may not be representative of all the affected tissues. Another limitation pertains to the MMC assay which detects homologous repair pathway defects and does not test deficiencies in other DNA repair pathways. Wider implications of the findings Our results provide evidence for functional impairment of DNA repair in idiopathic POI, which may predispose the patients to other DNA repair-related conditions such as accelerated aging and/or cancer susceptibility. Study funding/competing interest(s) Funding was provided by the National Institute of Child Health and Human Development. There were no competing interests to declare.

Mammalian embryo development is init iated by intracel lular Ca2+ oscillations that result in oocyte activation following gamete membrane fusion. It is widely believed that oocyte Ca2+ oscillations are triggered by a sperm-specific protein, phospholipase C-zeta (PLCζ) that activates InsP3 production leading to repetitive Ca2+ release from intracellular stores. However, a recent report in the FASEB Journal by Aarabi et al. challenges this view by proposing postacrosomal WW domain-binding protein (PAWP) as another sperm-derived protein that can also initiate Ca2+ oscillations and zygotic development at fertilization. Here we discuss these new findings and examine the evidence suggesting PAWP as the "real" sperm factor.

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like ( WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

Supplementation with high doses of folic acid, an important mediator of one-carbon transfers for DNA methylation, is used clinically to improve sperm parameters in infertile men. We recently detected an unexpected loss of DNA methylation in the sperm of idiopathic infertile men after 6 months of daily supplementation with 5 mg folic acid (>10× the daily recommended intake—DRI), exacerbated in men homozygous for a common variant in the gene encoding an important enzyme in folate metabolism, methylenetetrahydrofolate reductase (MTHFR 677C>T). To investigate the epigenomic impact and mechanism underlying effects of folic acid on male germ cells, wild-type and heterozygote mice for a targeted inactivation of the Mthfr gene were fed high-dose folic acid (10× the DRI) or control diets (CDs) for 6 months. No changes were detected in general health, sperm counts or methylation of imprinted genes. Reduced representation bisulfite sequencing revealed sperm DNA hypomethylation in Mthfr(+/−) mice on the 10× diets. Wild-type mice demonstrated sperm hypomethylation only with a very high dose (20×) of folic acid for 12 months. Testicular MTHFR protein levels decreased significantly in wild-type mice on the 20× diet but not in those on the 10× diet, suggesting a possible role for MTHFR deficiency in sperm DNA hypomethylation. In-depth analysis of the folic acid-exposed sperm DNA methylome suggested mouse/human susceptibility of sequences with potential importance to germ cell and embryo development. Our data provide evidence for a similar cross-species response to high dose folic acid supplementation, of sperm DNA hypomethylation, and implicate MTHFR downregulation as a possible mechanism.

Postacrosomal sheath WW domain-binding protein (PAWP) is a novel sperm protein identified as a candidate sperm-borne, oocyte-activating factor (SOAF). However, regulation of PAWP gene expression is poorly understood. Therefore, we examined the PAWP gene expression across different stages of mouse embryonic stem cell (ESC)-based spermatogenesis in vitro and compared this expression at different stages of mouse testis development in vivo. Expression of PAWP was also examined in mouse embryonic fibroblasts (MEF), Sertoli cell, and the NIH3T3 cancerous cell line. We used a transgenic mouse ESC line C57BL/6J expressing Stra8-EGFP that was plated in murine ESC medium. To induce differentiation, cells were cultured on gelatin-coated medium with Retinoic Acid (RA) treatment. We applied reverse transcription-PCR and real-time PCR to analyze the differential expression of PAWP mRNA during different stages of mouse ESC differentiation in vitro parallel with mouse testis development in vivo and in cell lines. We found that expression of PAWP is increased during testis development in vivo with greatest expression at postmeiotic phase. It is also highly expressed in mouse ESC-derived germ-like cells after 30 d of RA induction in vitro. PAWP is remarkably expressed in mouse ESC and NIH3T3 cell line. These results indicate that PAWP plays a role in spermatogenesis and germ cell development. Moreover, we suggest PAWP as one of the markers that could be looked in ESC studies as a confirmed testis-specific gene. We also suggest an additional possible role for PAWP in proliferation of cancerous cell in general.

We investigated polymorphisms of plasminogen activator inhibitor-1 (PAI-1), angiotensin converting enzyme (ACE ) and coagulation factor XIII (FXIII) genes and their association with recurrent spontaneous abortion (RSA) in Iranian patients and normal healthy controls. Ten (18.5%) patients were homozygote (4G/4G) for PAI-1 polymorphism, in contrast with two (2%) controls (p = 0.001). Patients with homozygote 4G mutation were significantly more prone to RSA in contrast to others (odds ratio: 11.0, 95% CI: 2.3-52.4). Nineteen (30.2%) patients and 25 (26.6%) controls were homozygote (DD) for ACE polymorphism. We observed only two patients and one control with homozygosity (34leu) for FXIII polymorphism. 4G/4G polymorphism for PAI-1 gene could be a thrombophilic mutation leading to abortion in Iranian population.

Background Cancer–testis genes are a group of genes expressed in testicular germinal cells and a range of human cancers. Testis-specific gene A10 (TSGA10) is expressed in testis and actively dividing and fetal differentiating tissues. Mouse homologue (Tsga10) mRNA is translated to a 65kDa protein and appears to be processed to a major fibrous sheath protein of sperm tail. SYCP3 gene is supposed to be a testis-specific gene and constitutes the core of the lateral elements of synaptonemal complex. It has role in regulating DNA binding to the chromatid axis, sister chromatid cohesion, synapsis, and recombination. Methods In this study expression of TSGA10 and SYCP3 were investigated in different cancers (156 tumor samples) using RT-PCR. Diagnosis of cancer was based on histopathological reports. The association with histopathological characteristics of tumors was analyzed using statistical programs. Results TSGA10 expression was observed in 83% of brain tumors, 66% of breast cancers, 58% of gastrointestinal tumors, 66% of skin tumors and 53% of soft tissue tumors. But, SYCP3 transcripts were found in four tumor samples (moderately differentiated gemistocytic astrocytoma, pituitary adenoma, glioma and an ovarian tumor). Conclusion These results may get further insight into TSGA10, but not SYCP3, potential role as a cancer marker and a cancer testis gene implicated in tumorogenesis of cancers.

Dietary folate is a major source of methyl groups required for DNA methylation, an epigenetic modification that is actively maintained and remodeled during spermatogenesis. While high-dose folic acid supplementation (up to 10 times the daily recommended dose) has been shown to improve sperm parameters in infertile men, the effects of supplementation on the sperm epigenome are unknown. To assess the impact of 6 months of high-dose folic acid supplementation on the sperm epigenome, we studied 30 men with idiopathic infertility. Blood folate concentrations increased significantly after supplementation with no significant improvements in sperm parameters. Methylation levels of the differentially methylated regions of several imprinted loci (H19, DLK1/GTL2, MEST, SNRPN, PLAGL1, KCNQ1OT1) were normal both before and after supplementation. Reduced representation bisulfite sequencing (RRBS) revealed a significant global loss of methylation across different regions of the sperm genome. The most marked loss of DNA methylation was found in sperm from patients homozygous for the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, a common polymorphism in a key enzyme required for folate metabolism. RRBS analysis also showed that most of the differentially methylated tiles were located in DNA repeats, low CpG-density and intergenic regions. Ingenuity Pathway Analysis revealed that methylation of promoter regions was altered in several genes involved in cancer and neurobehavioral disorders including CBFA2T3, PTPN6, COL18A1, ALDH2, UBE4B, ERBB2, GABRB3, CNTNAP4 and NIPA1. Our data reveal alterations of the human sperm epigenome associated with high-dose folic acid supplementation, effects that were exacerbated by a common polymorphism in MTHFR.

Objective To determine the expression of the synaptonemal complex protein-3 ( SYCP3 ) gene in men with nonobstructive azoospermia. Design Cross-sectional case study. Setting Avesina Infertility Clinic, Tehran, Iran. Patient(s) One hundred and ten consecutive infertile men presenting nonobstructive azoospermia. Intervention(s) Testicular biopsies for histopathological assessment and analyses of SYCP3 expression level by semiquantitative nested reverse transcription–polymerase chain reaction (RT-PCR). The SYCP3 levels were normalized to expression of the housekeeping phosphoglucomutase 1 gene. Main Outcome Measure(s) Expression of SYCP3 messenger ribonucleic acid (mRNA). Correlation of the histopathological findings with SYCP3 expression levels. Results(s) Testicular SYCP3 mRNA expression was observed in 67/110 (60.9%) patients. The expression level correlated with the degree of spermatogenic failure. Although it was expressed in patients with spermatogenesis and maturation arrest, a lack of expression was seen in all of those men with spermatogonial arrest, Sertoli cell-only syndrome, and testicular atrophy. Conclusion(s) These data indicate that SYCP3 is expressed in human testis and is restricted to germ cells. Our findings, in association with those obtained in experimental animals, shows that lack of SYCP3 expression in human testis may have a negative effect on spermatogenesis and male fertility.

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like ( WBP2NL ) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

Breast cancer is the most frequent cause of mortality in women all around the world; therefore, study on molecular aspects of breast cancer is necessary for finding new biomarkers. Recent studies have shown that WW Binding Protein 2 (WBP2) is an important protein for the oncogenic property of cancer. We have previously evaluated the WW Binding Protein 2 N-Terminal Like (WBP2NL) gene expression in cancerous cell line and breast tumor tissues, and reported changes in expression, which could increase tumorigenic cell growth. However, the molecular mechanisms of WBP2NL and its clinical relevance have not been investigated. In this study, the expression of WBP2NL-related genes in the invasive breast carcinoma and normal breast tissues was evaluated for the first time. Analysis of WBP2NL-related genes expression was performed with reverse transcription-PCR and real time-PCR detection method. The target genes studied were as follow: WW domain containing E3 ubiquitin protein ligase 1(WWP1), membrane associated guanylatekinase containing WW and PDZ domain-1 (MAGI1), neural precursor cell expressed developmentally down-regulated 4 (NEDD4), formin binding protein-4 (FNBP4), BCL2-associated athanogene-3 (BAG3), WW domain-containing oxidoreductase (WWOX), yes-associated protein-1 (YAP1), WW domain containing transcription regulator (WWTR1), member RAS oncogene family (RAB2A), and small G protein signaling modulator 3 (SGSM3). The expression of WWP1, BAG3, and WWTR1 was significantly increased in breast cancer. In contrast, the expression of WWOX, YAP1, RAB2A, and SGSM3 was significantly decreased. The MAGI1 and NEDD4 expression was increased, while the expression of FNBP4 was unchanged. These findings lead us to suggest that WBP2NL might play roles as an anti-apoptotic factor or co-activator to promote breast cancer cell survival and proliferation.

Objective To determine the levels of postacrosomal WW binding protein (PAWP) in the spermatozoa of men that were used clinically for intracytoplasmic sperm injection (ICSI) and to correlate them with infertility treatment outcomes. Design Prospective clinical and laboratory study. Setting University-based laboratory and infertility clinic. Patient(s) Men undergoing ICSI for the treatment of couples' infertility (n = 110). Intervention(s) Quantitative analysis of sperm PAWP levels by flow cytometry and developmental analysis of PAWP expression by immunoblotting, immunofluorescence, and immunohistochemistry. Main Outcome Measure(s) PAWP flow-cytometric levels and immunolocalization in spermatozoa. Result(s) A strong positive correlation was found between PAWP expression levels and fertilization rates after ICSI, with high levels of PAWP being associated with higher fertilization rates; the positive correlation was independent of age, DNA fragmentation index, and other sperm parameters. PAWP expression levels were correlated with embryonic development, with high levels of PAWP being associated with a lower number of arrested embryos within 3–5 days post-ICSI. PAWP expression was detected during the late stages of human spermiogenesis in elongating spermatids, confirming previous findings in various animal models. Conclusion(s) Our clinical data from infertile couples demonstrate significant correlations between sperm PAWP levels and both fertilization rates and normal embryonic development after ICSI. Considering its proposed role in the initiation of oocyte activation, we suggest that PAWP could have potential applications in the diagnosis and treatment of infertility.

Spermatogenesis-associated-19 (SPATA19) is a novel spermatogenesis related gene that has important biological functions in reproduction. However, the expression aspects of SPATA19 gene are not yet well understood. We analysed SPATA19 gene expression during mouse testis development in vivo (5, 15 and 25 days old mouse testis), in parallel with mouse embryonic stem cells (ESCs) differentiation into male germ-like cells in vitro (5, 11, 19 and 27 days after differentiation), as well as Sertoli cell, mouse embryonic fibroblasts (MEF), and NIH3T3 cancerous cell line. We cultured transgenic mouse ESCs line C57BL/6J expressing Stra8-EGFP in ESC's medium and induced their differentiation by retinoic acid (RA) treatment, and meiotic cells were then sorted by FACS. RT-PCR and real-time PCR were used for analysis of SPATA19 mRNA expression. The expression of SPATA19 mRNA is increased during mouse testis development in vivo and it is highest in 25-days-old mouse testis. SPATA19 is also expressed most highly in mouse ESC-derived germ-like cells after 27 days of RA induction and NIH3T3 cell line but no expression was found in mouse ESC and Sertoli cell line. These findings demonstrate that the expression of SPATA19 increases during male germ cells development. We also suggest an additional possible role for SPATA19 in male germ cell differentiation and tumourigenic process.

Phospholipase C zeta (PLCζ) is a candidate sperm-borne oocyte activating factor (SOAF) which has recently received attention as a potential biomarker of human male infertility. However, important SOAF attributes of PLCζ, including its developmental expression in mammalian spermiogenesis, its compartmentalization in sperm head perinuclear theca (PT) and its release into the ooplasm during fertilization have not been established and are addressed in this investigation. Different detergent extractions of sperm and head/tail fractions were compared for the presence of PLCζ by immunoblotting. In both human and mouse, the active isoform of PLCζ was detected in sperm fractions other than PT, where SOAF is expected to reside. Developmentally, PLCζ was incorporated as part of the acrosome during the Golgi phase of human and mouse spermiogenesis while diminishing gradually in the acrosome of elongated spermatids. Immunofluorescence localized PLCζ over the surface of the postacrosomal region of mouse and bull and head region of human spermatozoa leading us to examine its secretion in the epididymis. While previously thought to have strictly a testicular expression, PLCζ was found to be expressed and secreted by the epididymal epithelial cells explaining its presence on the sperm head surface. In vitro fertilization (IVF) revealed that PLCζ is no longer detectable after the acrosome reaction occurs on the surface of the zona pellucida and thus is not incorporated into the oocyte cytoplasm for activation. In summary, we show for the first time that PLCζ is compartmentalized as part of the acrosome early in human and mouse spermiogenesis and is secreted during sperm maturation in the epididymis. Most importantly, no evidence was found that PLCζ is incorporated into the detergent-resistant perinuclear theca fraction where SOAF resides.

Perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) and anti-Saccharomyces Cerevisiae antibody (ASCA) are potential markers for diagnosis of inflammatory bowel disease (IBD). The aim of the present study was to evaluate the diagnostic value of pANCA and ASCA in Iranian patients with IBD.Serum samples were collected from 144 patients with IBD (113 ulcerative colitis and 31 Crohn's disease) and patients with non-IBD problems were assayed for ASCA by Enzyme-Linked Immunosorbent Assay (ELISA) and for pANCA by indirect immunofluorescence assay.Sensitivity and specificity of pANCA in UC were 39.8% and 82.1%, respectively. For CD, pASCA test provided the sensitivity of 58% and specificity of 70%. A combination of pANCA+/ASCA- for diagnosis of UC showed a sensitivity of 31.9% and specificity of 89.1%. In addition the combination of pANCA-/ASCA+ showed a sensitivity of 35.5% and specificity of 79.8% for diagnosis of CD.Due to low sensitivity of pANCA and ASCA alone or in combination, they are not valuable serological markers for diagnosis of UC or CD.

We previously reported postacrosomal sheath WW domain binding protein (PAWP) as a candidate sperm borne, oocyte-activating factor. PAWP enters the oocyte during fertilization and induces oocyte activation events including meiotic resumption, pronuclear formation, and egg cleavage. However, in order to provide proof that PAWP is a primary initiator of zygotic development it is imperative to show that PAWP initiates intracellular calcium signaling, which is considered essential for oocyte activation. Utilizing Xenopus laevis as our model, we injected recombinant PAWP or Xenopus sperm into metaphase II-arrested oocytes and observed a significant rise in intracellular calcium levels over controls. Concurring intensities and durations of PAWP and sperm-induced calcium waves, detected by infrared two-photon laser-scanning fluorescence microscopy, were prevented by coinjection of a competitive PPGY-containing peptide derived from PAWP but not by the point-mutated form of this peptide. This study also correlates PAWP and sperm-induced calcium release with meiotic resumption in Xenopus. The similar mode of oocyte activation, and the ability of the competitive peptide in blocking both sperm- and PAWP-induced calcium release, provide evidence for the first time that sperm-anchored PAWP is a primary initiator of zygotic development.

HOX genes are well-known to encode transcriptional regulatory proteins that play essential roles in directing embryonic development. TGIFLX/Y contains two genes, TGIFLX (X-linked) and TGIFLY (Y-linked), which are specifically expressed in human adult testes. The function(s) of these genes in normal and abnormal development are unknown. To investigate the potential role(s) of the TGIFLX/Y gene in infertile males, a nested reverse transcriptase polymerase chain reaction (RT-PCR) was performed on testicular samples from 110 patients with nonobstructive azoospermia. Although the only 51 (46.4%) of the 110 patients had detectable levels of TGIFLY expression, none of the patients with various spermatogenesis defects showed any of the TGIFLX gene expression found in normal testes. These results suggest that the function of TGIFLX may be required for the regulation of spermatogonial stem cell specification and proliferation. While functional similarity has been demonstrated among some homeobox genes, these results may refute the suggestion of redundancy between TGIFLX and TGIFLY. Furthermore, TGIFLX might be a potential biomarker candidate for male infertility assessment.

Factor V Leiden (FVL) G1691A, methylenetetrahydrofolate reductase (MTHFR) C677T, and factor II (FII) G20210A mutations are three important causes of thrombophilia, the condition that might be related to infertility and recurrent spontaneous abortion (RSA). In this study we evaluated the presence of these three mutations in 36 female patients with unexplained infertility, 65 female patients with unexplained RSA, and 62 healthy fertile women as control group. DNA was extracted from peripheral blood samples and PCR-RFLP was performed for the molecular diagnosis of each mutation. In addition, activated protein C resistance (APC-R) was also evaluated. The frequencies of FVL, MTHFR, and FII mutations (heterozygous and homozygous) in the control group were 0.0%, 38.7%, and 3.2%, respectively. The frequency of FVL mutation in patients with infertility (30.6%) or RSA (20.0%) was significantly higher than that of the control group. A significantly higher MTHFR mutation rate was also observed in patients with RSA (63.1%) as compared to controls. However, the mutation rate of MTHFR in patients with infertility (50.0%) was not statistically different from that in controls. No significant difference was observed in the frequencies of FII mutations between the patients and controls. Decreased levels of APC-R were observed in 25.0% of infertile patients and 18.9% of patients with RSA. In conclusion, our results show a skew towards higher mutation frequencies of FVL and MTHFR in patients that may necessitate detection of such mutations in these Iranian patients.

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry (FC). A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies (ART) and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA (Lens culinaris agglutinin; reveals altered sperm surface) and PNA (Arachis hypogaea/peanut agglutinin; reveals acrosomal malformation or damage). The Postacrosomal Sheath WWI Domain Binding Protein (PAWP), implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery., Work summarized in this review was supported by grants 2013-67015-20961 and 2011-67015-20025 from USDA-NIFA, award #1R21HD066333-01 from NIH-NICHD and award #13324-2007 from Missouri Life Science Research Board, and by seed funding from the F21C Program, University of Missouri to PS. AMV was supported by the Instituto de Salud Carlos III [Projects PI050065 and PI080557, co-financed by the Fondo Social Europeo, FEDER] and Junta de Andalucía [Projects P07-CVI-02697 and P08-CVI-03629}, Spain. This study was also supported by a Canadian Institutes of Health Research grant (MOP-84440 to R.O.).