Activin A promotes human trophoblast invasion by upregulating integrin ß3 via ALK4-SMAD4 signaling.

INTRODUCTION: Activin A has been widely regarded as an important promoter of trophoblast invasion during the first trimester of pregnancy. However, whether integrin ß3 is involved in activin A-upregulated trophoblast invasion and the underlying molecular mechanisms remain largely unknown. METHODS: We utilized immortalized (HTR8/SVneo) and primary human extravillous trophoblast (EVT) cells, as well as first-trimester chorionic villous explants as study models to investigate the function and underlying molecular mechanisms of integrin ß3 in activin A-promoted human trophoblast invasion. RESULTS: We found that activin A increased integrin ß3 mRNA and protein levels in both HTR8/SVneo and primary EVT cells, and knockdown of integrin ß3 significantly decreased basal and activin A-upregulated trophoblast invasion. Moreover, SB431542 (a specific inhibitor of TGF-ß type Ι receptor kinase) abolished activin A-upregulated integrin ß3 expression and SMAD2/3 phosphorylation. In addition, siRNA-mediated knockdown of ALK4 or SMAD4 both abolished activin A-upregulated integrin ß3 expression in HTR8/SVneo cells, while knockdown of ALK4 or SMAD4 attenuated activin A-upregulated integrin ß3 expression in primary EVTs. DISCUSSION: Our findings reveal the mediation role of integrin ß3 in activin A-upregulated human trophoblast invasion and that activin An upregulates integrin ß3 expression in an ALK4-SMAD4 signaling-dependent manner.

A Novel Exon Polymorphism of TP53 Associated with Recurrent Pregnancy Loss in a Chinese Han Population.

BACKGROUND: To identify recurrent pregnancy loss (RPL)-related genetic variants in exons of TP53 gene in a population of Chinese Han women. METHODS: This study is a case control study. The cases comprised 90 Chinese Han women with RPL. Another 90 women with at least one child and not more than one miscarriage were recruited as the controls. All exons of TP53 were amplified from genomic DNA and sequenced. RESULTS: A total of five single-nucleotide polymorphisms (SNPs) were identified in both RPL and control women, namely rs1642785 G>C, rs1042522 G>C, rs4968187 G>A, rs17884306 G>A, and rs55817367 A>G. A significant difference was only observed for rs17884306 between cases and controls. The wild type G allele was associated with an increased risk of RPL. AA+GA genetic variants significantly decreased the risk of RPL compared with GG variant (odds ratio [OR] = 0.315, 95% confidence interval [CI]: 0.125 - 0.793, p = 0.014). Linkage disequilibrium exits between rs17884306 and rs1642785 and A-C double mutant haplotype showed significantly lower risk of RPL compared with G-G wild type haplotype (OR = 0.303, 95% CI: 0.117 - 0.786, p = 0.014). Model based-multifactor dimensionality reduction indicated that the influence of rs17884306 had interaction with the genotypes of four other loci (all p < 0.05). However, rs17884306 G>A did not cause amino acid substitution. CONCLUSIONS: Our study showed that rs17884306 c.826G>A was a novel polymorphism associated with RPL in Chinese Han women. Moreover, the influence of this SNP on RPL is not associated with p53 amino acid sequence.

Paternal USP26 mutations raise Klinefelter syndrome risk in the offspring of mice and humans.

Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26-mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring.

PCOS-GWAS Susceptibility Variants in THADA, INSR, TOX3, and DENND1A Are Associated With Metabolic Syndrome or Insulin Resistance in Women With PCOS.

Polycystic ovary syndrome is characterized by reproductive and metabolic disturbances throughout the female lifespan. Therefore, this study aimed to determine whether genome-wide association studies (GWAS)-identified risk variants for PCOS could confer risk of metabolic syndrome (MS) or insulin resistance (IR). Fifteen independent SNPs mapping to 11 GWAS loci genotyped in a total of 2,082 Han Chinese women independent of previous GWAS and phenotype-genotype correlations were assessed. The CC group for rs12478601 in THADA was associated with decreased rate of MS after adjustment for age (23.2 vs. 27%, P = 0.042, OR = 0.81). Using a dominant model, the GG+AG group for rs2059807 in INSR was associated with increased risk of MS after adjustment for age (26.8 vs. 22.5%, P = 0.023, OR = 1.27). The GG + GT group for rs4784165 in TOX3 was found to be associated with an increased rate of IR after adjustment for age and BMI(53.3 vs. 48.5%, P = 0.027, OR = 1.27). The GG+AG group for rs2479106 in DENND1A was associated with a decreased rate of IR (48.3 vs. 53.6%, adjusted P = 0.039, OR = 0.80). After exclusion of PCOS cases with a family history of diabetes, hypertension, or dyslipidemia, the phenotype-genotype correlations between the genes INSR and TOX3 and MS or IR were still significant (P < 0.05). Three SNPs (rs13429458 in THADA, rs10818854 in DENND1A, and rs2059807 in INSR) were significantly associated with IR; however, their association was not significant after adjustment for age and BMI. This genotype-phenotype study thus provides clues that THADA, INSR, TOX3, and DENND1A play a role in PCOS possibly through a metabolic disorder-related pathway.

Variation analysis of tousled like kinase 1 gene in patients with sporadic premature ovarian insufficiency.

Tousled like kinase 1 (TLK1), a member of DNA repair family, participates in the regulation of chromatin assembly and is associated with early menopause and premature ovarian insufficiency (POI) in European women. However, whether the sequence variant in the TLK1 gene was causative for POI is still elusive. Here we performed direct sequencing of the TLK1 gene in 192 patients with sporadic POI. All exons and exon-intron boundaries of TLK1 were amplified and sequenced. Six known single-nucleotide polymorphisms were identified in POI, including rs149844334, rs11553951, rs757600673, rs2277339, rs113416007 and rs17283147. No novel variant was identified, which indicates that sequence variants in the coding region of TLK1 might be uncommon in Chinese women with POI. The role of TLK1 in POI pathogenesis needs to be further explored in larger cohorts from Chinese and other ethnic populations.

Novel WEE2 gene variants identified in patients with fertilization failure and female infertility.

OBJECTIVE: To determine whether variants in the WEE2 (WEE1 homolog 2, also known as WEE1B) gene, which has been known to function in the formation of pronuclei during fertilization, contribute to fertilization failure. DESIGN: Case-control genetic study. SETTING: University hospital. PATIENT(S): Ninety infertile women with repeated cycles of pronucleus formation failure undergoing in vitro fertilization and/or intracytoplasmic sperm injection treatment as well as 200 fertile control women. INTERVENTION(S): Genomic DNA was extracted from the peripheral blood. The whole exons of WEE2 were amplified by means of polymerase chain reaction and then Sanger sequencing was performed. MAIN OUTCOME MEASURE(S): Variants analysis of WEE2 gene. RESULT(S): We identified five subjects that were subjected to homozygous or compound-heterozygous variants of WEE2: case 1 (from a consanguineous family) with homozygous frameshift variant: c.293_294insCTGAGACACCAGCCCAACC (p.Pro98Pro fsX2); case 2 with homozygous missense variant: c.1576T>G (p.Tyr526Asp); and three cases with compound-heterozygous variants: case 3: c.991C>A (p.His331Asn) and c.1304_1307delCCAA (p.Thr435Met fsX31); case 4: c.341_342 del AA (p.Lys114Asn fsX20) and c.864G>C (p.Gln288His); and case 5: c.1A>G (p.0?) and c.1261G>A (p.Gly421Arg). Besides c.1576T>G (from case 2) and c.864G>C (from case 4), which have been previously reported as rare single nucleotide polymorphisms (SNPs), the other six variants were novel and predicted by software to be deleterious. The parental genotypes of case 1 and case 2 indicated that the detected homozygous variants were inherited in an autosomal recessive mode. All of the detected variants were absent from the control cohort. CONCLUSION(S): Novel variants found in WEE2, which is autosomal-recessive inherited, may be related to recurrent pronucleus formation failure and female infertility.

Mutational analysis of IZUMO1R in women with fertilization failure and polyspermy after in vitro fertilization.

PURPOSE: The etiology of fertilization failure and polyspermy during assisted reproductive technology (ART) remains elusive. The aim of this study was to determine whether mutations in the IZUMO1 receptor (IZUMO1R) gene, which is essential for mammalian fertilization, contribute to the pathogenesis of fertilization failure or polyspermy in humans. METHODS: We recruited 215 female subjects with fertilization failure/poor fertilization, 330 females with polyspermy, and 300 matched controls. All subjects underwent IVF treatment. Peripheral blood DNA of cases was extracted and screened for mutations in IZUMO1R gene. RESULTS: Four rare single nucleotide polymorphisms (SNPs) of the IZUMO1R were identified among specimens from patients with fertilization failure and polyspermy but were absent in the 300 control subjects. These included a missense SNP (rs76779571 in exon 4), which was found in two fertilization failure patients, and a nonsynonymous SNP (rs61742524 in exon 1) and two synonymous SNPs (rs76781645 in exon 1 and rs377369966 in intron 2), which were found among three polyspermy cases. CONCLUSIONS: The variations in IZUMO1R might play a role in the pathogenesis of fertilization failure and polyspermy, and the putative functions and effects of these rare variants require further studies.

Novel zona pellucida gene variants identified in patients with oocyte anomalies.

OBJECTIVE: To detect ZP (zona pellucida) gene (ZP1-ZP4) mutations in patients with oocyte anomalies. DESIGN: Case-control genetic study. SETTING: University-based reproductive medicine center. PATIENT(S): A total of 92 infertile patients with repeated cycles of oocyte maturation arrest (group I, n = 49) or oocyte morphologic defect (group II, n = 43) as well as 373 healthy controls. INTERVENTION(S): Genomic DNA extracted from peripheral blood and coding regions of ZP genes amplified by polymerase chain reaction and sequenced by a DNA analyzer. MAIN OUTCOME MEASURE(S): Variant prediction of ZP genes with software. RESULT(S): In group I with oocyte maturation arrest, no novel variants were found. In group II with oocyte morphologic defects, four novel variants, two in the ZP1 gene [c.247T>C (p.W83R) and c.1413G>A (p.W471X)] and two in the ZP2 gene [c.1599G>T (p.R533S) and c.1696T>C (p.C566R)] were detected in 4 of 43 patients (approximately 9%) but were absent from the controls. Protein alignments showed that the four variants were highly conserved among different species, and all four variants were predicted to be deleterious by gene software predictions. CONCLUSION(S): ZP gene variants may account for patients with oocyte morphologic abnormalities but not for those with oocyte maturation arrest.

Kisspeptin: a new marker for human pre-ovulation.

Kisspeptin is a polypeptide that plays an important role in reproductive endocrine regulation. The aim of present study was to investigate the dynamic trend of kisspeptin levels during the menstrual cycle and to elucidate the relationship between kisspeptin ovulation. First, 15 female volunteers with regular menstrual cycles were recruited to detect the change in serum and urine kisspeptin levels over one menstrual cycle within each individual. Subsequently, 114 serum samples and 79 urine samples from 114 individuals were randomly collected at the outpatient department to better ascertain the results. Kisspeptin levels showed a distinctive stage-specific pattern during the normal menstrual cycle in both serum and urine. It was low during the first 5 days, while the first surge appeared on the 11th day (the diameter of the dominant follicle was approximately 1.2 cm). Later, a second smaller surge appeared around the 14th day and the same changes were identified in serum and urine. Furthermore, serum kisspeptin levels were positively related to 17-ß estradiol (E2) level increase. Thus, kisspeptin surge in serum and urine may be used as a marker for dominant follicle development and pre-ovulation. Moreover, kisspeptin may also play a vital role in female reproduction through regulating hormonal state.

ERBB4 Confers Risk for Polycystic Ovary Syndrome in Han Chinese.

A recent genome-wide association study (GWAS) of polycystic ovary syndrome (PCOS) in European cohorts has identified six susceptibility loci mapping to 11q22.1 (YAP1), 2p21 (THADA), 11p14.1 (FSHB), 2q34 (ERBB4), 12q21.2 (KRR1), and 5q31.1 (RAD50). The loci of 11q22.1, 2p21 and 11p14.1 have been confirmed to be associated with PCOS in Chinese; whereas the other three new loci (2q34, 12q21.2, and 5q31.1) still need to be evaluated in Chinese. This study was aimed to determine if the three new loci identified in European PCOS also confer risks for PCOS in Han Chinese. We performed a case-control genetic association study comprising 1500 PCOS cases and 1220 age-matched control subjects. Marker SNPs rs1351592 (2q34, ERBB4), rs1275468 (12q21.2, KRR1) and rs13164856 (5q31.1, RAD50) were genotyped using TaqMan-MGB probe assay. Genotyping analysis showed the allele frequency of rs1351592 in gene ERBB4 was significantly different (P = 1.05E-03) between PCOS cases and control group, and remained significant even after BMI adjustment (Padjusted = 2.09E-04). However, the allele frequencies of the other two risk variants, rs1275468 (12q21.2, KRR1) and rs13164856 (5q31.1, RAD50), were not significantly different in the replication cohort. Our results demonstrate that ERBB4, with the strongest association in European PCOS, also confers risk for PCOS in Han Chinese.

Variation analysis of EXO1 gene in Chinese patients with premature ovarian failure.

Exonuclease 1 (EXO1) is required for both DNA repair and meiosis. Inactivation of EXO1 gene in mice leads to infertility. This study aimed to investigate whether variants in the EXO1 gene contribute to human premature ovarian failure (POF). The coding region of EXO1 was sequenced in 186 Han Chinese patients with non-syndromic POF. No plausible mutation was detected. The results suggest that mutations in the coding region of EXO1 may not be responsible for POF in Han Chinese women.

Family-based analysis of eight susceptibility loci in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that is proposed to have a genetic basis. A recent genome-wide association study (GWAS) identified eight new risk loci that are independently associated with PCOS. To further validate the findings, a total of 321 case-parent trios (963 participants) who had a proband affected with PCOS were recruited for the family-based study. The transmission disequilibrium test (TDT) was used to analyze associations between PCOS and ten single nucleotide polymorphisms (SNPs) mapped to eight new susceptibility loci. Significant differences in transmission were observed for the SNPs rs2349415 (located in the FSHR gene, P = 0.0001) and rs3802457 (located in the C9orf3 gene, P = 0.0001), even after correction for multiple testing bias. The present data provides further evidence for an association between two susceptibility loci, 2p16.3 and 9q22.32, and PCOS. Follow-up functional studies on the FSHR and C9orf3 genes are required to understand their roles in PCOS development.

Polycystic ovary syndrome susceptibility single nucleotide polymorphisms in women with a single PCOS clinical feature.

STUDY QUESTION: What is the direct genetic contribution of the polycystic ovary syndrome (PCOS) susceptibility single nucleotide polymorphisms (SNPs), identified by previous genome-wide association studies (GWAS) to the definitive clinical features of the syndrome? SUMMARY ANSWER: Each single PCOS clinical feature had a specific genetic association, and rs4385527 in the chromosome 9 open reading frame 3 (C9orf3) conferred a particular risk to the three defined PCOS clinical features in this study, which suggested its fundamental role in the etiology of PCOS. WHAT IS KNOWN ALREADY: PCOS is a heterogeneous disorder characterized by anovulation (OA), hyperandrogenism (HA) and polycystic ovary morphology (PCOM). Two previous GWAS in China have identified 15 independent susceptibility SNPs related to PCOS (PCOS-SNPs). However, little is known about the candidate gene of each clinical feature. STUDY DESIGN, SIZE, DURATION: Case-control study. Three independent groups of women were recruited from 2010 to 2012: 746 subjects with OA only, 278 subjects with HA only and 536 subjects with PCOM only. A total of 1790 healthy women with none of the above pathological characteristics were also enrolled as control subjects during the same time period. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants were women of reproductive age. Genotype and allelic frequencies of 15 PCOS-SNPs were determined in all subjects using direct sequencing and Sequenom Arrays. The allelic frequencies of each case group were compared with the controls. MAIN RESULTS AND THE ROLE OF CHANCE: After adjustment for age and BMI, variants in luteinizing hormone/choriogonadotropin receptor (LHCGR) (rs13405728), C9orf3 (rs4385527) and insulin receptor gene (INSR) (rs2059807) were strongly associated with OA (Padjust < 0.01, <0.001 and <0.05, respectively); rs4385527 in C9orf3 was strongly associated with HA (Padjust< 0.001); variants in the thyroid adenoma associated gene (THADA) (rs13429458 and rs12478601), DENN/MADD domain containing 1A (DENND1A)(rs10818854), and C9orf3 (rs4385527) were significantly associated with PCOM (Padjust < 0.01, <0.001, <0.05 and <0.001, respectively). LIMITATIONS, REASONS FOR CAUTION: The sample size of some case groups was relatively small, which therefore limited the statistical power of the analysis to a certain extent. WIDER IMPLICATIONS OF THE FINDINGS: The present study indicates a potential common genetic basis of three PCOS clinical features. Other specific associated genes may play a synergistic role, leading to heterogeneous pathophysiological changes. Additionally, the increased frequency of PCOS-risk alleles in women with single PCOS clinical features suggests that these subjects have an elevated risk of developing the syndrome, although they cannot be currently diagnosed. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Basic Research Program of China (973 Program) (2012CB944700, 2011CB944502), the National Key Technology Research and Development Program(2011BAI17B00), the National Natural Science Foundation of China (81430029, 81201441, 81490743, 31371453), the Scientific Research Foundation of Shandong Province of Outstanding Young Scientist (2012BSE27089) and the Fundamental Research Funds of Shandong University(2014GN025). There were no competing interests.

Gene therapy of malignant solid tumors by targeting erbB2 receptors and by activating T cells.

One of the strategies to improve the outcome of anti-erbB2-mediated immunotherapy is to combine anti-erbB2 antibodies with T-cell-based adoptive immunotherapy, which can be achieved by expressing anti-erbB2 mAb on the surface of T cells. A single-chain variable fragment (scFv) from an anti-erbB2 mAb has been expressed on T cell surface to bind to erbB2-positive cells, and CD3ζ has been expressed as a fusion partner at C terminus of this scFv to transduce signals. T cells grafted with this chimeric scFv/CD3ζ were able to specifically attack target tumor cells with no MHC/Ag restriction. To test the effects of CD28 signal on cellular activation and antitumor effectiveness of chimeric scFv/CD3ζ-modified T cells, we constructed a recombinant anti-erbB2 scFv/Fc/CD28/CD3ζ gene in a retroviral vector. T cells expressing anti-erbB2 scFv/Fc/CD28/CD3ζ specifically lyzed erbB2-positive target tumor cells and secreted not only interferon-γ (IFN-γ) but also IL-2 after binding to their target cells. Our data indicate that CD3 and CD28 signaling can be delivered in one molecule, which is sufficient for complete T cell activation without exogenous B7/CD28 co-stimulation.