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Objective: To investigate whether LHX1 gene mutations exist in Han Chinese patients with müllerian duct abnormalities (MDAs)., Design: Mutation screening., Setting: University hospital., Patient(s): Ninety-six MDA patients and 105 control subjects from a Han Chinese population. The parents of the patients carrying the genetic variation were also screened., Intervention(s): Gene sequencing., Main Outcome Measure(s): Karyotype, LHX1 gene sequencing., Result(s): We found no significant mutation in coding regions of LHX1. However, there is a new rare polymorphism of LHX1 gene, c.1070-1081del, found in 1 out of 77 incomplete müllerian fusion patients and 1 out of 105 control individuals in the Han Chinese population (thus affecting ∼1% of Han Chinese)., Conclusion(s): No causative perturbation was identified in the LHX1 gene. Mutations in the coding regions of LHX1 may not be a common genetic etiologic factor involved in Han Chinese MDA patients., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To search for WNT7A gene mutations in a cohort of 191 Chinese Han patients with müllerian duct abnormalities (MDAs)., Design: Phenotypic and mutational study., Setting: University hospital., Patient(s): A total of 191 Chinese Han patients with MDAs and 192 healthy control individuals., Intervention(s): Genomic DNA extracted from blood samples, all coding regions amplified by polymerase chain reaction (PCR) then directly sequenced to screen variants., Main Outcome Measure(s): Not applicable., Result(s): The sequence analysis revealed one novel synonymous variant and three known single-nucleotide polymorphisms (SNPs)., Conclusion(s): The results indicate that mutations in the coding sequence of WNT7A are not responsible for müllerian duct abnormalities in the Chinese population., (Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Premature ovarian failure (POF) is a complex and heterogeneous disorder that is influenced by multiple genetic components. Numerous candidate gene studies designed to identify POF susceptibility loci have been published, but most positive findings have not been confirmed in follow up studies. We sought to determine if sequence variants previously associated with age at natural menopause (AANM) or early menopause (EM) contribute as well to genetic susceptibility to POF., Methods: Our study was performed on 371 unrelated idiopathic women with POF and 800 women controls, all Chinese Han. Thirty six SNPs from previous genome-wide association studies (GWAS) responsible for AANM or EM and 3 additional SNPs in ESR1, and 2 additional SNPs in PTHB1 were tested using the Sequenom MassARRAY iPLEX platform for genotyping., Results: Three SNPs - rs2278493 in HK3, rs2234693 in ESR1 and rs12611091 in BRSK1 - showed nominally significant association with POF. Thus, a plausible relationship could exist between ESR1, BRSK1, HK3 and POF., Conclusions: This largest association study undertaken to determine correlation between POF and AANM/EM revealed three significant SNPs (rs2278493, rs2234693, and rs12611091). All are associated with not only AAWM and EM but also POF. Insights into shared genetic susceptibility between POF and AANM/EM will provide novel entry points for unraveling genetic mechanism involved in ovarian reserve and oocyte aging processes.

Premature ovarian failure (POF) is a complex heritable disorder known to be caused by chromosomal abnormalities and to date a limited number of known mutations, often autosomal. We sought to identify additional genetic loci associated with POF by performing the first large-scale genome-wide association study (GWAS). GWAS, using Affymetrix SNP 6.0 chip, was conducted in an initial discovery set of 391 well-documented (follicle-stimulating hormone >40 IU/ml) Chinese Han POF patients, compared with 895 unrelated Chinese female controls. A replication study on the most significant loci was then performed in an independent set of 400 cases and 800 controls. Suggestive significant associations were observed at 8q22.3. Replication of eight single-nucleotide polymorphisms (SNPs) (rs10464815, rs10808365, rs3847152, rs3847153, rs3847154, rs3843552, rs10955242, rs3843555) (P ≤ 3.86 × 10(-6)) was confirmed in verification sets. No specific candidate gene was found in the immediate region of 8q22.3. This GWAS, involving by far the largest sample of POF cases accumulated to date, revealed heretofore unrecognized association between POF and a novel genetic locus or region of unknown nature on 8q22.3. We speculate existence of a long-distance regulatory region that has relevance to the control of ovarian differentiation or oogenesis. Given failure to find association with any of the other autosomal regions known to harbor genes causing ovarian failure, our findings also underscore the likelihood of considerable genetic and etiologic heterogeneity in POF and the need for additional approaches like whole-genome sequencing., (© The Author 2011. Published by Oxford University Press. All rights reserved.)

We examined the PBX1 gene in 192 Chinese women with Müllerian duct abnormalities and revealed 2 known single nucleotide polymorphisms: c.61 > A in exon 1 and c.998-1330A>G in intron 7. Future studies in large cohorts of different ethnic populations are warranted to establish definite associations between the PBX1 gene and Müllerian duct abnormalities., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: To evaluate basal testosterone (T) levels during follicular phase of the menstrual cycle as a predictor for ovarian response and in vitro fertilization (IVF) outcome., Method: We analyzed data retrospectively from hospital-based IVF center including one thousand two hundred and sixty Chinese Han women under their first IVF cycle reached the ovum pick-up stage, without polycystic ovary syndrome (PCOS) or endometriosis undergoing long IVF protocol. Patients were divided into 2 groups. Group 1: patients with diminished ovarian reserve (basal FSH >10 IU/L) (n = 187); Group 2: patients with normal ovarian reserve (basal FSH < = 10 IU/L) (n = 1073). We studied the association of basal T levels with ovarian response and IVF outcome in the two groups. Long luteal down-regulation protocol was used in all patients, that is, the gonadotropin releasing hormone agonist was administered in the midluteal phase of the previous cycle and use of recombinant FSH was started when satisfactory pituitary desensitization was achieved., Results: Basal T levels were markly different between pregnant and non-pregnant women in Group 1; whereas not in Group 2. A testosterone level of 47.85 ng/dl was shown to predict pregnancy outcome with a sensitivity of 52.8% and specificity of 65.3%; and the basal T was correlated with the numbers of large follicles (> 14 mm) on HCG day in Group 1. Significantly negative correlations were observed between basal T, days of stimulation and total dose of gonadotropins after adjusting for confounding factors in both groups., Conclusion: In women with diminished ovarian reserve, basal T level was a predictor for the number of large follicles on HCG day and pregnancy outcome; but could not in those with normal serum FSH. Basal T levels were associated with both days of stimulation and total dose of gonadotropins, indicating that lower level of T might relate with potential ovarian poor response.

Polycystic ovary syndrome (PCOS) is a common metabolic disorder in women. To identify causative genes, we conducted a genome-wide association study (GWAS) of PCOS in Han Chinese. The discovery set included 744 PCOS cases and 895 controls; subsequent replications involved two independent cohorts (2,840 PCOS cases and 5,012 controls from northern Han Chinese; 498 cases and 780 controls from southern and central Han Chinese). We identified strong evidence of associations between PCOS and three loci: 2p16.3 (rs13405728; combined P-value by meta-analysis P(meta) = 7.55 × 10⁻²¹, odds ratio (OR) 0.71); 2p21 (rs13429458, P(meta) = 1.73 × 10⁻²³, OR 0.67); and 9q33.3 (rs2479106, P(meta) = 8.12 × 10⁻¹⁹, OR 1.34). These findings provide new insight into the pathogenesis of PCOS. Follow-up studies of the candidate genes in these regions are recommended.

The newborn ovary homeobox gene (NOBOX) is an oocyte-specific homeobox gene that plays a critical role in early folliculogenesis, thus representing an attractive candidate gene for nonsyndromic ovarian failure. We investigated whether perturbation in the homeodomain region of NOBOX was present in Chinese women with premature ovarian failure (POF). We sequenced 200 Chinese patients with POF, and discovered only two known single nucleotide polymorphisms: in intron 6 (c.1154+11 T>C and c.1155-22 G>A); neither offers plausible explanations for POF. Failing to find causative mutation contrasts with our previous study in a caucasian sample, in which we found plausible homeobox mutation in 1 of 96 POF cases. Mutations in the homeobox domain of NOBOX are not common explanations for POF in Chinese women.

Premature Ovarian Failure (POF) is a genetically heterogenous disorder that leads to hypergonadotropic ovarian failure and infertility. We screened 100 Chinese women with POF for mutations in the oocyte-specific gene FIGLA and identified three variants in four women: missense mutation c.11C --> A (p.A4E) was found in two women; deletion c. 15-36 del (p.G6fsX66), resulting in a frameshift that leads to haploinsufficiency, was found in one woman; and deletion c.419-421 delACA (p.140 delN) was found in one. Functional analyses by the yeast two-hybrid assay demonstrated that the p.140 delN mutation disrupted FIGLA binding to the TCF3 helix-loop-helix (HLH) domain. Our findings show that a subset of Chinese women with sporadic, premature ovarian failure harbor mutations in FIGLA.

The LHX8 (LIM homeobox 8) gene encodes a LIM homeodomain transcriptional regulator that is preferentially expressed in germ cells and critical for mammalian oogenesis. The authors investigated whether nucleotide changes were present in the LHX8 gene of Caucasian women with premature ovarian failure (POF), as compared with control women. When the authors sequenced 95 Caucasian women with POF, they discovered two novel single-nucleotide polymorphisms (SNPs) in intron 3 (c.769+10G>T) and 3' untranslated region (c.1787A>G) of the LHX8 gene. These polymorphisms also were found in controls (n = 94), at frequencies that were not statistically different from those in POF women. Mutations in the LHX8 exons are uncommon in Caucasian women with POF.

NANOS3 encodes an RNA-binding protein and has a conserved function in germ cell development. Our objective was to investigate whether mutations in NANOS3 were present in Chinese and Caucasian women with premature ovarian failure. A known synonymous single-nucleotide polymorphism (rs 2016163) in exon 1 was identified through sequencing 80 Chinese and 88 Caucasian women with premature ovarian failure. No additional single-nucleotide polymorphisms or mutations were found in exons encoding for NANOS3. Our findings suggest that mutations in NANOS3 exons are rare in both Chinese and Caucasian women with premature ovarian failure.

We screened growth differentiation factor 9 coding regions for mutations in a Chinese sample of 100 women with premature ovarian failure and discovered four novel single-nucleotide polymorphisms: c.436C>T (p.Arg146Cys), c.588A>C (silent), c.712A>G (p.Thr238Ala), and c.1283G>C (p.Ser428Thr). Nonsynonymous single-nucleotide polymorphisms c.436C>T and c.1283G>C were also detected in the control population. The c.712A>G perturbation results in a missense mutation (p.Thr238Ala) and was not present in any of 96 controls. Substitution of the hydrophobic amino acid residue alanine for hydrophilic threonine may disrupt growth differentiation factor 9 function.

In order to further understand the role of fibrillin-1 (FBN1, OMIM 134797) perturbations in the pathogenesis of Marfan syndrome (MFS, OMIM 154700) we studied a Han Chinese family in which MFS was segregating. In the Chinese family with 5 affected members, mutation screening for FBN1 was performed using direct sequencing. A novel non-synonymous mutation in the transforming growth factor beta binding protein-like (TB) domain of the FBN1 gene was found. The missense mutation c.3022T>C (C1008R) located in exon 24. This mutation was present in the proband and in two other affected family members, but in neither unaffected family members nor unrelated control subjects. The novel non-synonymous mutation, c.3022T>C (C1008R) in the TB domain of FBN1 gene, may be involved in the pathogenesis of MFS in a Han Chinese family.

NOBOX (newborn ovary homeobox gene) is an oocyte-specific homeobox gene that plays a critical role in early folliculogenesis and represents a candidate gene for nonsyndromic ovarian failure. We investigated whether mutations in the NOBOX gene cause premature ovarian failure (POF). We sequenced the NOBOX gene in 96 white women with POF and discovered seven known single-nucleotide polymorphisms and four novel variations, two of which, p.Arg355His and p.Arg360Gln, cause missense mutations in the homeobox domain. Electrophoretic mobility shift assay (EMSA) confirmed that the missense mutation, p.Arg355His, disrupted NOBOX homeodomain binding to NOBOX DNA-binding element (NBE) and had a dominant negative effect on the binding of wild-type NOBOX to DNA. Our findings demonstrate that NOBOX mutations can cause POF.

Nobox is a homeobox gene expressed in oocytes and critical in oogenesis. Nobox deficiency leads to rapid loss of postnatal oocytes. Early oocyte differentiation is poorly understood. We hypothesized that lack of Nobox perturbs global expression of genes preferentially expressed in oocytes as well as microRNAs. We compared Nobox knockout and wild-type ovaries using Affymetrix 430 2.0 microarray platform. We discovered that 28 (74%) of 38 of the genes downregulated more than 5-fold in the absence of Nobox were preferentially expressed in oocytes, whereas only 5 (15%) of 33 genes upregulated more than 5-fold in the absence of Nobox were preferentially expressed in oocytes. Protein-binding microarray helped identify nucleotide motifs that NOBOX binds and that several downregulated genes contain within putative promoter regions. MicroRNA population in newborn ovaries deficient of Nobox was largely unaffected. Genes whose proteins are predicted to be secreted but were previously unknown to be significantly expressed in early oogenesis were downregulated in Nobox knockouts and included astacin-like metalloendopeptidase (Astl), Jagged 1 (Jag1), oocyte-secreted protein 1 (Oosp1), fetuin beta (Fetub), and R-spondin 2 (Rspo2). In addition, pluripotency-associated genes Pou5f1 and Sall4 are drastically downregulated in Nobox-deficient ovaries, whereas testes-determining gene Dmrt1 is overexpressed. Our findings indicate that Nobox is likely an activator of oocyte-specific gene expression and suggest that the oocyte plays an important role in suppressing expression of male-determining genes, such as Dmrt1.

Objective: The objective was to examine the separation of X- and Y-bearing spermatozoa in a modified swim-up procedure using fluorescent in situ hybridization (FISH), and to find out the influence of swim-up time on the ratio of X- and Y-bearing spermatozoa., Study Design: Prospective study., Setting: Reproductive testing laboratory in a university hospital., Patients: Normal spermatozoa samples were obtained from 10 volunteers by masturbation after sexual abstinence for 3-5 days., Interventions: Spermatozoa were put into 18 tubes with 0.25 ml in each, then mixed with HTF medium and centrifuged for 5 min (400 x g). The supernatant was removed and discarded and 0.5 ml HTF was added slowly along the tube wall. Motile spermatozoa were collected after swimming up in different times (from 5 up to 150 min, with a total of 17 intervals). The X- and Y-bearing spermatozoa were determined using the FISH technique. The X/Y dual-color CEP probes that were marked by fluorescein isothiocyanate (FITC) and Texas red were applied to analyze the ratio of X- and Y-bearing spermatozoa. The FISH staining slides were analyzed under an immunofluorescence microscope. About 1000-1500 spermatozoa were counted per slide., Main Outcome Measures: The percentages of X- and Y-bearing spermatozoa were calculated., Results: The study results suggested that the total ratio of hybridization was 98.33%. The ratio of X-bearing spermatozoa after swimming up for different amounts of time is 50.03 +/- 0.91% at 0 min, 50.45 +/- 2.06% after 15 min, 50.61 +/- 2.47% after 30 min, 50.16 +/- 2.67% after 60 min, 50.72 +/- 2.64% after 90 min, and 50.56 +/- 2.20% after 150 min. The statistical analysis showed that there were no significant differences among different swim-up times in the ratio of X-bearing spermatozoa., Conclusions: There was no significant effect of swim-up time on the ratios of X- and Y-bearing spermatozoa using a modified swim-up procedure. No direct evidence was found that the swim-up procedure for separating motile spermatozoa to use for either intrauterine insemination (IUI) or in vitro fertilization (IVF) would lead to an imbalance of boys and girls.