Analysis of the MCL-1 gene in Chinese women with idiopathic premature ovarian insufficiency.

OBJECTIVE: Animal studies have demonstrated that myeloid cell leukemia-1 (Mcl-1) gene deficiency leads to premature ovarian failure and decreased reproductive ability in mice. This study investigated the relationship between MCL-1 gene variation and idiopathic premature ovarian insufficiency (POI) in Chinese women. METHODS: A total of 200 idiopathic POI patients and 100 healthy controls were recruited for this study, and peripheral blood was collected. First, genomic DNA was extracted from peripheral leukocytes. Then, the entire coding region and splice sites of the MCL-1 gene were amplified by polymerase chain reaction. Chi-squared tests were used to compare the genotype distribution and allele frequency of single nucleotide polymorphisms between the POI and control groups. RESULTS: Three mutations of the MCL-1 gene (c.-36C > T, c.-131C > T and c.78C > T) were identified. After data analysis, c.-36C > T and c.-131C > T in the 5'-untranslated region were both found in the POI group and the control group. No difference was found in the genotype distribution or allelic frequency of either variant between the POI group and the control group (p > 0.05). The synonymous variant (c.78C > T) in exon 1 was discovered in only one of the control subjects and did not result in a change in amino acid sequence (p.Gly26Gly). CONCLUSION: MCL-1 gene mutation may not be associated with idiopathic POI in Chinese women.

Evaluation of CD4+CD25+FOXP3+ regulatory T cells and FOXP3 mRNA in premature ovarian insufficiency.

Objective: T cell-mediated injury plays an important role in the pathogenesis of autoimmune premature ovarian insufficiency (POI). The purpose of this study was to assess the percentage of CD4+CD25+FOXP3+ regulatory T (Treg) cells and the level of forkhead box protein 3 (FOXP3) mRNA expression in POI patients.Methods: The case-control study compared 30 POI patients with 30 healthy subjects. Peripheral blood mononuclear cells were collected. The percentage of CD4+CD25+FOXP3+ Treg cells was measured by flow cytometry using specific monoclonal antibodies recognizing the CD4+, CD25+, and FOXP3+ markers. FOXP3 gene expression was evaluated by real-time polymerase chain reaction. In addition, the levels of transforming growth factor-ß1 (TGF-ß1), interferon-γ (IFN-γ), and adrenal cortex autoantibody (AAA) were determined by enzyme-linked immunosorbent assay.Results: The percentage of CD4+CD25+FOXP3+ Treg cells and the level of FOXP3 mRNA expression were significantly decreased in the POI patients compared with the control subjects. Moreover, the women with POI showed significantly increased levels of IFN-γ and AAA but reduced levels of TGF-ß1.Conclusions: Our study suggested that POI may be associated with an abrogated function of circulating CD4+CD25+FOXP3+ Treg cells and a decreased level of FOXP3 gene expression. However, these results require further investigation.

Genetic and morphology analysis among the pentaploid F1 hybrid fishes (Schizothorax wangchiachii ♀ × Percocypris pingi ♂) and their parents.

Triploid and pentaploid breeding is of great importance in agricultural production, but it is not always easy to obtain double ploidy parents. However, in fishes, chromosome ploidy is diversiform, which may provide natural parental resources for triploid and pentaploid breeding. Both tetraploid and hexaploid exist in Schizothorax fishes, which were thought to belong to different subfamilies with tetraploid Percocypris fishes in morphology, but they are sister genera in molecule. Fortunately, the pentaploid hybrid fishes have been successfully obtained by hybridization of Schizothorax wangchiachii (♀, 2n = 6X = 148) × Percocypris pingi (♂, 2n = 4X = 98). To understand the genetic and morphological difference among the hybrid fishes and their parents, four methods were used in this study: morphology, karyotype, red blood cell (RBC) DNA content determination and inter-simple sequence repeat (ISSR). In morphology, the hybrid fishes were steady, and between their parents with no obvious preference. The chromosome numbers of P. pingi have been reported as 2n = 4X = 98. In this study, the karyotype of S. wangchiachii was 2n = 6X = 148 = 36m + 34sm + 12st + 66t, while that the hybrid fishes was 2n = 5X = 123 = 39m + 28sm + 5st + 51t. Similarly, the RBC DNA content of the hybrid fishes was intermediate among their parents. In ISSR, the within-group genetic diversity of hybrid fishes was higher than that of their parents. Moreover, the genetic distance of hybrid fishes between P. pingi and S.wangchiachii was closely related to that of their parental ploidy, suggesting that parental genetic material stably coexisted in the hybrid fishes. This is the first report to show a stable pentaploid F1 hybrids produced by hybridization of a hexaploid and a tetraploid in aquaculture.

Increased CD4+CD25-Foxp3+ T cells in Chinese systemic lupus erythematosus: correlate with disease activity and organ involvement.

OBJECTIVE: The increment of CD4+CD25-Foxp3+T cells has been reported in systemic lupus erythematosus (SLE) patients. However, the exact identity of this T cell subset is still unclear. Thus, we analyzed CD4+CD25-Foxp3+T cells and Treg cells (CD4+CD25+Foxp3+ T cells) in a large sample of Chinese SLE patients in different disease states. METHODS: A total of 280 SLE patients and 38 healthy volunteers were enrolled, which included 21 patients with untreated new-onset lupus (UNOL), 13 patients with drug withdrawal more than 6 months and 246 patients with treatments. Phenotypic and functional analysis of peripheral blood CD4+CD25-Foxp3+ T cells and Treg cells were performed by flow cytometry. The correlation of CD4+CD25-Foxp3+T cells and Treg cells with disease activity, clinical indicators and organ involvement were analyzed. RESULTS: CD4+CD25-Foxp3+ T cells and Treg cells were significantly increased in SLE patients and showed significantly positive correlations with disease activity. CD4+CD25-Foxp3+ T cells were significantly increased in patients with skin and hematologic involvement as well as arthritis. Diverse changes between CD4+CD25-Foxp3+ T cells and Treg cells when faced with different medications, especially HCQ and MMF. CD4+CD25-Foxp3+ T cells expressed more IFN-γ and less CTLA-4 than CD4+CD25+Foxp3+ T cells, which were similar to CD4+CD25+Foxp3- T cells, and expressed similar IL-17, ICOS and Helios to CD4+CD25+Foxp3+ T cells. The synthesis capacity of IL-10 of CD4+CD25-Foxp3+ T cells and the expression of GITR on CD4+CD25-Foxp3+ T cells were between CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- T cells. CONCLUSIONS: Our results indicate that increased CD4+CD25-Foxp3+ T cells in lupus patients, which combined the features of suppression and pro-inflammatory, may serve as a biomarker for disease activity and organ involvement in SLE.

Research on application value of combined detection of serum CA125, HE4 and TK1 in the diagnosis of ovarian cancer.

OBJECTIVE: To explore the diagnostic value of joint examination of cancer antigen 125 (CA125), thymidine kinase-1 (TK1) and human epididymis protein 4 (HE4) in the serum of patients with ovarian cancer. PATIENTS AND METHODS: A total of 75 ovarian cancer specimens (ovarian cancer group), 40 benign ovarian specimens (benign group) and 35 ovarian specimens of healthy women (normal control group) were collected. The serum levels of HE4, CA125 and TK1 and the positive detection rates in the three groups were compared. Meanwhile, the sensitivity and specificity of the three tumor markers in the diagnosis of ovarian cancer in the three groups were compared. RESULTS: The levels of HE4, CA125 and TK1 in the ovarian cancer group were significantly higher than those in the control group (p<0.05), and those in the ovarian cancer group were significantly higher than those in the benign group (p<0.05). The positive rates of CA125 as well as TK1 in the ovarian cancer group and the benign group were significantly higher than those in the control group (p<0.05), and those in the ovarian cancer group were significantly higher than those in the benign group (p<0.05). In the detection of an individual tumor marker, the sensitivity of CA125 was the highest, followed by HE4. The specificity of HE4 was the highest, followed by TK1. For the combination of two tumor markers, the sensitivity of CA125+HE4 ranked the first (92.18%), and the specificity of TK1+HE4 ranked the first (88.37%). The sensitivity and specificity of the joint detection of CA125+HE4+TK1 were 94.18% and 79.53%, respectively. The sensitivity of the joint detection of CA125+HE4+TK1 was significantly higher than that of the detection of a single tumor marker and that of joint detection of two tumor markers (p<0.05). CONCLUSIONS: Combined detection of CA125, HE4 and TK1 can significantly improve the sensitivity in the diagnosis of ovarian cancer.

Metabolic syndrome in menopause and associated factors: a meta-analysis.

OBJECTIVE: Metabolic syndrome (MetS) is a cluster of risk factors for cardiovascular disease and diabetes. Menopause is associated with an increased risk for MetS. The purpose of this meta-analysis is to better understand the relationship between MetS and menopause. METHODS: MEDLINE and EMBASE were searched for all the associated articles on (1) MetS components in postmenopausal women vs. premenopausal women, (2) comparison of MetS incidence between surgical menopause and natural menopause, (3) the effect of hormone therapy (HT) with 17ß-estradiol (E2) compared to conjugated equine estrogen (CEE) on MetS components among postmenopausal women. A meta-analysis was applied by Review Manager 5.3 software. RESULTS: All comparable indicators were significantly unfavorably changed in postmenopausal women compared to premenopausal women except for high density lipoprotein cholesterol. Women who underwent surgical menopause suffered a 1.51-fold higher risk for MetS compared to those with natural menopause. HT with E2 provided more benefits for levels of triglyceride and diastolic blood, while CEE showed a better effect on both high and low density lipoprotein cholesterol levels. CONCLUSIONS: Menopause nearly adversely affects all components of MetS, and surgical menopause may lead to a higher incidence of MetS compared to natural menopause. HT with various preparations may have different effects on MetS components. These results may clarify the management of menopause-related MetS in clinical practice.

TBP2 gene may not be associated with primary ovarian insufficiency.

OBJECTIVE: It has previously been reported that TATA-binding protein 2 (TBP2) knockout female mice are sterile. Our objective was to assess the association between the TBP2 gene and primary ovarian insufficiency (POI) in a Chinese population. METHODS: A cohort of 60 POI patients matched with 60 fertile controls was recruited by Nanjing Medical University. There were no interventions. The complete TBP2 exon regions were analyzed by direct sequencing in all POI patients and controls. RESULTS: A known single nucleotide polymorphism (rs8019270) was identified in both POI and control groups. There was no difference in the genotype distribution or allelic frequencies between the control group and the POI group. No plausible pathogenic mutations were identified. CONCLUSION: Our study indicates that the TBP2 gene is not responsible for idiopathic POI in the Chinese Han population.

Association analysis between HFM1 variation and primary ovarian insufficiency in Chinese women.

HFM1 is a meiosis-specific gene and expressed in germ-line tissues. More recently, evidence has indicated that variations in HFM1 gene could be causative for primary ovarian insufficiency (POI), also known as premature ovarian failure. The aim of this study was to investigate the association between HFM1 gene variants and sporadic POI in Chinese women. A total of 138 POI patients and 316 healthy controls (matched for ethnic background, sex, and age of the patients) were recruited in this study. We screened the entire HFM1 coding region by direct sequencing in all subjects and identified six variants of HFM1 gene in POI group, namely c.148G>A/p.Glu50Lys, c.1241A>C/p.His414Pro, c.2325C>A/p.Phe775Leu, c.3367T>C/p.Ser1123Pro, c.3580C>T/p.Arg1194Cys, and c.1686-1G>C. The variation rate of HFM1 in POI group is significantly higher than control group (p < 0.01). The p.His414Pro and p.Arg1194Cys were predicted to be probably damaging to the HFM1 protein function, while p.Glu50Lys, p.Phe775Leu and p.Ser1123Pro mutants might not have any deleterious effect on the structure or function of the protein by online predictors. Taken together, our data suggested that HFM1 gene might be associated with primary ovarian insufficiency in Chinese population.

Gene variation and premature ovarian failure: a meta-analysis.

OBJECTIVE: Premature ovarian failure (POF) is a complex, heterogeneous disorder that is influenced by multiple genetic components. This meta-analysis aimed to investigate the association between gene variants and susceptibility to POF. STUDY DESIGN: MEDLINE and CNKI were searched for studies published from inception (1950) to June 2014. Meta-analysis was performed when three or more studies reported genetic data on the same polymorphism or mutation. Additive and dominant models were analyzed using RevMan Version 5.1. RESULTS: The literature search yielded 575 articles, of which 59 studies on the association between POF and gene variants were identified for meta-analysis. Five genes were selected for analysis, including 10 common gene polymorphisms [BMP15 (-9C>G, 788insTCT and 852C>T), ESR1 (-351A>G and -397C>T), FMR1 CGG repeat, FSHR (919A>G and 2039A>G), INHA (-16C>T and -124A>G)] and two mutations (BMP15 538G>A and INHA 769G>A). BMP15 538G>A was found to be significantly more common in patients with POF compared with controls. No significant associations were found between the other variants of BMP15 and POF. With respect to ESR1, the accumulative results were not significant, although the findings of the individual studies were controversial. The incidence of FMR1 premutation was significantly higher in patients with POF compared with controls [odds ratio (OR) 9.2, 95% confidence interval (CI) 5.42-15.61; p<0.001] in the overall population, as well as in both Caucasian and Asian subgroups. Stratified analysis was applied for INHA 769G>A by ethnicity; a significant association with POF was only found in the Asian subgroup (allelic frequency: OR 8.89, 95% CI 2.1-5.52; p=0.004). No significant associations were found between the other variants of INHA and POF. CONCLUSIONS: BMP15 538A, FMR1 premutation and INHA 769A (in Asians alone) may indicate susceptibility to POF. Further well-designed studies and larger samples are required to confirm the association between gene variants and POF.

Isolation and identification of epithelial and stromal stem cells from eutopic endometrium of women with endometriosis.

OBJECTIVE: The recent characterization of possible stem/progenitor cells in the endometrium has shed new light on the origins of ectopic endometrial tissue and the mechanism for the pathogenesis of endometriosis, but has raised new questions. Is it possible that abnormal endometrial stem/progenitor cells increase their capacity to implant and establish themselves as ectopic tissue, or that normal stem cells implant in abnormal peritoneum? This study investigated key stem cell properties in cologenic epithelial and stromal cells obtained from eutopic endometrium of women with endometriosis. STUDY DESIGN: Single cell suspensions of endometrial epithelial and stromal cells were cultured at densities of 20, 50, 100 and 200cells/cm(2). Cloning efficiency (CE) was determined, and stem cell phenotypic surface markers were detected using Western blotting and quantitative real-time polymerase chain reaction. RESULTS: CE was significantly higher in cells cultured at a density of 50cells/cm(2) compared with the other groups. After 15 days of culture, small and large colonies were observed. Large-colony-derived epithelial and stromal cells had high proliferative potentials, producing millions of cells in vitro, with strong expression of epithelial and stromal stem cell phenotypic surface markers EMA, CK, CD49f, THY-1(CD90), collagen type I, 5B5 and vimentin. CONCLUSION: Adult stem cells were found in eutopic endometrium of women with endometriosis, and this may play an important role in disease development.

An economical and practical method for whole-mount in situ hybridization for mouse embryos and organs.

Whole-mount in situ hybridization (WISH) is a useful method for detecting specific gene expression patterns at their site of action during embryonic development. Traditional WISH methods are costly and suitable only for mouse embryos younger than 11.5 days. We present here an economical and practical in situ hybridization method using DIG-labeled RNA probes. We changed the conditions in several steps to make the WISH method suitable for whole mouse embryos from embryonic days 9.5 to 12.5 and for older stage mouse embryonic organs. We performed all steps in one microcentrifuge tube up to the staining steps to avoid losing or damaging the mouse embryos. We re-used the solutions and materials to make the method more economical and suitable for less sophisticated laboratories. We also performed ß-galactosidase staining on Tb × 18 Cre/Rosa26/LacZ mouse embryos; the results agreed with the in situ hybridization results. Finally, we sectioned the specimens after hybridization and ß-galactosidase staining; the results agreed with the literature.

Characterization of tetrathiofulvalene compounds using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Tetrathiofulvalene compounds are important components of charge-transfer complexes, which may be applied in various fields of scientific research and practical applications. Some of these compounds cannot be characterized by mass spectrometry. Here, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for the characterization of tetrathiofulvalenes. The samples could be easily desorbed and ionized to form singly charged ions, and mass spectra with isotopic resolution readily obtained. The mass spectrometric results for 26 compounds have shown that MALDI-TOF is more effective and convenient than other mass spectrometry methods, and resolves the problem of mass spectrometric characterization of tetrathiofulvalene compounds.

[Study on mutations in the connexin 26 gene among Chinese with nonsyndromic hearing loss].

OBJECTIVE: To study the relation between nonsyndromic hearing loss in Chinese and mutations in connexin 26 (Cx 26) gene and to explore the pathogenic mechanism. METHODS: One hundred and thirty-eight individuals from thirty-five pedigrees with nonsyndromic hearing loss, 99 children with sporadic nonsyndromic hearing loss and 100 normal adults as control were collected in present studies. The Cx 26 coding sequence was screened by single strand conformational polymorphism (SSCP) and analyzed by direct sequencing when SSCP shifts were observed. RESULTS: Five SSCP shifts in 2 pedigrees were observed. Homozygous deletion C at position 233-235 of Cx 26 cDNA, which resulted in frameshift mutation, was found in 2 pedigrees with nonsyndromic hearing loss. CONCLUSION: The hot-spot mutations of Cx 26 gene in Chinese with nonsyndromic hearing loss may be different from other ethnic groups. The 233-235 delC homozygous mutation of Cx 26 cDNA can result in autosomal recessive nonsyndromic hearing loss in Chinese population.

[Clinical analysis of 15 patients with primary malignant melanoma in the genital tract].

OBJECTIVE: To analyse the clinical characteristics and prognosis of primary malignant melanoma in female genital tract. METHODS: Clinical data of 15 patients with primary malignant genital melanoma were analysed retrospectively. The tumors occurred in the vulva (4 patients), in vagina (9) and in uterine cervix (2). 11 of them were treated by surgery combined with chemotherapy and immunotherapy, the remaining 4 by chemotherapy and immunotherapy only. RESULTS: All cases except one were followed up. The survival periods were shorter than 1 year in 5 patients, shorter than 2 years in 7. Two were alive for more than 3 years and 1 for more than 19 years. CONCLUSIONS: The prognosis of primary malignant melanoma is rather poor. Surgery combined of chemotherapy and immunotherapy is the main therapeutic approach.