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Featured researches published by Qing Sang.


The Journal of Clinical Endocrinology and Metabolism | 2013

Identification of MicroRNAs in Human Follicular Fluid: Characterization of MicroRNAs That Govern Steroidogenesis in Vitro and Are Associated With Polycystic Ovary Syndrome in Vivo

Qing Sang; Zhongyuan Yao; Huan Wang; Ruizhi Feng; Haojue Wang; Xinzhi Zhao; Qinghe Xing; Li Jin; Lin He; Lingqian Wu; Lei Wang

CONTEXT Human follicular fluid is a combination of proteins, metabolites, and ionic compounds that is indicative of the general state of follicular metabolism and is associated with maturation and quality of oocytes. Deviations in these components are often associated with reproductive diseases. There has been no report of microRNAs (miRNAs) in human follicular fluids. OBJECTIVE We hypothesized that human follicular fluid may contain miRNAs. We sought to identify cell-free miRNAs in human follicular fluid and to investigate the function of these miRNAs in vitro and any roles they play in polycystic ovary syndrome (PCOS). DESIGN Genome-wide deep sequencing and TaqMan miRNA arrays were used to identify miRNAs, and the roles of the highly expressed miRNAs in steroidogenesis were investigated in KGN cells. Quantification of candidate miRNAs in follicular fluids of PCOS and controls was performed using TaqMan miRNA assays. RESULTS We identified miRNAs in microvesicles and the supernatant of human follicular fluid. Bioinformatics analysis showed that the most highly expressed miRNAs targeted genes associated with reproductive, endocrine, and metabolic processes. We found that miR-132, miR-320, miR-520c-3p, miR-24, and miR-222 regulate estradiol concentrations and that miR-24, miR-193b, and miR-483-5p regulate progesterone concentrations. Finally, we showed that miR-132 and miR-320 are expressed at significantly lower levels in the follicular fluid of polycystic ovary patients than in healthy controls (P = .005 and P = .0098, respectively). CONCLUSION These results demonstrate that there are numerous miRNAs in human follicular fluids, some of which play important roles in steroidogenesis and PCOS. This study substantially revises our understanding of the content of human follicular fluid and lays the foundation for the future investigation of the role of miRNAs in PCOS.


The New England Journal of Medicine | 2016

Mutations in TUBB8 and Human Oocyte Meiotic Arrest

Ruizhi Feng; Qing Sang; Yanping Kuang; Xiaoxi Sun; Zheng Yan; Shaozhen Zhang; Juanzi Shi; Guoling Tian; Anna Luchniak; Yusuke Fukuda; Bin Li; Min Yu; Junling Chen; Yao Xu; Luo Guo; Ronggui Qu; Xueqian Wang; Zhaogui Sun; Miao Liu; Huijuan Shi; Hongyan Wang; Yi Feng; Ruijin Shao; Renjie Chai; Qiaoli Li; Qinghe Xing; Rui Zhang; Eva Nogales; Li Jin; Lin He

Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).


Scientific Reports | 2015

MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro

Ruizhi Feng; Qing Sang; Yan Zhu; Wei Fu; Miao Liu; Yan Xu; Huijuan Shi; Yao Xu; Ronggui Qu; Renjie Chai; Ruijin Shao; Li Jin; Lin He; Xiaoxi Sun; Lei Wang

Previous work from our laboratory demonstrated the existence of miRNAs in human follicular fluid. In the current study, we have sought to identify miRNAs that might affect oocyte/embryo quality in patients undergoing intracytoplasmic sperm injection and to investigate their roles in in vitro fertilization outcomes in mouse oocytes. 53 samples were classified as Group 1 (high quality) if the day-3 embryos had seven and more cells or as Group 2 (low quality) if the embryos had six and fewer cells. TaqMan Human microRNAs cards and qRT-PCR were performed to verify differently expressed miRNAs. The function of the corresponding miRNA was investigated in mouse oocytes by injecting them with miRNA-inhibitor oligonucleotides. We found that hsa-miR-320a and hsa-miR-197 had significantly higher expression levels in the Group 1 follicular fluids than in Group 2 (p = 0.0073 and p = 0.008, respectively). Knockdown of mmu-miR-320 in mouse oocytes strongly decreased the proportions of MII oocytes that developed into two-cell and blastocyst stage embryos (p = 0.0048 and p = 0.0069, respectively). Wnt signaling pathway components had abnormal expression level in miR-320 inhibitor-injected oocytes. This study provides the first evidence that miRNAs in human follicular fluid are indicative of and can influence embryo quality.


Molecular Human Reproduction | 2011

A common polymorphism in the human aromatase gene alters the risk for polycystic ovary syndrome and modifies aromatase activity in vitro

Hui Wang; Qiaoli Li; Teng Wang; Guizhong Yang; Ying Wang; Xiaoping Zhang; Qing Sang; Huan Wang; Xinzhi Zhao; Qinghe Xing; Juanzi Shi; Lin He; Lei Wang

Aromatase is a key enzyme involved in estradiol and estrone biosynthesis. Given that polymorphisms of the CYP19A1 gene encoding aromatase have been correlated with plasma testosterone levels, CYP19A1 may therefore act as a genetic modifier of the hyperandrogenic phenotype of polycystic ovary syndrome (PCOS). However, no functional CYP19A1 polymorphisms that predict the risk of PCOS have been identified. We explored the role of CYP19A1 genetic variation in a large case-control study involving 1078 samples, in which five common genetic polymorphisms were scored. Human embryonic kidney 293 cells were transiently transfected with a vector encoding either the CYP19A1 wild-type (WT) allele or an Arg(264)Cys variant to evaluate aromatase activity. Cells were cultured with androstenedione and estrone levels were measured using a specific ELISA. The Arg(264)Cys variant of CYP19A1 (rs700519) is associated with PCOS (P= 0.004, corrected P = 0.02). In this functional study, when cells were cultured in varying concentrations of androstenedione (100, 400 and 500 nM), transfection with the Arg(264)Cys variant resulted in increased conversion of androstenedione to estrogen when compared with transfection with the WT construct (P< 0.001). Our data suggest that the common missense polymorphism rs710059 is associated with susceptibility to PCOS and that the Arg(264)Cys variant may increase aromatase enzymatic activity. Overall, these findings imply that aromatase plays an important role in PCOS.


The Journal of Clinical Endocrinology and Metabolism | 2014

A Possible New Mechanism in the Pathophysiology of Polycystic Ovary Syndrome (PCOS): The Discovery That Leukocyte Telomere Length Is Strongly Associated With PCOS

Qiaoli Li; Jing Du; Ruizhi Feng; Yao Xu; Haojue Wang; Qing Sang; Qinghe Xing; Xinzhi Zhao; Li Jin; Lin He; Lei Wang

CONTEXT Telomeres are specialized chromatin structures located at the ends of eukaryotic chromosomes, and telomere length plays a clear role in various diseases. However, it is not known whether telomere length is related to polycystic ovary syndrome (PCOS). OBJECTIVE We hypothesized that leukocyte telomere length (LTL) plays an important role in the pathophysiology of PCOS. DESIGN We used an established and validated quantitative PCR technique to measure the mean LTL in a large sample of PCOS patients and controls. We used logistic regression and multiple linear regression to analyze the association of PCOS and several related clinical parameters with the age-adjusted ratio of the telomere repeat length to the copy number of a single-copy gene (T/S). RESULTS Individuals with PCOS (n = 698) exhibited significantly shorter LTLs than the controls (n = 611) after adjusting for age (0.764 ± 0.016 vs 0.876 ± 0.023; P = .001; odds ratio = 1.403; 95% confidence interval, 1.150-1.712). The mean telomere length in the leukocytes of PCOS patients was comparable to that of control individuals who were on average 6.16 years older. Individuals having shorter telomere lengths (middle and lowest tertile) had significantly higher disease risk than those having the longest telomere length (highest tertile) (odds ratio = 1.614; 95% confidence interval, 1.262-2.066; P = .0001) after adjusting for age. In addition, a significant correlation between the LTL and the level of dehydroepiandrosterone sulfate was observed in controls (r = -0.185; P = .01). CONCLUSION We provide the first report that LTL is strongly associated with PCOS. This study suggests a new role for LTL in the pathophysiology of PCOS and might have important implications for our understanding of the etiology of the disease.


Journal of Medical Genetics | 2016

Mutations in TUBB8 cause a multiplicity of phenotypes in human oocytes and early embryos

Ruizhi Feng; Zheng Yan; Bin Li; Min Yu; Qing Sang; Guoling Tian; Yao Xu; Biaobang Chen; Ronggui Qu; Zhaogui Sun; Xiaoxi Sun; Li Jin; Lin He; Yanping Kuang; Nicholas J. Cowan; Lei Wang

Background TUBB8 is a primate-specific β-tubulin isotype whose expression is confined to oocytes and the early embryo. We previously found that mutations in TUBB8 caused oocyte maturation arrest. The objective was to describe newly discovered mutations in TUBB8 and to characterise the accompanying spectrum of phenotypes and modes of inheritance. Methods and results Patients with oocyte maturation arrest were sequenced with respect to TUBB8. We investigated the effects of identified mutations in vitro, in cultured cells and in mouse oocytes. Seven heterozygous missense and two homozygous mutations were identified. These mutations cause a range of folding defects in vitro, different degrees of microtubule disruption upon expression in cultured cells and interfere to varying extents in the proper assembly of the meiotic spindle in mouse oocytes. Several of the newly discovered TUBB8 mutations result in phenotypic variability. For example, oocytes harbouring any of three missense mutations (I210V, T238M and N348S) could extrude the first polar body. Moreover, they could be fertilised, although the ensuing embryos became developmentally arrested. Surprisingly, oocytes from patients harbouring homozygous TUBB8 mutations that in either case preclude the expression of a functional TUBB8 polypeptide nonetheless contained identifiable spindles. Conclusions Our data substantially expand the range of dysfunctional oocyte phenotypes incurred by mutation in TUBB8, underscore the independent nature of human oocyte meiosis and differentiation, extend the class of genetic diseases known as the tubulinopathies and provide new criteria for the qualitative evaluation of meiosis II (MII) oocytes for in vitro fertilization (IVF).


PLOS ONE | 2014

Quantitative Methylation Level of the EPHX1 Promoter in Peripheral Blood DNA Is Associated with Polycystic Ovary Syndrome

Qing Sang; Xin Li; Haojue Wang; Huan Wang; Shaozhen Zhang; Ruizhi Feng; Yao Xu; Qiaoli Li; Xinzhi Zhao; Qinghe Xing; Li Jin; Lin He; Lei Wang

Steroid synthesis and metabolic pathways play important roles in the pathophysiology of PCOS, but until now there have been no studies on the methylation profiles of specific genes in steroid synthesis pathways that are known to be associated with PCOS. Here we used MassARRAY quantitative methylation analysis to determine the methylation levels of each CpG site or cluster in the promoters of EPHX1, SRD5A1, and CYP11A1 in 64 peripheral blood samples. We further examined the methylation level of EPHX1 in an independent cohort consisting of 116 people. Finally, we investigated the role of EPHX1 in steroidogenesis in the KGN cell line. For SRD5A1 and CYP11A1, there was no significant difference in methylation level between patients and controls. For EPHX1, however, the methylation levels of a few consecutive CpG sites and clusters were found to be significantly associated with PCOS. The methylation levels of a number of CpG clusters or sites were significantly lower in patients than in controls in the first cohort consisting of 64 people, such as clusters 13–14 (P<0.05), 15–16 (P<0.001), and 19–24 (P<0.001) and sites CpG_53 (P<0.01) and CpG_54 (P<0.05). Among differentiated methylation sites and clusters, the methylation levels of the CpG cluster 13–14 and CpG cluster 19–24 in PCOS patients were significantly lower than in controls in the second cohort of 116 people (P<0.05 for both). In addition, knockdown and overexpression experiments in KGN cells showed that EPHX1 can regulate estradiol concentrations, and this indicates a role for EPHX1 in steroidogenesis. Our study has demonstrated that methylation of the EPHX1 promoter might be associated with PCOS. This study provides direct evidence that methylation plays an important role in PCOS and demonstrates a novel role for EPHX1 in female reproduction.


Human Reproduction | 2017

Novel mutations and structural deletions in TUBB8: expanding mutational and phenotypic spectrum of patients with arrest in oocyte maturation, fertilization or early embryonic development.

Biaobang Chen; Bin Li; Da Li; Zheng Yan; Xiaoyan Mao; Yao Xu; Jian Mu; Qiaoli Li; Li Jin; Lin He; Yanping Kuang; Qing Sang; Lei Wang

STUDY QUESTION Are there any new type of mutations and novel phenotypes in patients with arrest in oocyte maturation, fertilization or early embryonic development having tubulin beta eight class VIII (TUBB8) mutations? SUMMARY ANSWER We identified new types of mutations in TUBB8 associated with maturation, fertilization and developmental arrest. WHAT IS KNOWN ALREADY We previously found heterozygous mutations and a homozygous frameshift/internal seven amino acid deletion in TUBB8 that are responsible for oocyte maturation arrest. STUDY DESIGN, SIZE, DURATION We recruited 10 new primary infertility patients from 9 families from December 2015 to May 2016, most of which exhibited failures in oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS Ten primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA samples from the affected individuals, their family members and healthy controls were extracted from peripheral blood. TUBB8 in the DNA samples were sequenced by Sanger sequencing. TUBB8 sequence was then aligned by CodonCode software to identify rare variants. ExAC database was used to search frequency of corresponding mutations. In silico analysis of mutations was used by Polyphen and PROVEAN. Phenotypes of oocytes and embryos were evaluated by light microscopy, polarization microscopy or immunolabeling. MAIN RESULTS AND THE ROLE OF CHANCE Besides several novel heterozygous missense mutations, we also identified other new types of genetic variants, including homozygous mutations and a de novo compound heterozygous mutation. We also found a patient with a homozygous deletion of the whole TUBB8 gene, which is the first reported case of a large structural variation in this gene. In addition, we found different mutations in TUBB8 that could result in variability in oocyte/embryo phenotypes, including oocyte maturation arrest, first polar body (PB1) oocytes that cannot be fertilized, and PB1 oocytes that can be fertilized but arrest at an early embryonic stage. LIMITATIONS, REASONS FOR CAUTION The exact molecular mechanism has not been analyzed and should be further investigated in the future. In addition, immunostaining of more oocytes with mutations and checking spindle status of oocytes with mutations non-invasively by polarization microscopy needs to be done in order to determine exact stage of PB1 oocytes and the functional differences of these mutations. WIDER IMPLICATIONS OF THE FINDINGS The results not only emphasize the important role of TUBB8 in oocyte maturation, fertilization and early embryonic development but they also provide a basis for determining the genetic variations in TUBB8 as a potential additional criterion for evaluating the quality of patients’ functional PB1 oocytes. STUDY FUNDING/COMPETING INTEREST(S) National Key R&D Program of China (2016YFC1000600); Basic Research Program of China (2015CB943300); National Natural Science Foundation of China (81270747 and 81571501). No competing interests declared.


PLOS ONE | 2013

Identification and Functional Study of a New Missense Mutation in the Motor Head Domain of Myosin VIIA in a Family with Autosomal Dominant Hearing Impairment (DFNA11)

Qing Sang; Xukun Yan; Huan Wang; Ruizhi Feng; Xiang Fei; Duan Ma; Qinghe Xing; Qiaoli Li; Xinzhi Zhao; Li Jin; Lin He; Huawei Li; Lei Wang

The MYO7A encodes a protein classified as an unconventional myosin. Here, we present a family with non-syndromic autosomal dominant hearing impairment that clinically resembles other previously published DFNA11 families. Affected members of the family present with an ascending audiogram affecting low and middle frequencies at young ages and then affecting all frequencies with increasing age. Genome-wide linkage analysis using Illumina Cyto-12 Chip mapped the disease locus to the DFNA11 interval in the family. A c.2003G→A (p.R668H) mutation of the MYO7A, is heterozygous in all affected family members and absent in 100 healthy individuals. Arg668His is located in a region of the myosin VIIA motor domain that is highly conserved among different species. Molecular modeling predicts that the conserved R668 residue plays important structural role in linking different lobes of motor domain together. In the actin-activated ATPase activity assay, the rate of NADH oxidation was higher in the wild-type myosin VIIA, indicating that the ATPase activity in the p.R668H mutant myosin VIIA was significantly destroyed.


Human Molecular Genetics | 2014

Ildr1b is essential for semicircular canal development, migration of the posterior lateral line primordium, and hearing ability in zebrafish: Implications for a role in the recessive hearing impairment DFNB42

Qing Sang; Junyu Zhang; Ruizhi Feng; Xu Wang; Qiaoli Li; Xinzhi Zhao; Qinghe Xing; Weiyu Chen; Jiu-lin Du; Shan Sun; Renjie Chai; Dong Liu; Li Jin; Lin He; Huawei Li; Lei Wang

Immunoglobulin-like domain containing receptor 1 (ILDR1) is a poorly characterized gene that was first identified in lymphoma cells. Recently, ILDR1 has been found to be responsible for autosomal recessive hearing impairment DFNB42. Patients with ILDR1 mutations cause bilateral non-progressive moderate-to-profound sensorineural hearing impairment. However, the etiology and mechanism of ILDR1-related hearing loss remains to be elucidated. In order to uncover the pathology of DFNB42 deafness, we used the morpholino injection technique to establish an ildr1b-morphant zebrafish model. Ildr1b-morphant zebrafish displayed defective hearing and imbalanced swimming, and developmental delays were seen in the semicircular canals of the inner ear. The gene expression profile and real-time PCR revealed down-regulation of atp1b2b (encoding Na(+)/K(+) transporting, beta 2b polypeptide) in ildr1b-morphant zebrafish. We found that injection of atp1b2b mRNA into ildr1b-knockdown zebrafish could rescue the phenotype of developmental delay of the semicircular canals. Moreover, ildr1b-morphant zebrafish had reduced numbers of lateral line neuromasts due to the disruption of lateral line primordium migration. In situ hybridization showed the involvement of attenuated FGF signaling and the chemokine receptor 4b (cxcr4b) and chemokine receptor 7b (cxcr7b) in posterior lateral line primordium of ildr1b-morphant zebrafish. We concluded that Ildr1b is crucial for the development of the inner ear and the lateral line system. This study provides the first evidence for the mechanism of Ildr1b on hearing in vivo and sheds light on the pathology of DFNB42.

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Lin He

Shanghai Jiao Tong University

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Bin Li

Shanghai Jiao Tong University

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Yanping Kuang

Shanghai Jiao Tong University

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Zheng Yan

Shanghai Jiao Tong University

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