Yingpu Sun

Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3, 2p21 and 9q33.3

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 Pmeta = 7.55 × 10−21, odds ratio (OR) 0.71); 2p21 (rs13429458, Pmeta = 1.73 × 10−23, OR 0.67); and 9q33.3 (rs2479106, Pmeta = 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.

Genotype–phenotype correlations of PCOS susceptibility SNPs identified by GWAS in a large cohort of Han Chinese women

STUDY QUESTION Are there any correlations between the phenotypes of polycystic ovary syndrome (PCOS) and the genotypes of the PCOS susceptibility single nucleotide polymorphisms (SNPs) in THADA, DENND1A and LHCGR? SUMMARY ANSWER The PCOS susceptibility genes, THADA and DENND1A, carry risk alleles that are associated with endocrine and metabolic disturbances in patients with PCOS. WHAT IS KNOWN ALREADY PCOS is a heterogeneous endocrinopathy characterized by oligo-anovulation, hyperandrogenism and polycystic ovaries. In a previous genome-wide association study, the SNP variants rs13429458, rs12478601, rs2479106, rs10818854 and rs13405728 in the THADA, DENND1A and LHCGR genes were identified as being independently associated with PCOS. The aim of this study was to identify any additional correlations between the phenotypes of PCOS and genotypes of the five SNPs described in the previous study. STUDY DESIGN, SIZE, DURATION In the present cross-sectional study, a total of 1731 PCOS patients and 4964 controls were enrolled. PARTICIPANTS/MATERIALS, SETTING, METHODS Patients were diagnosed according to Rotterdam criteria. Clinical information was collected from the patients and controls. Endocrine and metabolic parameters were evaluated for phenotype-genotype correlation analyses. MAIN RESULTS AND THE ROLE OF CHANCE Using a recessive model, the AA group for rs13429458 in THADA was associated with increased luteinizing hormone (LH) (P < 0.01) and testosterone (T) (P = 0.02) levels in subjects with PCOS; the LH/follicle-stimulating hormone ratio was also higher in the AA group (P < 0.01). Also using a recessive model, the CC genotype of rs12478601, also in THADA, was associated with increased levels of low-density lipoprotein (P = 0.02). Using a dominant model, the GG + AG group for rs2479106 in DENND1A was associated with elevated serum insulin levels 2 h after a glucose load in the patients with PCOS (P = 0.02). All of the comparisons were adjusted for age and BMI. LIMITATIONS, REASONS FOR CAUTION The relatively younger age of the participants may represent a considerable bias when evaluating metabolic alterations as a function of different genotypes, as significant metabolic disturbances may emerge later in life. Furthermore, the sample sizes of several sub-genotype groups were relatively small; to some extent this limited the statistical power of the analysis. WIDER IMPLICATIONS OF THE FINDINGS The PCOS susceptibility genes, THADA and DENND1A, carry risk alleles that are associated with endocrine and metabolic disturbances in PCOS patients of Han Chinese descent. The findings have shown genuine heterogeneity, stratified on the basis of both clinical findings and genotypes. Replication of these results is expected in other ethnic groups.

Birth defects after assisted reproductive technologies in China: analysis of 15,405 offspring in seven centers (2004 to 2008)

This first large-scale report of birth defects in 15,405 offspring conceived by assisted reproductive technologies in China found infants born after IVF alone to have a birth defect frequency comparable to that in the general Chinese population; rates were nonsignificantly higher in infants conceived with use of intracytoplasmic sperm injection compared with those conceived after IVF alone.

Sperm telomere length is positively associated with the quality of early embryonic development

STUDY QUESTION What is the relationship between telomere length in sperm and early embryonic development in in vitro fertilization (IVF)? SUMMARY ANSWER Sperm telomere length (STL) is positively associated with embryo quality in IVF. WHAT IS KNOWN ALREADY Previous studies have shown that STL differs among human males. STUDY DESIGN, SIZE, DURATION In order to determine the associations between STL, fertilization laboratory parameters and clinical pregnancy in IVF, 418 couples were recruited from August 2013 to August 2014. MATERIALS, SETTING, METHODS We collected semen samples and used quantitative PCR technique to detect the mean STL for each patient. These data were compared with the IVF outcomes. MAIN RESULTS AND THE ROLE OF CHANCE The mean STL was positively correlated with the age of patient (rP = 0.100; P = 0.041) and total sperm count/ejaculate (rp = 0.28; P < 0.001). Analysis of the age-adjusted mean STL in relation to the male patients paternal and maternal ages at the time of his conception showed significant positive relationships between STL and both paternal (r = 0.16; P = 0.003) and maternal (r = 0.19; P < 0.001) ages at the time of conception. In addition, significant correlations were found between STL and good quality embryo (regression coefficient: 1.63; P < 0.001) and transplantable embryo rates (regression coefficient: 1.57; P < 0.001), but clinical pregnancy rates were not affected (odds ratio = 1.00 [95% CI: 0.93-1.07]; P = 0.90). LIMITATIONS, REASONS FOR CAUTION This study showed that STL was positively associated with embryo quality in IVF. Additional studies are needed to confirm these observations. WIDER IMPLICATION OF THE FINDINGS STL has the potential to be used as a marker for the prediction of embryonic quality. STUDY FUNDING/COMPETING INTERESTS This work was supported by the National Natural Science Foundation of China (Grants 31271605 and 31471404), and the National Science Foundation for Young Scientists of China (Grant 31401274), and Science Foundation of First Affiliated Hospital of Zhengzhou University for Yong Scientists. The authors have declared that no competing interests exist.

TGF-β1 Up-Regulates Connective Tissue Growth Factor Expression in Human Granulosa Cells through Smad and ERK1/2 Signaling Pathways

Connective tissue growth factor (CTGF), which is also called CCN2, is a secreted matricellular protein. CTGF regulates various important cellular functions by interacting with multiple molecules in the microenvironment. In the ovary, CTGF is mainly expressed in granulosa cells and involved in the regulation of follicular development, ovulation and luteinization. TGF-β1 has been shown to up-regulate CTGF expression in rat and hen granulosa cells. However, the underlying molecular mechanisms of this up-regulation remain undefined. More importantly, whether the stimulatory effect of TGF-β1 on CTGF expression can be observed in human granulosa cells remains unknown. In the present study, our results demonstrated that TGF-β1 treatment up-regulates CTGF expression in both immortalized human granulosa cells and primary human granulosa cells. Using a siRNA-mediated knockdown approach and a pharmacological inhibitor, we demonstrated that the inhibition of Smad2, Smad3 or ERK1/2 attenuates the TGF-β1-induced up-regulation of CTGF. This study provides important insights into the molecular mechanisms that mediate TGF-β1-up-regulated CTGF expression in human granulosa cells.

Effects of growth differentiation factor 8 on steroidogenesis in human granulosa-lutein cells

OBJECTIVE To investigate the biological role of growth differentiation factor 8 (GDF8) in the regulation of steroidogenesis in human granulosa-lutein (hGL) cells. DESIGN Experimental study. SETTING Academic medical center. PATIENT(S) In vitro fertilization patients who provided hGL cells. INTERVENTION(S) Cultured hGL cells treated with recombinant human GDF8 for 24 hours. MAIN OUTCOME MEASURE(S) Expression of steroidogenic enzymes and steroid production in primary cultures of hGL cells used to investigate the effects of GDF8 via specific mRNA and protein levels examined using real time-quantitative polymerase chain reaction and Western blot analysis, respectively, and levels of estradiol and progesterone measured by enzyme immunoassays. RESULT(S) Extracts were prepared from cultured hGL cells after exposure to GDF8. The levels of cytochrome P450 aromatase (aromatase), the FSH receptor, and estradiol were increased, whereas steroidogenic acute regulatory protein (StAR), luteinizing hormone (LH) receptor, and progesterone levels were decreased after treatment with GDF8. In addition, follicle-stimulating hormone (FSH) stimulated the production of aromatase/estradiol, and LH induced the production of StAR/progesterone. Furthermore, pretreatment with GDF8 for 24 hours enhanced the effects of FSH on aromatase/estradiol induction, whereas GDF8 suppressed the effects of LH on StAR/progesterone stimulation. CONCLUSION(S) In human granulosa cells, GDF8 may play an important role in the modulation of cellular responsiveness to gonadotropins and in the regulation of ovarian steroid production, most likely as a luteinization inhibitor.

Risk factors for ectopic pregnancy in assisted reproductive technology: a 6-year, single-center study

OBJECTIVE To explore factors affecting the incidence of ectopic pregnancy (EP) in assisted reproductive technology (ART). DESIGN A retrospective cohort study on the incidence of EPs in IVF/intracytoplasmic sperm injection (ICSI) and IUI cycles from June 2009 to August 2015. Age of patients, tubal factor infertility, type of cycle (fresh or thawed), embryo being transferred (cleavage embryo or blastocyst), and number of embryos transferred were analyzed to explore their relationship with the incidence of EP. SETTING Teaching hospital. PATIENT(S) A total of 18,432 pregnancies resulting from ART treatment were retrospectively analyzed. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Ectopic pregnancy rate. RESULT(S) For IVF/ICSI cycles, the incidence of EP was different between cycles transferred with cleavage embryo and blastocyst (3.45% vs. 2.47%). In multivariate logistic regression analysis, tubal infertility was associated with EP (adjusted odds ratio 1.716, 95% confidence interval 1.444-2.039). For IUI cycles, EP was significantly higher in stimulated cycles compared with natural cycles (2.62% vs. 0.99%). The EP rate in cycles with sperm from donor and husband was 1.08% and 3.54%, respectively. However, when patients were stratified according to tubal infertility, the EP rate increased with level of peak estrogen. In thawed embryo transfer cycles, the EP rate was lower in blastocyst transfer cycles and in cycles transferred with fewer embryos. CONCLUSION(S) Irrespective of tubal infertility, for fresh IVF/ICSI cycles the rate of EP is positively associated with ovarian stimulation; for thawed IVF/ICSI cycles, blastocyst transfer or transfer with fewer embryos reduces the EP rate. In IUI cycles, EP is associated with sperm source.

TGF‐β1 up‐regulates connexin43 expression: A potential mechanism for human trophoblast cell differentiation

Connexin43 (Cx43)‐mediated gap junctional intercellular communication (GJIC) are required for human trophoblast differentiation. To date, whether Cx43 mediates TGF‐β1‐induced trophoblast differentiation has not been determined. We showed that treatment with TGF‐β1 increased Cx43 expression and GJIC in HTR‐8/SVneo human trophoblast cells. In addition, Smad and ERK1/2 signaling pathways were involved in TGF‐β1‐induced up‐regulation of Cx43. Moreover, TGF‐β1 increased the expression of the syncytiotrophoblast marker, β‐hCG. Importantly, knockdown of Cx43 abolished the TGF‐β1‐induced up‐regulation of β‐hCG. Furthermore, overexpression of Cx43 up‐regulated β‐hCG expression. These results provide evidence that Cx43 and GJIC activity are up‐regulated by TGF‐β1 in human trophoblast cells, which subsequently contributes to TGF‐β1‐induced trophoblast differentiation. J. Cell. Physiol. 230: 1558–1566, 2015.

Live birth sex ratio after in vitro fertilization and embryo transfer in China--an analysis of 121,247 babies from 18 centers.

In order to study the impact of procedures of IVF/ICSI technology on sex ratio in China, we conducted this multi-center retrospective study including 121,247 babies born to 93,895 women in China. There were 62,700 male babies and 58,477 female babies, making the sex ratio being 51.8% (Male: Female  = 107∶100). In univariate logistic regression analysis, sex ratio was imbalance toward females of 50.3% when ICSI was preformed compared to 47.7% when IVF was used (P<0.01). The sex ratio in IVF/ICSI babies was significantly higher toward males in transfers of blastocyst (54.9%) and thawed embryo (52.4%) when compared with transfers of cleavage stage embryo (51.4%) and fresh embryo (51.5%), respectively. Multiple delivery was not associated with sex ratio. However, in multivariable logistic regression analysis after controlling for related factors, only ICSI (adjusted OR = 0.90, 95%CI: 0.88–0.93; P<0.01) and blastocyst transfer (adjusted OR = 1.14, 95% CI: 1.09–1.20; P<0.01) were associated with sex ratio in IVF/ICSI babies. In conclusion, the live birth sex ratio in IVF/ICSI babies was influenced by the use of ICSI, which may decrease the percentage of male offspring, or the use of blastocyst transfer, which may increase the percentage of male offspring.

Amphiregulin mediates hCG-induced StAR expression and progesterone production in human granulosa cells.

Progesterone plays critical roles in maintaining a successful pregnancy at the early embryonic stage. Human chorionic gonadotropin (hCG) rapidly induces amphiregulin (AREG) expression. However, it remains unknown whether AREG mediates hCG-induced progesterone production. Thus, the objective of this study was to investigate the role of AREG in hCG-induced progesterone production and the underlying molecular mechanism in human granulosa cells; primary cells were used as the experimental model. We demonstrated that the inhibition of EGFR and the knockdown of AREG abolished hCG-induced steroidogenic acute regulatory protein (StAR) expression and progesterone production. Importantly, follicular fluid AREG levels were positively correlated with progesterone levels in the follicular fluid and serum. Treatment with AREG increased StAR expression and progesterone production, and these stimulatory effects were abolished by EGFR inhibition. Moreover, activation of ERK1/2, but not PI3K/Akt, signaling was required for the AREG-induced up-regulation of StAR expression and progesterone production. Our results demonstrate that AREG mediates hCG-induced StAR expression and progesterone production in human granulosa cells, providing novel evidence for the role of AREG in the regulation of steroidogenesis.