Androgen Excess Increases Food Intake in a Rat Polycystic Ovary Syndrome Model by Downregulating Hypothalamus Insulin and Leptin Signaling Pathways Preceding Weight Gain.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common reproductive and metabolic disorder characterized by high androgen levels. The aim of this study was to evaluate the effects of hyperandrogenism on the hypothalamus and subsequently on the food intake and obesity in females. METHODS: A dihydroxy testosterone (DHT)-induced rat model was established to recapitulate the hyperandrogenism features of PCOS patients. Body weight and food intake of the rats were recorded. The food intake of DHT-induced rats was restricted by pair feeding to exclude possible effects of weight gain on the hypothalamus. The expression levels of relevant proteins and mRNAs in the hypothalamus and primary hypothalamic neurons exposed to DHT were analyzed by Western blotting and RT-PCR, respectively. The leptin levels in the serum and cerebrospinal fluid (CSF) were measured, and leptin was injected via the intracerebroventricular (ICV) route to test the leptin sensitivity of the hypothalamus. RESULTS: The excessive prepuberty androgen levels in the DHT-induced rats markedly elevated food intake prior to weight gain. Consistent with this, the expression of neuropeptide Y and agouti-related peptide mRNAs was upregulated, which occurred prior to obesity and even with restricted food intake. In addition, the hypothalamic sensitivity to insulin and leptin was also impaired in the DHT-induced rats before obesity and with restricted food intake. DHT significantly reduced the leptin levels in the CSF, and ICV injection of leptin inhibited the DHT-induced increase in food intake. CONCLUSIONS: Androgen excess increased food intake in rats and promoted obesity by downregulating insulin and leptin signaling in the hypothalamus, most likely by suppressing leptin levels in the CSF.

Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, we detected kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly downregulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Collectively, the present study has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and has provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent.

[Peroxiredoxin 4 protects the testis from heat stress].

OBJECTIVE: To explore the protective effect of Peroxiredoxin 4 (PRDX4) on the testes undergoing heat stress in PRDX4 knockout mice. METHODS: Twenty-four C57BL/6 mice underwent CRISPR/Cas9-mediated total knockout of the PRDX4 gene and another 24 wild-type mice were used as controls. At 9 weeks of age, the rats were subjected to 15-minute testicular heat stress in 43℃ water once a day for 3 days, or in 25℃ water as the control. Before and at 1 day and 5 weeks after treatment, 4 from each group were sacrificed respectively and their testes harvested for observation of histological changes by HE staining, detection of the apoptosis of spermatogenic cells by TUNEL and determination of the expression of PRDX4 by Western blot and those of the oxidative stress factors hydroxynonenal (HNE) and 8-OHdG by immunohistochemistry. RESULTS: No statistically significant differences were observed in testicular histology, the apoptosis rate of spermatogenic cells, and the expressions of HNE and 8-OHdG between the PRDX4 knockout mice and wild-type controls (P > 0.05). After 1-day 43℃ heat stress, the PRDX4 knockout mice showed a significantly increased apoptosis rate of spermatogenic cells as compared with the baseline (ï¼»38.65 ± 2.57ï¼½% vs ï¼»0.46 ± 0.06ï¼½%, P < 0.01), and so did the wild-type controls (ï¼»13.21 ± 1.43ï¼½% vs ï¼»0.33 ± 0.01ï¼½%, P < 0.01), higher in the PRDX4 knockout than in the wild-type control group even at 5 weeks after heat stress (ï¼»3.09 ± 0.16ï¼½% vs ï¼»1.45 ± 0.11ï¼½%, P < 0.01). The PRDX4 knockout mice also exhibited a markedly upregulated expression of 8-OHdG (38.25 ± 1.19 vs 19.54 ± 1.13, P < 0.01), and so did the wild-type controls (24.30 ± 1.65 vs 18.22 ± 1.18, P < 0.01), higher in the PRDX4 knockout than in the wild-type control group even at 5 weeks after heat stress (25.40 ± 1.57 vs 23.25 ± 1.48, P < 0.01). The expression of HNE, however, showed no statistically significant difference before and at 1 day after 43℃ heat stress either in the PRDX4 knockout mice or in the wild-type controls (P > 0.05), though remarkably higher in the former than in the latter group at 5 weeks after treatment (28.57 ± 0.56 vs 19.00 ± 1.35, P < 0.01). The expression of 8-OHdG was also higher in the PRDX4 knockout than in the wild-type control group at 5 weeks, but with no statistically significant difference (P > 0.05). CONCLUSIONS: PRDX4 can effectively protect the testis from heat stress and promote the restoration of its spermatogenic function.

[High-level androgen enhances the differentiation of hESCs into male primordial germ cells].

OBJECTIVE: To observe the effects of different concentrations of testosterone on the differentiation of human embryonic stem cells (hESCs) into early male germ cells and investigate the potential impact of high-level androgen exposure in early pregnancy in women with polycystic ovary syndrome (PCOS) on the fertility and primordial germ cell reserve of the male offspring in adulthood. METHODS: We used 2 µmol/L retinoic acid to induce the differentiation of hESCs (46, XY) into male germ cells in vitro and meanwhile treated them with testosterone (T) at 0 mol/L, 3×10－7 mol/L, 5×10－7 mol/L, 15×10－7 mol/L, 45×10－7 mol/L, and 135×10－7 mol/L, respectively. We collected the cell samples at 0, 4, 7 and 14 days to determine the expressions of the specific genes and compare the differentiation process and efficiency of the male germ cells in different stages. RESULTS: There was no difference in the morphology of the hESCs treated with different concentrations of testosterone in the same differentiation stage. The expression of the marker gene DAZL in the primordial germ cells peaked on the 4th day of differentiation, significantly higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.05), and that of the specific gene SCP3 in the early-meiosis germ cells began to increase on the same day, more significantly in the 45×10－7mol/L than in the 3×10－7 mol/L and 5×10－7 mol/L groups (P < 0.01), and peaked on the 7th day, dramatically higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.01). Immunofluorescence staining and flow cytometry showed a T concentration-dependent increase in the expression of DAZL at 4 days and those of SCP3 and VASA at 7 days. Moreover, the expression of the androgen receptor (AR) in the hESCs began to rise on the 4th day and kept going up till the 14th day, higher in the high-concentration than in the low-concentration T groups in the same stage of differentiation, though with no statistically significant difference (P > 0.05). CONCLUSIONS: Exposure to high-level androgen during the differentiation of hESCs into early male germ cells can induce earlier expression of AR and earlier differentiation of hESCs into early male germ cells, which may result in insufficient reserve of male primary germ cells in the male offspring of PCOS women and affect their fertility after adulthood. hESCs can be used as an in vitro model to study the effects of intrauterine hyperandrogen on the reproductive development of male offspring in PCOS patients, which is also contributive to researches on the etiology of male infertility.

Peroxiredoxin 4, a new oxidative stress marker in follicular fluid, may predict in vitro fertilization and embryo transfer outcomes.

BACKGROUND: Peroxiredoxin 4 (Prdx4), a member of the Prdx family, can catalyze the reduction of reactive oxygen species. This study aims to explore whether Prdx4 can serve as an effective marker in follicular fluid (FF) for predicting in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycle outcomes. METHODS: In this prospective study, all participants were recruited from the center of clinical reproductive medicine from 2017 September to 2018 December. Women with tubal or male factor infertility undergoing their first IVF/ICSI cycle were recruited (n=138). FF samples from each patient were collected on the day of oocyte retrieval. Prdx4 concentrations were measured, and the correlation between Prdx4 levels and IVF outcomes was analyzed. RESULTS: The results showed that pregnant women had higher levels of Prdx4 than nonpregnant women. Prdx4 was positively correlated with the oocyte fertilization rate (r =0.334; P=0.011) and good quality embryo rate (r =0.326; P=0.013). Furthermore, we found that the clinical pregnancy rate was positively correlated with Prdx4 levels in a concentration-dependent manner in the Prdx4 quartiles (<13.38, 13.83-16.93, 16.93-22.93, >22.93 ng/mL). The fertilization rates, clinical pregnancy rates and live pregnancy rates were all significantly higher in the highest Prdx4 quartile group than in the lowest quartile. Moreover, the results indicated that Prdx4 had an area under the receiver operating characteristic curve (AUC) of 0.754, corresponding to an optimal cutoff point of 22.30 ng/mL. CONCLUSIONS: Our results provide evidence that higher expression of antioxidants, such as Prdx4, in the FF of IVF patients tends to indicate a higher likelihood of pregnancy through an oocyte quality mechanism.

Hypoxia-induced and HIF1α-VEGF-mediated tight junction dysfunction in choriocarcinoma cells: Implications for preeclampsia.

INTRODUCTION: Accumulated data indicate that placental hypoxia is implicated in the pathogenesis of preeclampsia (PE). Tight junction (TJ) is important structure that sustains normal placental barrier function, its dysregulation under hypoxia has been observed. This study was designed to explore hypoxia-induced TJ dysfunction in trophoblast cells and its possible involvement in PE pathophysiology. METHODS: Choriocarcinoma cells were grown in a monolayer and treated with cobalt chloride (CoCl2) to induce hypoxia. TJ architecture was assessed using transmission electron microscopy, and locations of TJ proteins were determined by immunofluorescence. TJ functions were assessed by transepithelial electrical resistance (TER) and increased cell paracellular permeability (CPP), and the expression of TJ-related proteins, HIF-1α and VEGF was measured. RESULTS: The TJ functions of trophoblast cells were significantly altered by hypoxia; TER decreased and CPP increased in a time- and concentration-dependent manner. Significant alterations in TJ protein expression and increases in HIF1α and VEGF expression were observed in hypoxic cells, and these effects were attenuated by pretreatment with YC-1. Moreover, corresponding changes in TJ protein expression were also detected in preeclamptic placentas. CONCLUSION: These data demonstrate that trophoblast cells undergo significant changes in TJ protein expression under hypoxic conditions and highlight the potential significance of the HIF1α-VEGF axis in the regulation of TJ structure and function in the preeclamptic placenta.

Serum levels of trace elements in children born after assisted reproductive technology.

INTRODUCTION: The health and development of newborn children born via assisted reproductive technology (ART), as well as their health in adulthood, have raised great concern. This study was designed to investigate whether ART children have differences in the levels of trace elements compared with naturally conceived children. METHODS: This study included those ART children and controls aged 1 to 12 years assessed with a follow-up protocol. Serum levels of the trace elements zinc, copper, iron, calcium, magnesium and lead were determined and analyzed. RESULTS: There were no significant differences in age, gender or body weight between the ART and control groups. There were no significant differences in the rates of deficiency or excess of trace elements between the two groups. Serum lead levels in children born via ART were significantly higher than those in the controls, whereas the levels of zinc and iron were significantly decreased in the ART group, although these levels were still within the normal ranges. DISCUSSION: These results indicate the need to monitor the blood levels of zinc, iron and lead in ART children aged 1-6 years old. These findings contribute to our understanding on the long-term safety of ART and may facilitate screening for potential diseases related to trace elements.

[Preimplantation genetic diagnosis for infertile males with autosomal dominant polycystic kidney disease].

OBJECTIVE: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic renal diseases, which may cause oligoasthenospermia and azoospermia and result in male infertility. This study aimed to analyze the outcomes of preimplantation genetic diagnosis (PGD) in male patients with ADPKD-induced infertility. METHODS: We retrospectively analyzed the clinical data on 7 male patients with ADPKD-induced infertility undergoing PGD from April 2015 to February 2017, including 6 cases of oligoasthenospermia and 1 case of obstructive azoospermia, all with the PKD1 gene heterozygous mutations. Following intracytoplasmic sperm injection (ICSI), we performed blastomere biopsy after 5 or 6 days of embryo culture and subjected the blastomeres to Sureplex whole-genome amplification, followed by haplotype linkage analysis, Sanger sequencing, array-based comparative genomic hybridization to assess the chromosomal ploidy of the unaffected embryos, and identification of the unaffected euploid embryos for transfer. RESULTS: One PGD cycle was completed for each of the 7 patients. Totally, 26 blastocysts were developed, of which 12 were unaffected and diploid. Clinical pregnancies were achieved in 6 cases following 7 cycles of frozen embryo transplantation, which included 5 live births and 1 spontaneous abortion. CONCLUSIONS: For males with ADPKD-induced infertility, PGD may contribute to high rates of clinical pregnancy and live birth and prevent ADPKD in the offspring as well. This finding is also meaningful for the ADPKD patients with normal fertility.

[Differentiation of embryonic stem cells from the embryos of the couples with male asthenozoospermia and Robertsonian translocation into germ cells].

OBJECTIVE: To assess the risk of male infertility in the offspring conceived through assisted reproductive technology (ART) byin vitroinductionof the differentiation of embryonic stem cells (ESCs) derived from the embryos of the couples with male asthenozoospermia and Robertsonian translocation (RT) into germ cells. METHODS: We established a CCRM16ESC line with the karyotype of 46, XY, +14, rob(13; 14) (q10; q10) from the embryo donated by a patientwithasthenozoospermiaand RT and his wife by isolation of the inner cell mass of blastula, culturing, passaging, and amplification,followed by in vitro induction and differentiationof the ESCs into germ cells with ratinoic acid(RA) at 2 mol/L. Then, we analyzed the process of differentiation and the expressions of its related genes and compared them with those in the normal CCRM23ESCs. RESULTS: CCRM16 showed the typical characteristics of ESCs, expressing the pluripotency makers of NANOG, OCT4, TRA-1-181 and SSEA4, forming embryoid bodies, and differentiating into three germlayer tissues in vitro and in vivo. Intervention with 2 mol/LRAinduced direct differentiation of the ESCs into germ cells. The expressions of the primordial germ cell marker geneDAZLand the meiosis marker geneSCP3were markedly decreased in the CCRM16 as compared with those in the normal CCRM23 ESCs. CONCLUSIONS: The CCRM16ESC linewith the karyotype of46, XY, +14, rob(13; 14) (q10; q10) has thetypical characteristics of ESCs but an abnormal process of differentiation into germ cells in the early stage. In vitroinductionof the differentiation of ESCs into germ cells can be used for assessing the risk of male infertility in the offspring conceived through ART for asthenozoospermia patients.

Low dose clomiphene citrate as a mild stimulation protocol in women with unsuspected poor in vitro fertilization result can generate more oocytes with optimal cumulative pregnancy rate.

BACKGROUND: The use of long protocol during controlled ovarian stimulation for assisted reproduction attracts high dosage of gonadotropins. High dose of gonadotropins can be detrimental to oocyte development, which affects its quality and compromises the treatment outcome. Mild stimulation protocols that attract low dose gonadotropins could be useful alternative regimen to address such problems. This study evaluated the efficacy of low dose clomiphene citrate based protocol plus low dose gonadotropins on predicted normal responder patients who had unsuspected poor in vitro fertilization (IVF) result, following an initial stimulation with long gonadotropin-releasing hormone (GnRH) agonist protocol. METHODS: This a retrospective study of 65 infertile women who underwent 130 cycles in our center from January 2011 to December 2014. The initial IVF cycle (Group 1) was treated with long GnRH-a protocol plus a high dose of gonadotropins (≥150 IU/d), while second IVF cycle (Group 2) had low dose clomiphene citrate based protocol plus low dose gonadotropins (75-112.5 IU/d). RESULTS: The rate of cumulative pregnancy/started cycle (9.2% [6/65] vs. 51% [33/65]; P < 0001) was significantly better in CC protocol than the long GnRH agonist protocol. The number of oocytes retrieved was also higher in CC protocol compared to the long protocol (7.26 ± 1.95 vs. 5.98 ± 1.31; P = 0.03). There was a lower number of patients without embryos (12.31% vs. 33.85%; p < 0.0001) in CC protocol than long protocol. CONCLUSIONS: This study showed a better cumulative pregnancy rate in the low dose CC based protocol. There was a higher number of oocytes retrieved after using a lower total dose of recombinant FSH in CC protocol. Thus, clomiphene treatment plus low dose rFSH can be an alternative option for such patients in second cycle stimulation instead of repeating long protocol regimen. Randomized controlled studies with larger number of patients will be needed for more accurate evidence.

Abnormal CFTR Affects Glucagon Production by Islet α Cells in Cystic Fibrosis and Polycystic Ovarian Syndrome.

Glucagon, produced by islet α cells, functions to increase blood glucose. Abnormal glucose levels are often seen in cystic fibrosis (CF), a systematic disease caused by mutations of the CF transmembrane conductance regulator (CFTR), and in polycystic ovarian syndrome (PCOS), an endocrine disorder featured with hyperandrogenism affecting 5-10% women of reproductive age. Here, we explored the role of CFTR in glucagon production in α cells and its possible contribution to glucagon disturbance in CF and PCOS. We found elevated fasting glucagon levels in CFTR mutant (DF508) mice compared to the wildtypes. Glucagon and prohormone convertase 2 (PC2) were also upregulated in CFTR inhibitor-treated or DF508 islets, as compared to the controls or wildtypes, respectively. Dihydrotestosterone (DHT)-induced PCOS rats exhibited significantly lower fasting glucagon levels with higher CFTR expression in α cells compared to that of controls. Treatment of mouse islets or αTC1-9 cells with DHT enhanced CFTR expression and reduced the levels of glucagon and PC2. The inhibitory effect of DHT on glucagon production was blocked by CFTR inhibitors in mouse islets, and mimicked by overexpressing CFTR in αTC1-9 cells with reduced phosphorylation of the cAMP/Ca2+ response element binding protein (p-CREB), a key transcription factor for glucagon and PC2. These results revealed a previously undefined role of CFTR in suppressing glucagon production in α-cells, defects in which may contribute to glucose metabolic disorder seen in CF and PCOS.

[Differentiation of pluripotent stem cells into male germ cells: An update].

Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential of differentiating into all types of adult cells. Today, mature functional sperm can be derived from mouse PSCs in vitro, and meanwhile primordial germ cells (PGCs) and meiotic prophase sperm cells can be generated from human ESCs/iPSCs (hESCs/hiPSCs). It is proposed that non-genetic azoospermia might be cured if functional sperm could be obtained from human PSCs (hPSCs) in vitro. It is also possible that healthy functional sperm could be derived from the patient with genetic factor-induced azoospermia by combining iPSCs and gene editing technology. IPSC-derived functional sperm have a higher clinical value for the avoidance of the sperm source and the issue of medical ethics. This article summarizes recent advances in the differentiation of PSCs into male germ cells in vitro, aiming to provide some reference for the treatment of male infertility with PSCs.

Therapeutic effect of acupuncture on the outcomes of in vitro fertilization: a systematic review and meta-analysis.

PURPOSE: Controversial results have been reported concerning the effect of acupuncture on in vitro fertilization (IVF) outcomes. The current review was conducted to systematically review published studies of the effects of acupuncture on IVF outcomes. METHODS: Women undergoing IVF in randomized controlled trials (RCTs) were evaluated for the effects of acupuncture on IVF outcomes. The treatment groups involved traditional, electrical, laser, auricular, and other acupuncture techniques. The control groups consisted of no, sham, and placebo acupuncture. The PubMed, Embase, and Web of Science databases were searched. The pregnancy outcomes data are expressed as odds ratios (ORs) with 95% confidence intervals (CIs) based on a fixed model or random model depending on the heterogeneity determined by the Q test and I2 statistic. The major outcomes were biochemical pregnancy rate (BPR), clinical pregnancy rate (CPR), live birth rate (LBR), and ongoing pregnancy rate (OPR). Heterogeneity of the therapeutic effect was evaluated by a forest plot analysis, and publication bias was assessed by a funnel plot analysis. RESULTS: Thirty trials (a total of 6344 participants) were included in this review. CPR data showed a significant difference between the acupuncture and control groups (OR 1.26, 95% CI 1.06-1.50, p = 0.01), but there was significant statistical heterogeneity among the studies (p = 0.0002). When the studies were restricted to Asian or non-Asian area trials with a sensitivity analysis, the results significantly benefited the CPR in Asian group (OR 1.51, 95% CI 1.04-2.20, p = 0.03). Based on the area subgroup analysis, we found that in the Asian group, the IVF outcomes from the EA groups were all significantly higher than those from the control groups (CPR: OR 1.81, 95% CI 1.20-2.72, p = 0.005; BPR: OR 1.84, 95% CI 1.12-3.02, p = 0.02; LBR: OR 2.36, 95% CI 1.44-3.88, p = 0.0007; OPR: OR 1.94, 95% CI 1.03-3.64, p = 0.04). Meanwhile, compared with other acupuncture time, the IVF outcome results were significantly superior in the acupuncture group when acupuncture was conducted during controlled ovarian hyperstimulation (COH) (CPR: OR 1.71, 95% CI 1.27-2.29, p = 0.0004; LBR: OR 2.41, 95% CI 1.54-3.78, p = 0.0001; BPR: OR 1.50, 95% CI 1.02-2.20, p = 0.04; OPR: OR 1.88, 95% CI 1.06-3.34, p = 0.03). However, when acupuncture was conducted at the time of embryo transfer, the BPR and OPR from the acupuncture groups were significantly lower than those of the controls in the Asian group (BPR: OR 0.67, 95% CI 0.48-0.92, p = 0.01; OPR: OR 0.68, 95% CI 0.49-0.96, p = 0.03). CONCLUSIONS: Based on an analysis of the studies, acupuncture improves the CPR among women undergoing IVF. When the studies were restricted to Asian or non-Asian area patients, compared with traditional acupuncture and other methods, electrical acupuncture yielded better IVF outcomes. Optimal positive effects could be expected using acupuncture in IVF during COH, especially in Asian area. However, as a limitation of this review, most of the included studies did not mention the number of embryos transferred.

Association of cardiac development with assisted reproductive technology in childhood: a prospective single-blind pilot study.

AIMS: To examine the pattern and extent of cardiovascular developmental alterations among children conceived by assisted reproductive technology (ART) and its association with potential confounders. METHODS: The present study was a prospective single-blind pilot design lasting 15 months. The ART group was recruited by a non-random, consecutive sample on the basis of the unique personal identification number assigned to ART children, whereas spontaneous conception controls were recruited by a population-based random sample from the same hospital by age. Echocardiography was available for the measurement of 128 ART children and 100 controls with respect to cardiovascular geometric morphology and cardiac function. RESULTS: The majority of cardiac geometric morphology parameters were comparable among the study groups (P>0.05), except for significant increases in left ventricular (LV) relative wall thickness (P=0.038), LV mass index (P=0.005) and LV remodeling index (P=0.005) in ART children after adjustment for age, gender, body surface area and heart rate. The results showed similarity in LV systolic function characterized by ejection fraction (P=0.140) and shortening fraction (P=0.167) between the groups. However, ART children had a significant tendency toward a decrease in mitral A (P=0.008) and mitral E' (P=0.012) compared with controls after adjusting for confounders. Additionally, Cox analysis suggested an independent association (P<0.05) of anthropometrics and perinatal outcomes in addition to the ART procedure itself with the differences in cardiac developmental status. CONCLUSION: Our findings support the presence of remodeling in the left cardiac geometric morphology and diastolic dysfunction and the absence of any change to the aortocoronary morphometry or systolic function in ART children compared with controls, which may be independently associated with the anthropometrics and perinatal outcomes in addition to the ART procedure.

Expression of the SET protein in testes of mice at different developmental stages.

SET is a multifunctional protein involved in regulating many biological processes of the cell cycle. It is also a regulator of steroidogenesis in the ovary. However, the expression of SET protein in testis, and its function, still remains ambiguous. In this study, we observed the expression of SET in the testes of mice at different developmental stages, and have discussed its potential function in regulating spermatogenesis and androgen production. Forty-eight male mice at different developmental stages (1 week old as the infancy group; 4 weeks old as the prepubertal group; 12 weeks old as the adult group; over 12 months old as the ageing group) were used. Cellular location of SET protein in the testes was observed by immuno-histochemistry. Expression levels of Set mRNA and SET protein were analyzed by quantitative polymerase chain reaction and Western blotting. SET protein was expressed in spermatogonial cells and spermatocytes; the highest level was mainly in haploid and tetraploid cells of the prepubertal and adult groups, and Leydig cells of the adult and ageing groups. There was a low expression in Sertoli cells. Expression of Set mRNA in the prepubertal group was significantly higher than that in the adult group (P < 0.05), while expression of SET protein was at the highest level in the adult group (P < 0.05). SET protein is mainly expressed in spermatogonial cells and spermatocytes, and poorly expressed in Sertoli cells, suggesting that it is involved in spermatogenesis. Expression of SET protein in Leydig cells suggests a possible role in steroidogenesis.

Downregulated expression of peroxiredoxin 4 in granulosa cells from polycystic ovary syndrome.

Peroxiredoxin 4 (PRDX4), a member of Peroxiredoxin (PRDX) family, is a typical 2-Cys PRDX. PRDX4 monitors the oxidative burden within cellular compartment and reduces hydrogen peroxide and alkyl hydroperoxide related to oxidative stress and apoptosis. Antioxidant, like PRDX4, may promote follicle development and participate in the pathophysiology of PCOS. In our previous study, we found that PRDX4 was expressed in mice oocyte cumulus oophorus complex, and that PRDX4 could be associated with follicle development. In this study, we explored the expression of PRDX4 in human follicles and possible role of PRDX4 in PCOS pathophysiology. Our data showed that PRDX4 was mainly expressed in granulosa cells in human ovaries. When compared to control group, both PRDX4 mRNA level and protein level decreased in PCOS group. The lowered levels of PRDX4 may relate to oxidative stress in the pathophysiologic progress of PCOS. Furthermore, expression of PRDX4 in the granulosa cells of in vivo or in vitro matured follicles was higher than that in immatured follicles, which suggested that PRDX4 may have a close relationship with follicular development. Altogether, our findings may provide new clues of the pathophysiologic mechanism of PCOS and potential therapeutic strategy using antioxidant, like PRDX4.

Effect of HSP10 on apoptosis induced by testosterone in cultured mouse ovarian granulosa cells.

OBJECTIVE: To investigate the effect of heat shock protein 10 (HSP10) on apoptosis induced by testosterone in granulosa cells (GCs) of mouse ovaries in order to define the possible roles of HSP10 in ovarian pathological development of polycystic ovarian syndrome (PCOS) and hyperandrogenic conditions. STUDY DESIGN: Cultured mouse ovarian GCs were treated with testosterone (10(-5) mol/l). Apoptosis was assessed using flow cytometry, and proliferation was assessed using the MTT assay. HSP10 expression in the treated GCs was detected by real-time polymerase chain reaction (PCR). HSP10 gene was downregulated in the cultured GCs by AdCMV-H1-SiRNA/HSP10 or overexpressed by AdCMV-HSP10. PD98059 [phosphorylated ERK (p-ERK) inhibitor] was used to treat GCs to induce a high apoptosis index. Critical apoptotic factors and proliferation factors, including P-ERK, Bcl-2, Bax, caspase 9, caspase 3 and Ki67, were monitored by real-time reverse transcriptase PCR (RT-PCR) and Western blot. RESULTS: Compared with the control group, the apoptosis index was higher (p<0.05) and HSP10 expression was lower (p<0.05) in the testosterone-treated groups. In the AdCMV-H1-SiRNA/HSP10-treated group, cell viability was decreased (p<0.05) and the cell cycle was arrested at G2. Expression of p-ERK, Bcl-2 and Ki67, and the Bcl-2:Bax ratio were lower, while expression of apoptotic factors, including Bax, caspase 9 and caspase 3, was higher (p<0.05). Compared with the control group, Bcl-2 expression in the GCs that overexpressed HSP10 was increased (p<0.05), while the reduction of p-ERK and Bcl-2 and the elevation of caspase 9 and caspase 3 induced by PD98059 were significantly suppressed (p<0.05). CONCLUSIONS: Hyperandrogenic conditions induced apoptosis of mouse GCs. Testosterone may have reduced HSP10 expression in GCs, leading to reduced Bcl-2 expression and increased Bax expression.

SET/PP2A system regulates androgen production in ovarian follicles in vitro.

SET has multiple cell functions including nucleosome assembly, histone binding, transcription control, and cell apoptosis. In ovaries SET is predominantly expressed in theca cells and oocytes. In our study, SET overexpression in theca cells stimulated testosterone production whereas SET knockdown decreased testosterone production. Moreover, SET negatively regulated PP2A activity. Treatment with PP2A inhibitor okadaic acid (OA) led to increased testosterone synthesis, while treatment with PP2A activators resulted in the decreased testosterone synthesis. Furthermore, PP2A knockdown confirmed the key role of PP2A in the testosterone synthesis, and OA was able to block the AdH1-SiRNA/SET-mediated inhibition of testosterone production. The central role of PP2A in SET-mediated regulation of testosterone production was confirmed by the finding that SET promoted the lyase activity of P450c17 and that PP2A inhibited its lyase activity. Taken together, these results reveal a specific, SET-initiated, PP2A-mediated, pathway that leads to the increased lyase activity of P450c17 and testosterone biosynthesis.

[Impact of sperm DNA fragmentation index and sperm malformation rate on the clinical outcome of ICSI].

OBJECTIVE: To evaluate the sperm DNA fragmentation index (DFI) and sperm malformation rate (SMR) before intracytoplasmic sperm injection (ICSI) and their impact on the clinical outcome of ICSI. METHODS: This study included 79 cycles of ICSI because of oligoasthenozoospermia. We detected the sperm concentration, percentage of progressively motile sperm, DFI and SMR at 3 to 6 months before ICSI, and analyzed the relationship of DFI and SMR with the outcome parameters. RESULTS: Of the 79 oligoasthenozoospermia cases, DFI was found to be normal (< or = 25%) in 51 and abnormal (> 25%) in the other 28, significantly increased in the latter (14.18% vs 41.47%), and coincidently, SMR, too, was normal (< or = 96%) in 51 cases and abnormal (> 96%) in 28, significantly higher in the abnormal than in the normal cases (87.88% vs 98.46%). There were no significant differences between the normal and abnormal DFI groups in age, females'BMI, number of oocytes retrieved, and number of embryos transferred, nor between the normal and abnormal SMR groups in the number of fertilized oocytes and quality embryos, biochemical pregnancy, clinical pregnancy, and early pregnancy loss. Sperm DFI was significantly positively correlated with SMR (r = 0.231, P < 0.05). CONCLUSION: ICSI may reduce the rates of biochemical pregnancy and clinical pregnancy for men with increased sperm DFI (> 25%) and SMR (> 96%) by strict detection criteria, but with no statistically significant difference from normal males. Our findings need to be supported by further studies with larger sample sizes.