Integrative multi-omics analysis depicts the methylome and hydroxymethylome in recurrent bladder cancers and identifies biomarkers for predicting PD-L1 expression.

BACKGROUND: Urinary bladder cancer (UBC) is a common malignancy of the urinary tract; however, the mechanism underlying its high recurrence and responses to immunotherapy remains unclear, making clinical outcome predictions difficult. Epigenetic alterations, especially DNA methylation, play important roles in bladder cancer development and are increasingly being investigated as biomarkers for diagnostic or prognostic predictions. However, little is known about hydroxymethylation since previous studies based on bisulfite-sequencing approaches could not differentiate between 5mC and 5hmC signals, resulting in entangled methylation results. METHODS: Tissue samples of bladder cancer patients who underwent laparoscopic radical cystectomy (LRC), partial cystectomy (PC), or transurethral resection of bladder tumor (TURBT) were collected. We utilized a multi-omics approach to analyze both primary and recurrent bladder cancer samples. By integrating various techniques including RNA sequencing, oxidative reduced-representation bisulfite sequencing (oxRRBS), reduced-representation bisulfite sequencing (RRBS), and whole exome sequencing, a comprehensive analysis of the genome, transcriptome, methylome, and hydroxymethylome landscape of these cancers was possible. RESULTS: By whole exome sequencing, we identified driver mutations involved in the development of UBC, including those in FGFR3, KDMTA, and KDMT2C. However, few of these driver mutations were associated with the down-regulation of programmed death-ligand 1 (PD-L1) or recurrence in UBC. By integrating RRBS and oxRRBS data, we identified fatty acid oxidation-related genes significantly enriched in 5hmC-associated transcription alterations in recurrent bladder cancers. We also observed a series of 5mC hypo differentially methylated regions (DMRs) in the gene body of NFATC1, which is highly involved in T-cell immune responses in bladder cancer samples with high expression of PD-L1. Since 5mC and 5hmC alternations are globally anti-correlated, RRBS-seq-based markers that combine the 5mC and 5hmC signals, attenuate cancer-related signals, and therefore, are not optimal as clinical biomarkers. CONCLUSIONS: By multi-omics profiling of UBC samples, we showed that epigenetic alternations are more involved compared to genetic mutations in the PD-L1 regulation and recurrence of UBC. As proof of principle, we demonstrated that the combined measurement of 5mC and 5hmC levels by the bisulfite-based method compromises the prediction accuracy of epigenetic biomarkers.

Association between preconception serum lipid concentrations and treatment outcomes in women with PCOS who underwent ovulation induction.

RESEARCH QUESTION: What is the association between preconception serum lipid concentrations and reproductive outcomes after ovulation induction in women with polycystic ovary syndrome (PCOS)? DESIGN: A secondary analysis of a randomized controlled trial with 1000 PCOS women undergoing ovulation induction with clomiphene with or without acupuncture. Preconception serum total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides, apolipoprotein A1 (ApoA1), apolipoprotein B (ApoB) were measured. Outcomes were ovulation, conception, pregnancy, live birth and miscarriage. Logistic regression was used to calculate odds ratios (OR) with 95% confidence intervals (CI). RESULTS: In total, 780 women ovulated; 320 women achieved conception, 218 had a clinical pregnancy, 205 had a live birth and 115 had a miscarriage. Serum lipid concentrations per one unit increment were independently associated with reproductive outcomes after controlling for confounders. Increasing LDL-C (OR 0.79, 95% CI 0.63-0.99) was independently associated with a lower chance of ovulation. Increasing total cholesterol (OR 0.76, 95% CI 0.62-0.92), LDL-C (OR 0.73, 95% CI 0.57-0.93), triglycerides (OR 0.74, 95% CI 0.58-0.95) and ApoB (OR 0.34, 95% CI 0.16-0.74) were independently associated with a lower chance of clinical pregnancy. Increased total cholesterol (OR 0.78, 95% CI 0.64-0.96), LDL-C (OR 0.77, 95% CI 0.60-0.99), triglycerides (OR 0.76, 95% CI 0.59-0.96) and ApoB (OR 0.39, 95% CI 0.18-0.86) were independently associated with a lower chance of live birth. Furthermore, increased total cholesterol (OR 1.43, 95% CI 1.06-1.93), LDL-C (OR 1.51, 95% CI 1.04-2.19) and ApoB (OR 3.82, 95% CI 1.17-12.41) were independently associated with a higher chance of miscarriage. CONCLUSIONS: Increased serum lipids were negatively associated with the reproductive outcomes of PCOS women undergoing ovulation induction with clomiphene with or without acupuncture.

Associations of Serum Magnesium With Insulin Resistance and Testosterone in Women With Polycystic Ovary Syndrome.

Objective: This article aimed to investigate whether serum magnesium is associated with insulin resistance index and testosterone level in women with polycystic ovary syndrome (PCOS). Materials and Methods: Overall 1000 women with PCOS were enrolled in a randomized controlled trial and a cross-sectional analysis of the association of serum magnesium with glucose metabolism markers and testosterone was performed. Serum magnesium, glucose metabolism markers and testosterone were measured. Insulin resistance was evaluated by homeostatic model assessment of insulin resistance (HOMA-IR) and quantitative insulin-sensitivity check index (QUICKI). Multivariable linear regression and logistic regression models were used to estimate the association between serum magnesium, insulin resistance and testosterone. Results: In comparative analyses, women with higher quartile of serum magnesium had significantly lower fasting glucose, HOMA-IR and testosterone. Multiple linear regression showed serum magnesium was independently negatively associated with insulin, glucose, HOMA-IR, testosterone and positively associated with QUICKI (P for trend <0.05) after adjusting confounding covariates. Logistic regression showed serum magnesium in quartile 1 and 2 were independently associated with insulin resistance status (Quartile 1: OR: 2.15, 95%CI: 1.35-3.40, P = 0.001; Quartile 2: OR: 1.90, 95%CI: 1.20-3.02, P = 0.006), while quartile 1 was marginally associated with hyperandrogenemia status (Quartile 1: OR: 1.45, 95%CI: 0.99-2.11, P = 0.055) after adjusting confounding covariates. Conclusion: The current findings suggest that lower serum magnesium was associated with aggravated insulin resistance and higher testosterone levels among women with PCOS.

Predictive value of circulating sex hormone-binding globulin for gestational diabetes: a meta-analysis.

Aim: To detect predictive value of preconception or early pregnancy sex hormone-binding globulin (SHBG) for subsequent gestational diabetes mellitus (GDM). Materials & methods: We searched Embase, Medline, PubMed, Web of Science and Cochrane library up to January 2020. Studies assessing diagnostic performance of SHBG for GDM diagnosed by well-defined diagnostic criteria using oral glucose tolerance test. Results: Totally seven studies with 1947 women were included and 247 were diagnosed as GDM. SHBG had a combined diagnostic odds ratio of 6.68 (95% CI: 4.58-9.74), sensitivity of 0.70 (95% CI: 0.51-0.84), specificity of 0.74 (95% CI: 0.52-0.88), positive likelihood ratio of 2.49 (95% CI: 1.73-3.57) and negative likelihood ratio of 0.37 (95% CI: 0.23-0.61). The area under the summary receiver operating characteristic curve was 0.78 (95% CI: 0.74-0.82). Conclusion: SHBG had a predictive value for GDM and might improve GDM screening. However, heterogeneity between studies warrants more research into this topic.

Impact of Low Frequency Electro-acupuncture on Glucose and Lipid Metabolism in Unmarried PCOS Women: A Randomized Controlled Trial.

OBJECTIVE: To explore the effect of electro-acupuncture (EA) on glucose and lipid metabolism in unmarried patients with polycystic ovary syndrome (PCOS). METHODS: Fifty-four PCOS patients were equally randomized into true acupuncture group and sham acupuncture group (control) for totally 16 weeks of treatment by random method with a computerized randomization program. Patients in true acupuncture group accepted traditional acupuncture methods with EA and two sets of acupoint groups were used alternatively. The first set consisted of Zhongji (CV 3), Qihai (CV 6), Guilai (ST 29), Sanyinjiao (SP 6), Yinlingquan (SP 9), Hegu (LI4) and Baihui (GV 20), and the second set consisted of Tianshu (ST 25), ST 29, CV 3, CV 6, SP 6, Taichong (LR 3), Neiguan (PC) 6 and GV 20. Patients in the sham acupuncture group accepted shallow acupuncture methods through EA without electricity at 4 non-meridian points in each shoulder and upper arm. Outcome measures included body mass index (BMI), waist-hip-ratio (WHR), oral glucose tolerance test (OGTT), insulin release test, glucose and lipid metabolism indicators such as total cholesterol (TC), triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, adiponectin, leptin, visfatin, resistin, and interleukin (IL-6). RESULTS: Twenty-six subjects in the true acupuncture group and 20 subjects in the sham group completed the clinical trial. After 16 weeks of treatment, no significant difference in the outcome measures were observed between the two groups (P>0.05). However, as compared with baseline data, a reduction in weight, BMI, hipline, WHR, fasting glucose, homeostatic model assessment of insulin sensitivity, visfatin and HDL-C, and an increase in resistin and IL-6 were observed in the true acupuncture group (P<0.05). In addition, a reduction in visfatin and an increase in TC were also observed in the sham group (P<0.05). CONCLUSIONS: Acupuncture may have a beneficial effect in the treatment of PCOS by improving glucose and lipid metabolism. Moreover, the sham acupuncture may be not completely ineffective. Sham acupuncture may improve some of the aspects of the glucose and lipid metabolism of PCOS patients through a placebo effect. (Registration Nos. ChiCTR-TRC-12002529 and NCT01812161).

[Mechanism of transcutaneous auricular vagus nerve stimulation on polycystic ovary syndrome].

Non-invasive transcutaneous auricular vagus nerve stimulation (ta-VNS) is established on the basis of auricular acupuncture and the principle of vagus nerve stimulation (VNS). The results of the comparative study of 4 indications of VNS show that ta-VNS achieves almost the equivalent effect as VNS. Moreover, the ta-VNS is advantageous at safety, economy and portability. Regarding the clinical prevention and treatment of polycystic ovary syndrome (PCOS), it is still controversial for the effect of acupuncture and moxibustion therapy including auricular acupuncture. Moreover, it is the difficulty for the principle of acupoint selection and the treatment mechanism of this therapy to be in line with modern medicine. As neuroendocrine disease, PCOS and the ovary, its target organ are all associated with the vagus nerve. It is a new idea to introduce the ta-VNS in treatment of PCOS by regulating the reproductive and endocrinal disturbance of PCOS in terms of vagus nerve and improving the acupuncture and moxibustion therapy, such as the auricular acupuncture. In the paper, the potential mechanism of the ta-VNS in treatment of PCOS is presented: 1)the ta-VNS treats PCOS by improving insulin resistance; 2)the ta-VNS relieves PCOS-induced psychological disorders through the treatment of depression; 3)the ta-VNS is applicable in treatment of PCOS complications, such as hypertension and diabetes.

Endocrine characteristics, body mass index and metabolic syndrome in women with polycystic ovary syndrome.

RESEARCH QUESTION: The study aimed to evaluate the associations of endocrine and ultrasound characteristics with metabolic syndrome in women with polycystic ovary syndrome (PCOS), and whether these associations were modified by body mass index (BMI). DESIGN: The study was a secondary analysis of baseline data from a randomized controlled trial of induction of ovulation in women with PCOS. RESULTS: Among 947 Chinese women with PCOS, 153 (16.2%) were diagnosed with metabolic syndrome. The prevalence of metabolic syndrome in women with normal (<24 kg/m2) and high (≥24 kg/m2) BMI was 3.6% and 30.5%, respectively. In all women, a high free androgen index (FAI ≥5%) was positively associated with metabolic syndrome (adjusted odds ratio [OR] 2.06, 95% confidence interval [CI] 1.11-3.82). High FAI was positively associated with metabolic syndrome among women with high BMI (adjusted OR 3.37, 95% CI 1.78-6.37), but the association was not significant in women with normal BMI (adjusted OR 1.27, 95% CI 0.34-4.70). The presence of polycystic ovary morphology was negatively associated with metabolic syndrome (adjusted OR 0.52, 95% CI 0.26-1.03) in all women (normal BMI adjusted OR 0.42, 95% CI 0.11-1.67; high BMI adjusted OR 0.54, 95% CI 0.23-1.28). LH, sex hormone-binding globulin (SHBG) and anti-Müllerian hormone (AMH) were negatively associated with metabolic syndrome. The associations of FAI, SHBG and AMH in relation to metabolic syndrome were significantly modified by BMI. CONCLUSION(S): The associations of endocrine characteristic with metabolic syndrome were modified by BMI in women with PCOS. Women with PCOS and normal BMI did not have an increased risk of metabolic syndrome.

Hyperandrogenism and insulin resistance-induced fetal loss: evidence for placental mitochondrial abnormalities and elevated reactive oxygen species production in pregnant rats that mimic the clinical features of polycystic ovary syndrome.

KEY POINTS: Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanisms remain unknown. Herein, pregnant rats chronically treated with 5α-dihydrotestosterone (DHT) and insulin exhibited hyperandrogenism and insulin resistance, as well as increased fetal loss, and these features are strikingly similar to those observed in pregnant PCOS patients. Fetal loss in our DHT+insulin-treated pregnant rats was associated with mitochondrial dysfunction, disturbed superoxide dismutase 1 and Keap1/Nrf2 antioxidant responses, over-production of reactive oxygen species (ROS) and impaired formation of the placenta. Chronic treatment of pregnant rats with DHT or insulin alone indicated that DHT triggered many of the molecular pathways leading to placental abnormalities and fetal loss, whereas insulin often exerted distinct effects on placental gene expression compared to co-treatment with DHT and insulin. Treatment of DHT+insulin-treated pregnant rats with the antioxidant N-acetylcysteine improved fetal survival but was deleterious in normal pregnant rats. Our results provide insight into the fetal loss associated with hyperandrogenism and insulin resistance in women and suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy. ABSTRACT: Women with polycystic ovary syndrome (PCOS) commonly suffer from miscarriage, but the underlying mechanism of PCOS-induced fetal loss during pregnancy remains obscure and specific therapies are lacking. We used pregnant rats treated with 5α-dihydrotestosterone (DHT) and insulin to investigate the impact of hyperandrogenism and insulin resistance on fetal survival and to determine the molecular link between PCOS conditions and placental dysfunction during pregnancy. Our study shows that pregnant rats chronically treated with a combination of DHT and insulin exhibited endocrine aberrations such as hyperandrogenism and insulin resistance that are strikingly similar to those in pregnant PCOS patients. Of pathophysiological significance, DHT+insulin-treated pregnant rats had greater fetal loss and subsequently decreased litter sizes compared to normal pregnant rats. This negative effect was accompanied by impaired trophoblast differentiation, increased glycogen accumulation, and decreased angiogenesis in the placenta. Mechanistically, we report that over-production of reactive oxygen species (ROS) in the placenta, mitochondrial dysfunction, and disturbed superoxide dismutase 1 (SOD1) and Keap1/Nrf2 antioxidant responses constitute important contributors to fetal loss in DHT+insulin-treated pregnant rats. Many of the molecular pathways leading to placental abnormalities and fetal loss in DHT+insulin treatment were also seen in pregnant rats treated with DHT alone, whereas pregnant rats treated with insulin alone often exerted distinct effects on placental gene expression compared to insulin treatment in combination with DHT. We also found that treatment with the antioxidant N-acetylcysteine (NAC) improved fetal survival in DHT+insulin-treated pregnant rats, an effect related to changes in Keap1/Nrf2 and nuclear factor-κB signalling. However, NAC administration resulted in fetal loss in normal pregnant rats, most likely due to PCOS-like endocrine abnormality induced by the treatment. Our results suggest that the deleterious effects of hyperandrogenism and insulin resistance on fetal survival are related to a constellation of mitochondria-ROS-SOD1/Nrf2 changes in the placenta. Our findings also suggest that physiological levels of ROS are required for normal placental formation and fetal survival during pregnancy.

Acupuncture and clomiphene for Chinese women with polycystic ovary syndrome (PCOSAct): statistical analysis approach with the revision and explanation.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrinopathy of reproductive-aged women. Clomiphene is regarded as the first-line medical treatment for ovulation induction in PCOS patients and acupuncture is often used as an alternative and complementary treatment for fertility issues such as those associated with PCOS. The efficacy of acupuncture alone or combined with clomiphene still lacks strong supporting evidence. Factorial 2 × 2 designs can be used for the evaluations of two treatments within a single study, to test the main effects of acupuncture and clomiphene and their interactions. METHODS: PCOSAct was designed to test the effect of clomiphene and acupuncture by three two-group comparisons in the original protocol. However, the trial was designed as a standard factorial trial and the factorial analysis approach for analyzing the data that were actually obtained during the trial was found to be more appropriate and more powerful than the three two-group comparisons described in the original protocol, so the statistical analysis approach and different datasets of PCOSAct in the primary publication were accordingly changed. DISCUSSION: Although the statistical analysis approach used in the primary publication deviated from the statistical analysis planned in the study protocol, focusing on the main effects of the two interventions and their interactions was a more standard approach to a factorial trial and proved to be more suitable and consistent with the characteristics of the trial data. Statistically, the revision is more powerful and precise and should be more useful to the journal and the readers. TRIAL REGISTRATION: Chinese clinical trial registry, ChiCTR-TRC-12002081 . Registered on 20 March 2012. Clinicaltrials.gov, NCT01573858 . Registered on 4 April 2012.

[Acupuncture treatment of oligoasthenozoospermia].

Studies show that acupuncture can significantly elevate the level of serum testosterone (T), reduce the concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2), initiate spermatogenesis, enhance testicular blood flow, maintain a relative low temperature in the testis, increase the concentration, motility and antioxidative injury capability of spermatozoa by raising the levels of seminal α-glucosidase, fructose and super oxide dismutase, and eventually improve semen quality and the rate of conception in the treatment of oligoasthenozoospermia. Currently, the quality of the clinical studies of acupuncture treatment of oligoasthenozoospermia is relatively poor, the existing evidence remains at a low level, its clinical application is limited, and its therapeutic effect has to be further verified. The present paper summarizes the literature from domestic and international databases about acupuncture treatment of oligoasthenozoospermia, and offers an overview of the effects of acupuncture on the reproductive endocrine system, testicular blood flow, semen quality, and rate of conception in the treatment of the patient.

Uterine progesterone signaling is a target for metformin therapy in PCOS-like rats.

Impaired progesterone (P4) signaling is linked to endometrial dysfunction and infertility in women with polycystic ovary syndrome (PCOS). Here, we report for the first time that elevated expression of progesterone receptor (PGR) isoforms A and B parallels increased estrogen receptor (ER) expression in PCOS-like rat uteri. The aberrant PGR-targeted gene expression in PCOS-like rats before and after implantation overlaps with dysregulated expression of Fkbp52 and Ncoa2, two genes that contribute to the development of uterine P4 resistance. In vivo and in vitro studies of the effects of metformin on the regulation of the uterine P4 signaling pathway under PCOS conditions showed that metformin directly inhibits the expression of PGR and ER along with the regulation of several genes that are targeted dependently or independently of PGR-mediated uterine implantation. Functionally, metformin treatment corrected the abnormal expression of cell-specific PGR and ER and some PGR-target genes in PCOS-like rats with implantation. Additionally, we documented how metformin contributes to the regulation of the PGR-associated MAPK/ERK/p38 signaling pathway in the PCOS-like rat uterus. Our data provide novel insights into how metformin therapy regulates uterine P4 signaling molecules under PCOS conditions.

Effect of exposure to second-hand smoke from husbands on biochemical hyperandrogenism, metabolic syndrome and conception rates in women with polycystic ovary syndrome undergoing ovulation induction.

STUDY QUESTION: Does second-hand smoke (SHS) exposure from husbands have adverse effects on sex hormones, metabolic profiles, clinical phenotypes and fertility outcomes in women with polycystic ovary syndrome (PCOS) undergoing ovulation induction? SUMMARY ANSWER: SHS exposure is associated with worsened biochemical hyperandrogenism, higher incidence of metabolic syndrome and reduced conception rates in women with PCOS. WHAT IS KNOWN ALREADY: Smoking in women impairs fecundity at some stages of the reproductive process including folliculogenesis, embryo transport, endometrial angiogenesis and uterine blood flow. Yet little is known about the hazard of SHS exposure in women with PCOS. STUDY DESIGN, SIZE, DURATION: This study was a secondary analysis of the Polycystic Ovary Syndrome Acupuncture and Clomiphene Trial (PCOSAct), a large randomized controlled trial conducted at 27 hospitals from 2012 to 2015 in mainland China. PARTICIPANTS/MATERIALS, SETTING, METHODS: Out of 1000 women with PCOS, SHS exposure status were available in 500 women, of whom 271 women were non-exposed and 229 exposed to cigarette smoke (170 women ≤10 cigarettes per day as low-SHS exposed and 59 women >10 cigarettes per day as high-SHS exposed). We compared circulating sex steroids, glucose and lipid metabolism, metabolic syndrome and phenotypes, fertility and obstetric outcomes between non-exposed and exposed women. MAIN RESULTS AND THE ROLE OF CHANCE: Women exposed to SHS, compared to non-exposed women, had a higher serum total testosterone (1.7 vs 1.5 nmol/L, P = 0.01), free androgen index (5.7 vs 4.0, P = 0.001) and lower sex hormone binding globulin (30.1 vs 35.6 nmol/L, P = 0.03). Metabolic syndrome, but not other phenotypes, was more frequent in exposed women as compared to non-exposed women (21.8 vs 13.3%, adjusted odds ratio (OR)=1.66; 95% CI, 1.02-2.71, P = 0.04). Ovulation rates between exposed and non-exposed groups were not significantly different (76.9 vs 82.9%, adjusted OR=0.72; 95% CI, 0.45-1.15, P = 0.17). Conception rates were significant lower in the exposed group (26.6 vs 36.9%; adjusted OR=0.61; 95% CI, 0.41-0.91; P = 0.01), while clinical pregnancy and live birth rates showed a similar trend that was not statistically significant. Gestational age, birth weight and other obstetric outcomes were not affected by SHS exposure. LIMITATIONS, REASONS FOR CAUTION: Data on SHS exposure were missing in 50% of the women. We did not assay serum nicotine or cotinine levels to quantify the SHS exposure status. WIDER IMPLICATIONS OF THE FINDINGS: These data suggest that smoking partners of infertile women with PCOS who seek treatment should be advised to quit smoking. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the National Public Welfare Projects for Chinese Medicine (201107005 and 200807002) and the National Clinical Trial Base in Chinese Medicine Special Projects (JDZX2012036 and 2015B009). There are no conflicts of interest. TRIAL REGISTRATION NUMBER: ClinicalTrial.gov number: NCT01573858 and chictr.org.cn number: ChiCTR-TRC-12002081.

Effect of Acupuncture and Clomiphene in Chinese Women With Polycystic Ovary Syndrome: A Randomized Clinical Trial.

Importance: Acupuncture is used to induce ovulation in some women with polycystic ovary syndrome, without supporting clinical evidence. Objective: To assess whether active acupuncture, either alone or combined with clomiphene, increases the likelihood of live births among women with polycystic ovary syndrome. Design, Setting, and Participants: A double-blind (clomiphene vs placebo), single-blind (active vs control acupuncture) factorial trial was conducted at 21 sites (27 hospitals) in mainland China between July 6, 2012, and November 18, 2014, with 10 months of pregnancy follow-up until October 7, 2015. Chinese women with polycystic ovary syndrome were randomized in a 1:1:1:1 ratio to 4 groups. Interventions: Active or control acupuncture administered twice a week for 30 minutes per treatment and clomiphene or placebo administered for 5 days per cycle, for up to 4 cycles. The active acupuncture group received deep needle insertion with combined manual and low-frequency electrical stimulation; the control acupuncture group received superficial needle insertion, no manual stimulation, and mock electricity. Main Outcomes and Measures: The primary outcome was live birth. Secondary outcomes included adverse events. Results: Among the 1000 randomized women (mean [SD] age, 27.9 [3.3] years; mean [SD] body mass index, 24.2 [4.3]), 250 were randomized to each group; a total of 926 women (92.6%) completed the trial. Live births occurred in 69 of 235 women (29.4%) in the active acupuncture plus clomiphene group, 66 of 236 (28.0%) in the control acupuncture plus clomiphene group, 31 of 223 (13.9%) in the active acupuncture plus placebo group, and 39 of 232 (16.8%) in the control acupuncture plus placebo group. There was no significant interaction between active acupuncture and clomiphene (P = .39), so main effects were evaluated. The live birth rate was significantly higher in the women treated with clomiphene than with placebo (135 of 471 [28.7%] vs 70 of 455 [15.4%], respectively; difference, 13.3%; 95% CI, 8.0% to 18.5%) and not significantly different between women treated with active vs control acupuncture (100 of 458 [21.8%] vs 105 of 468 [22.4%], respectively; difference, -0.6%; 95% CI, -5.9% to 4.7%). Diarrhea and bruising were more common in patients receiving active acupuncture than control acupuncture (diarrhea: 25 of 500 [5.0%] vs 8 of 500 [1.6%], respectively; difference, 3.4%; 95% CI, 1.2% to 5.6%; bruising: 37 of 500 [7.4%] vs 9 of 500 [1.8%], respectively; difference, 5.6%; 95% CI, 3.0% to 8.2%). Conclusions and Relevance: Among Chinese women with polycystic ovary syndrome, the use of acupuncture with or without clomiphene, compared with control acupuncture and placebo, did not increase live births. This finding does not support acupuncture as an infertility treatment in such women. Trial Registration: clinicaltrials.gov Identifier: NCT01573858.

Metformin Ameliorates Uterine Defects in a Rat Model of Polycystic Ovary Syndrome.

Adult rats treated concomitantly with insulin and human chorionic gonadotropin exhibit endocrine, metabolic, and reproductive abnormalities that are very similar to those observed in polycystic ovary syndrome (PCOS) patients. In this study, we used this rat model to assess the effects of metformin on PCOS-related uterine dysfunction. In addition to reducing androgen levels, improving insulin sensitivity, and correcting the reproductive cycle, metformin treatment induced morphological changes in the PCOS-like uterus. At the molecular and cellular levels, metformin normalized the androgen receptor-mediated transcriptional program and restored epithelial-stromal interactions. In contrast to glucose transport, uterine inflammatory gene expression was suppressed through the PI3K-Akt-NFκB network, but without affecting apoptosis. These effects appeared to be independent of AMPK subunit and autophagy-related protein regulation. We found that when metformin treatment partially restored implantation, several implantation-related genes were normalized in the PCOS-like rat uterus. These results improve our understanding of how metformin rescues the disruption of the implantation process due to the uterine defects that result from hyperandrogenism and insulin resistance. Our data provide insights into the molecular and functional clues that might help explain, at least in part, the potential therapeutic options of metformin in PCOS patients with uterine dysfunction.

The effect of berberine on insulin resistance in women with polycystic ovary syndrome: detailed statistical analysis plan (SAP) for a multicenter randomized controlled trial.

BACKGROUND: Although Traditional Chinese Medicine (TCM) has been widely used in clinical settings, a major challenge that remains in TCM is to evaluate its efficacy scientifically. This randomized controlled trial aims to evaluate the efficacy and safety of berberine in the treatment of patients with polycystic ovary syndrome. In order to improve the transparency and research quality of this clinical trial, we prepared this statistical analysis plan (SAP). METHODS: The trial design, primary and secondary outcomes, and safety outcomes were declared to reduce selection biases in data analysis and result reporting. We specified detailed methods for data management and statistical analyses. Statistics in corresponding tables, listings, and graphs were outlined. DISCUSSION: The SAP provided more detailed information than trial protocol on data management and statistical analysis methods. Any post hoc analyses could be identified via referring to this SAP, and the possible selection bias and performance bias will be reduced in the trial. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov, NCT01138930 , registered on 7 June 2010.

Molecular characterization of insulin resistance and glycolytic metabolism in the rat uterus.

Peripheral insulin resistance and hyperandrogenism are the primary features of polycystic ovary syndrome (PCOS). However, how insulin resistance and hyperandrogenism affect uterine function and contribute to the pathogenesis of PCOS are open questions. We treated rats with insulin alone or in combination with human chorionic gonadotropin (hCG) and showed that peripheral insulin resistance and hyperandrogenism alter uterine morphology, cell phenotype, and cell function, especially in glandular epithelial cells. These defects are associated with an aberration in the PI3K/Akt signaling pathway that is used as an indicator for the onset of insulin resistance in classical metabolic tissues. Concomitantly, increased GSK3ß (Ser-9) phosphorylation and decreased ERK1/2 phosphorylation in rats treated with insulin and hCG were also observed. We also profiled the expression of glucose transporter (Glut) isoform genes in the uterus under conditions of insulin resistance and/or hyperandrogenism. Finally, we determined the expression pattern of glycolytic enzymes and intermediates during insulin resistance and hyperandrogenism in the uterus. These findings suggest that the PI3K/Akt and MAPK/ERK signaling pathways play a role in the onset of uterine insulin resistance, and they also suggest that changes in specific Glut isoform expression and alterations to glycolytic metabolism contribute to the endometrial dysfunction observed in PCOS patients.

Randomized controlled trial of letrozole, berberine, or a combination for infertility in the polycystic ovary syndrome.

OBJECTIVE: To study whether a combination of berberine and letrozole results in higher live births than letrozole alone in infertile women with polycystic ovary syndrome (PCOS). DESIGN: A multicenter randomized double-blinded placebo-controlled trial. SETTING: Reproductive and developmental network sites. PATIENT(S): Eligible women had PCOS as defined by the Rotterdam criteria. We enrolled 644 participants randomized 1:1:1 among letrozole, berberine, and combination groups. INTERVENTIONS(S): Berberine or berberine placebo were administrated orally at a daily dose of 1.5 g for up to 6 months. Patients received an initial dose of 2.5 mg letrozole or placebo on days 3-7 of the first three treatment cycles. This dose was increased to 5 mg on the last three cycles if not pregnant. MAIN OUTCOMES MEASURE(S): Cumulative live births. RESULTS: The cumulative live births were similar between the letrozole and combination groups after treatment (36% and 34%), and were superior to those in the berberine group (22%). Likely, conception, pregnancy, and ovulation rates were similar between the letrozole and combination groups, and these were significantly higher than in the berberine group. There was one twin birth in the letrozole group, three twin births in the combination group, and none in the berberine group. CONCLUSION(S): Berberine did not add fecundity in PCOS when used in combination with the new ovulation agent letrozole. CLINICAL TRIAL REGISTRATION NUMBER: ChiCTR-TRC-09000376 (http://apps.who.int/trialsearch/).

[Effect of Brucea javanica fruit oil emulsion combined cisplatin on the growth inhibition of transplanted tumor in human ovarian cancer SKOV3 nude mice: an experimental study].

OBJECTIVE: To study the growth inhibition effect of Brucea javanica Fruit Oil Emulsion (BJFOE) on human ovarian caner SKOV3 cells and the transplanted tumor of SKOV3 nude mice. METHODS: Growth inhibition effects of different concentrations BJFOE alone or its combination with cisplatin on human ovarian cancer cell SKOV3 were measured using MTT method. The orthotopic transplantation tumor model of human ovarian cancer SKOV3 cell lines was established in nude mice. Totally 32 ovarian cancer nude mice were randomly divided into 4 groups, i.e., the blank control group (Group A), the BJFOE group (Group B), the BJFOE combined Cisplatin group (Group C), and the Cisplatin control group (Group D), 8 in each group. Mice in Group A were intraperitoneally injected with normal saline (0.2 mL/ 20 g), once per two days. Mice in Group B were intraperitoneally injected with BJFOE (0.2 mL/20 g), once per two days. Mice in Group C were intraperitoneally injected with cisplatin (3 mg/kg) 0.2 mL on the first day, and intraperitoneally injected with BJFOE on the second day. Mice in Group D were intraperitoneally injected with cisplatin (3 mg/kg) 0.2 mL, once per two days. All mice were injected for six times, and sacrificed 48 h after the last injection. The lesion formation of the abdominal tumor tissue was observed. Tumor specimens were obtained to perform HE staining. Expression levels of MRP-1/CD9 and integrinα-5 were detected using Western blot. RESULTS: The inhibition of BJFOE was time-dose depend- ently correlated with its inhibition effect of SKOV3 cells. The inhibition effect of BJFOE in combination of cisplatin was significantly superior to that of using any of the two drugs alone. Western blot results showed expression levels of MRP-1/CD9 and integrinα-5 were up-regulated in Group B and Group D with statistical difference (P < 0.05). But they were down-regulated in Group C with statistical difference (P < 0.05). CONCLUSIONS: Intraperitoneal injecting BJFOE was feasible and effective for treating ovarian cancer. BJFOE also could inhibit the invasion and migration of tumor cells targeting at MRP-1/CD9 and integrinα-5. But its specific anti-tumor mechanism was not clearly probed.

[Effect of Yangjing Zhongyu Decoction on mRNA and protein expression of PCNA, StAR, and FSHR in ovarian granulosa cells cultured by excess androgen].

OBJECTIVE: To observe the effect of Yangjing Zhongyu Decoction (YZD) on mRNA and protein expression of PCNA, StAR, and FSHR in ovarian granulose cells (GCs) cultured by excess androgen. METHODS: Ovarian GCs from porcine follicles were isolated and cultured in vitro. Follicular stimulating hormone (FSH) or YZD was added in the GCs treated by excess testosterone propionate. Totally 48 h later mRNA and protein expression of PCNA, StAR, and FSHR were detected by RT-PCR and Western blot. RESULTS: Excess androgen inhibited mRNA and protein expression of PCNA, StAR, and FSHR of GCs. FSH and YZD could antagonize inhibition of excess androgens, and promote mRNA and protein expression of PCNA, StAR, and FSHR in GCs. CONCLUSION: YZD could antagonize the inhibition of excess androgen on mRNA and protein expression of PCNA, StAR and FSHR in GCs. Thus, we inferred that YZD could improve the follicle dysplasia by promoting mRNA and protein expression of PCNA, StAR and FSHR in GCs.