The role of circulating miRNAs in mechanism of action and prediction of therapeutic responses of metformin in polycystic ovarian syndrome.

OBJECTIVE: To study the involvement of microribonucleic acids (miRNAs) in the pathogenesis of chronic anovulation and mechanism of metformin treatment in polycystic ovary syndrome (PCOS). DESIGN: Case-control and prospective validation cohort study. SETTING: Tertiary university hospital. PATIENT(S): A total of 146 patients with PCOS and chronic anovulation and 20 non-PCOS controls were enrolled. Patients who resumed ovulation after metformin treatment (MET-OV) and remained anovulatory after metformin treatment (MET-AO) were assigned to MET-OV and MET-AO groups, respectively. INTERVENTION(S): All patients with PCOS received metformin treatment for 6 months. MAIN OUTCOME MEASURE(S): Baseline and chronological changes in the plasma levels of 14 miRNAs (miR-21, 93, 132, 193b, 221, 222, 223, 27a, 125b, 200b, 212, 320a, 429, and 483) selected by literature review, anthropometric data, and hormonal as well as metabolic profiles were measured. Predictive modeling based on baseline circulatory miRNA levels and clinical parameters was performed to predict ovulation recovery after metformin treatment. RESULT(S): No significant differences were observed in the baseline hormonal and metabolic profiles between the MET-OV and MET-AO groups. However, the expression of miR-27a, miR-93, and miR-222 was significantly higher in the MET-OV group than that for the MET-AO and control groups. After 6 months of metformin treatment, the levels of insulin, luteinizing hormone, and 6 circulating miRNAs (miR-21, 27a, 93, 221, 222, and 223) and homeostatic model assessment for insulin resistance decreased significantly in the MET-OV group, but remained unchanged in the MET-AO group. The area under curve, sensitivity, and specificity of the adjusted prediction model, based on miRNA levels and clinical parameters using logistic regression analysis for predicting ovulatory response after metformin treatment, were 0.807, 0.892, and 0.632, respectively. CONCLUSION(S): The present study demonstrated a distinct pattern of baseline expression and chronological changes in the levels of several circulatory miRNAs between the MET-OV and MET-AO groups, suggesting that aberrantly overexpressed diabetogenic miRNAs are involved in the pathophysiology of chronic anovulation in PCOS, and their down-regulation might contribute toward the therapeutic effects of metformin. This could provide new insights into the mechanism of action and applicability of individualized metformin therapy in women with PCOS.

Metformin: a novel promising option for fertility preservation during cyclophosphamide-based chemotherapy.

Cyclophosphamide (CP) could cause severe gonadotoxicity via imbalanced activation of primordial follicles through PI3K/AKT/mTOR activation. Whether metformin, a widely prescribed anti-diabetes agent with mTOR inhibitory effect, could preserve ovarian function against CP toxicity is unknown. Female C57BL/6 mice were randomized into seven groups (n = 11), including control, CP-alone, CP + metformin, CP + sirolimus or everolimus, metformin-alone and sirolimus-alone groups. The duration of pharmaceutical treatment was 4 weeks. CP treatment significantly impaired ovarian function and fertility in mice. CP + metformin treatment significantly attenuated the gonadotoxicity comparing to CP-alone treatment (primordial follicle count: 17.6 ± 4.2 versus 10.3 ± 2.7 follicles/high-power field; P = 0.027). CP + metformin treatment also tended to increase antral follicular count (5.4 ± 1.1 versus 2.5 ± 1.6 follicles/section), serum AMH levels (4.6 ± 1.2 versus 2.0 ± 0.8 ng/ml) and the litter size (4.2 ± 1.3 versus 1.5 ± 1.0 mice per pregnancy), compared with CP-alone group. Expression of phospho-mTOR and the number of TUNEL-positive granulosa cells increased after CP treatment and decreased in the CP + metformin groups, suggesting the mTOR inhibitory and anti-apoptotic effects of metformin. In in-vitro granulosa cell experiments, the anti-apoptotic effect of metformin was blocked after inhibiting p53 or p21 function, and the expression of p53 mRNA was blocked with AMPK inhibitor, suggesting that the anti-apoptotic effect was AMPK/p53/p21-mediated. In conclusion, concurrent metformin treatment during CP therapy could significantly preserve ovarian function and fertility and could be a promising novel fertility preserving agent during chemotherapy. The relatively acceptable cost and well-established long-term safety profiles of this old drug might prompt its further clinical application at a faster pace.

Increased platelet factor 4 and aberrant permeability of follicular fluid in PCOS.

BACKGROUND/PURPOSE: Abnormal folliculogenesis is one of the cardinal presentations of polycystic ovarian syndrome (PCOS) and permeability of follicular wall has been proposed to be involved in the normal follicular growth. However, whether or not there is a change in intrafollicular permeability underlies PCOS is unknown. METHODS: This was a tertiary center-based case-control study. From 2014 to 2015, thirteen patients with PCOS who underwent in vitro fertilization-embryo transfer (IVF-ET) were enrolled. Eleven normo-ovulatory patients who underwent IVF-ET due to male factor and/or tubal factor infertility were enrolled as the control group. The influence of ovarian follicular fluid (FF) on endothelial cell permeability was evaluated using a human umbilical vein endothelial cell monolayer permeability assay. The intrafollicular expression profiles of angiogenesis-related proteins were analyzed using a Human Angiogenesis Protein Array Kit. RESULTS: The FF from PCOS patients caused significantly poorer endothelial cell permeability comparing with the effect of FF from the control group (46% ± 12% vs. 58% ± 9%, P = 0.023). Among the 55 angiogenesis-related proteins tested, there was a significantly higher level of intrafollicular platelet factor 4 (PF4) and PF4/IL-8 complex in the PCOS group (p = 0.004). The anti-permeability effect of PF4 was related to the decrease in the intercellular gaps and antagonistic binding with IL-8. CONCLUSION: Our study provides the first evidence of the pathophysiologic contribution of the well-known angiostatic protein, PF4, on human reproductive biology. The increase of the intrafollicular PF4 and its anti-permeability effect might affect the formation of FF and folliculogenesis in PCOS.

Early stop of progesterone supplementation after confirmation of pregnancy in IVF/ICSI fresh embryo transfer cycles of poor responders does not affect pregnancy outcome.

Previous studies indicated that progesterone can be withdrawn at the time of the first positive ß-hCG test without compromising the clinical pregnancy outcome in normal ovarian responder. However, the effect of early stop of progesterone supplementation for patients with poor ovarian response (POR) has not been investigated. This study retrospectively collected data from patients with POR in 156 IVF/ICSI fresh embryo transfer (ET) cycles in single tertiary center from January 2010 to June 2016. All the patients met ESHRE consensus, the Bologna criteria, of POR and had hCG injection for luteal phase support (LPS) on day 2, 5 and 8 after ovum pick-up. The pregnant patients were divided into two groups: early stop group represented those who stopped LPS from day of positive pregnancy test; control group represented those who kept progesterone supplementation till gestational age of 9 weeks. There were no significant differences in age, BMI, parity, hormone data, number of follicles>10(mm), endometrial thickness and number of embryos transferred between the two groups. After adjustment for possible confounders with multivariate logistic regression analysis, the clinical pregnancy rates (55.0% vs. 57.1%, P = 0.35), ongoing pregnancy rates (47.0% vs. 46.4%, P = 0.66), miscarriage rates (34.0% vs. 26.7%, P = 0.66) and live-birth rates (44.0% vs. 46.4%, P = 0.41) were not statistically different between early stop group and the control group. Our study indicates that early stop of progesterone supplementation on the day of positive pregnancy test for patients of POR using hCG as LPS in fresh ET cycles does not affect pregnancy outcome.

Advantages of cumulative pregnancy outcomes in freeze-all strategy in high responders - A case-control matching analysis of a large cohort.

BACKGROUND/PURPOSE: The freeze-all strategy in high responders is considered to be a safe and effective strategy for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment; however, the cumulative pregnancy outcomes have not been established. METHODS: A retrospective, single-center cohort study was conducted and 1311 high-responder patients (>20 oocytes retrieved and/or a serum estradiol level > 3000 pg/ml on the triggering day) were recruited from 2006 to 2015. The study group (n = 351) underwent the freeze-all strategy with subsequent thawed embryo transfer (ET), and the control group (n = 960) received fresh-cycle ET and subsequent thawed ET if needed. A case-control matching analysis was performed to match the two groups for the number of retrieved oocytes. The primary outcomes were the ongoing pregnancy rate (OPR) of the first ET cycle and the cumulative OPR. RESULTS: After matching, there was a significantly higher OPR in the first ET cycle (49.5% vs. 32.2%, p < 0.0001; n = 301 in each group) and the cumulative OPR (69.4% vs. 55.1%, p < 0.0001) in the study group, with significantly fewer total transferred embryos and cycles. The advantages of the freeze-all strategy for the OPR in the first ET cycle (OR: 1.97, p < 0.0001) and the cumulative OPR (OR: 1.49, p = 0.032) remained statistically significant after adjusting for other possible confounding factors in multivariate logistic regression analysis. CONCLUSION: For high responders, the freeze-all strategy with thawed ET achieved a significantly higher OPR in the first ET cycle and a higher cumulative OPR than the fresh ET strategy.

Feasibility of corifollitropin alfa/GnRH antagonist protocol combined with GnRH agonist triggering and freeze-all strategy in polycystic ovary syndrome patients.

BACKGROUND/PURPOSE: The long-acting corifollitropin alfa is comparable to FSH in terms of pregnancy outcomes in normal responders and poor responders. Corifollitropin alfa has never been studied in polycystic ovary syndrome (PCOS) patients because of concerns of excessive ovarian stimulation and ovarian hyperstimulation syndrome (OHSS). The purpose of the study was to evaluate if corifollitropin alfa can be used in PCOS patients. METHODS: Forty PCOS patients who were going to undergo in vitro fertilization were enrolled in this study. A single injection of corifollitropin alfa was administered on cycle day 2 or day 3. From stimulation day 8 onwards, daily FSH was administered until the day of final oocyte maturation. Cetrorelix was administered from stimulation day 5 to prevent premature LH surge. Final oocyte maturation was triggered by: acetate. All embryos were cryopreserved and replaced in subsequent cycles. RESULTS: All 40 patients were subjected to oocyte retrieval, and none developed moderate or severe ovarian hyperstimulation syndrome (0%, 95% CI 0-0.088). For each patient, an average of 23.4 (±7.4; 95% CI 21.0-25.7) oocytes were retrieved and a mean of 11.7 (±6.4; 95% CI 9.6-13.8) embryos were frozen. Mean serum estradiol level on the day of GnRHa triggering was 7829.9 pg/ml (±3297; 95% CI 6775-8885). The cumulated ongoing pregnancy rate after 3 frozen-thawed embryo transfers was 75.0% (95% CI 61.6%-88.4%). CONCLUSION: The results suggest that corifollitropin alfa/GnRH antagonist protocol can be used in PCOS patients, in combination with GnRHa triggering and embryo cryopreservation.

Preimplantation genetic diagnosis and screening: Current status and future challenges.

Preimplantation genetic diagnosis (PGD) is a clinically feasible technology to prevent the transmission of monogenic inherited disorders in families afflicted the diseases to the future offsprings. The major technical hurdle is it does not have a general formula for all mutations, thus different gene locus needs individualized, customized design to make the diagnosis accurate enough to be applied on PGD, in which the quantity of DNA is scarce, whereas timely result is sometimes requested if fresh embryo transfer is desired. On the other hand, preimplantation genetic screening (PGS) screens embryo with aneuploidy and was also known as PGD-A (A denotes aneuploidy) in order to enhance the implantation rates as well as livebirth rates. In contrasts to PGD, PGS is still under ferocious debate, especially recent reports found that euploid babies were born after transferring the aneuploid embryos diagnosed by PGS back to the womb and only very few randomized trials of PGS are available in the literature. We have been doing PGD and/or PGS for more than 10 years as one of the core PGD/PGS laboratories in Taiwan. Here we provide a concise review of PGD/PGS regarding its current status, both domestically and globally, as well as its future challenges.

Iron suppresses ovarian granulosa cell proliferation and arrests cell cycle through regulating p38 mitogen-activated protein kinase/p53/p21 pathway.

Iron is an essential nutrient that may exert toxic effects when it accumulates in tissues. Little is known regarding its effects on gonadal function. Both Fe2+ and Fe3+ could be released from iron deposition. We employed mouse nonluteinized granulosa cell for in vitro studies and human ovarian tissues for Prussian blue and immunohistochemical staining to identify the iron deposition and effect in vivo. After treatment with FeSO4-7H2O or FeCl3 in granulosa cell cultured with follicle-stimulating hormone (FSH) for 48 h, we found that Fe2+ significantly suppressed FSH-induced granulosa cell proliferation and arrested the cell cycle at the G2/M phase by cell proliferation assay and flow cytometry. Fe2+ significantly increased intracellular reactive oxygen species (ROS) and ferritin levels of mouse granulosa cells. The increases in p21 and p53 messenger RNA and protein expression facilitated by Fe2+ treatment in mouse granulosa cells were significantly suppressed by separate treatments with p53 small interfering RNA and p38 mitogen-activated protein kinase (MAPK) inhibitors. An ROS inhibitor downregulated Fe2+-induced increases in p38MAPK expression in mouse granulosa cells. Quantitative analysis of immunohistochemical staining revealed that human ovarian tissue sections with positive Prussian blue staining had lower levels of proliferating cell nuclear antigen expression, but higher levels of p21, p53, and CDC25C expression than those with negative Prussian blue staining. Conclusively, Fe2+ could directly arrest the cell cycle and inhibit granulosa cell proliferation by regulating the ROS-mediated p38MAPK/p53/p21 pathway. Therefore, iron can directly affect female gonadal function.

Correlation of serum anti-Mullerian hormone to follicular follicle stimulating hormone and implantation potential of the ensuing embryos.

Serum anti-Mullerian hormone (AMH) is a predictor of the pregnancy outcome in assisted reproductive technology (ART) cycles, particularly for older women. This study attempts to elucidate the effect of serum AMH on follicular hormone profiles and implantation outcome of the ensuing embryos in ART cycles. A total of 412 patients undergoing ART cycles at a private infertility center were included and 780 follicular fluid samples were collected. Levels of follicular hormones, including FSH, LH, inhibin B, AMH, estradiol, progesterone, and androstenedione were measured. The implantation outcome of the ensuing embryos was traced as a main outcome measure. We demonstrated that the follicular levels of estradiol, progesterone, and androstenedione were considerably similar, but the overall implantation rates increased as the serum AMH increased. Logistic regression analysis revealed that the best predictor for embryo implantation was follicular FSH (>9.64mIU/mL, odds ratio [OR] 0.9). Furthermore, serum AMH (Spearman's rho=-0.352) and female age (rho=0.369) are correlated with follicular FSH levels in those follicles. In conclusion, the serum AMH might affect follicular hormone profiles by interaction with gonadotrophin rather than with steroidogenesis. The follicular FSH levels are correlated with the implantation potential of the ensuing embryos.

Management of endometrial polyps incidentally diagnosed during IVF: a case-control study.

An endometrial polyp is a frequently encountered abnormality of the uterine cavity that may interfere with normal embryo implantation. In this case-control study, we enrolled 56 women in whom endometrial polyps were incidentally diagnosed by transvaginal ultrasound and office hysteroscopy during IVF (Group 1), and 112 age-matched IVF controls randomly selected from the same time period (group 2). Cryopreserved embryos were transferred in group 1 whereas fresh embryos were transferred in group 2, which is a limitation of the study. Hysteroscopic polypectomy was carried out for those in group 1, followed by vitrified-warmed embryo transfer 1-7 months later. Results revealed that the clinical pregnancy rate was higher in group 1 than in group 2 (63% versus 41%, P = 0.009), but the embryo implantation rates were not different between the two groups (26% versus 20%). In group 1, pregnancy rates (64%, 69%, and 53% respectively) and embryo implantation rates (30%, 24%, and 23%, respectively) were similar among women that received vitrified-warmed embryo transfer at 1, 2, and 3 months or over after hysteroscopic polypectomy. We conclude that, for women with endometrial polyps incidentally diagnosed during IVF, pregnancy outcomes are not compromised after hysteroscopic polypectomy followed by vitrified-warmed embryo transfer.

High Estradiol Concentrations Induce Heat Shock Protein 70 Expression and Suppress Nuclear Factor Kappa B Activation in Human Endometrial Epithelial Cells.

The high serum estradiol (E2) concentrations induced during in vitro fertilization are detrimental to endometrial receptivity and may result in lower embryo implantation rates. We have previously found that high E2 concentrations inhibit the activation of nuclear factor kappa B (NF-kappa B), which led to endometrial epithelial cells (EECs) apoptosis. The objective of this study is to investigate the signaling pathways through which high E2 results in NF-kappa B downregulation in EECs. Isolated human EECs were cultured in different concentrations of E2 (10-10, 10-9, 10-8, 10-7 M). The expression of heat shock protein 70 (Hsp70) and heat shock factor 1 (HSF-1) were upregulated under supraphysiological E2 (10-7 M) concentration, whereas phosphorylated inhibitory kappa B-alpha (pI kappa B-alpha) and NF-kappa B p65 subunits were downregulated. Immunohistochemistry of C57BL/6 mouse EECs, that were exposed in vivo to high serum E2 from the administration of 20 IUs of equine chorionic gonadotropin, also demonstrated the same increase in HSF-1 and Hsp70 expression, and decrease in NF-kappa B. Immunoprecipitation of the induced Hsp70 proteins was achieved with the addition of inhibitory kappa B kinase gamma (IKK-gamma) antibodies, and elimination of this reaction occurred after addition of hsp70 siRNA. In conclusion, high E2 concentrations enhance HSF-1 and Hsp70 expression in EECs. The induced Hsp70 forms a complex with IKK-gamma and inhibits pI kappa B-alpha, which consequently suppresses NF-kappa B activation.

Frequency of low serum LH is associated with increased early pregnancy loss in IVF/ICSI cycles.

The role of LH during ovarian stimulation remains uncertain. Previous studies defined the low LH group using a single LH measurement on a predefined day of stimulation possibly not reflecting the entire follicular phase. This study retrospectively collected data from 619 IVF/ICSI cycles with GnRH antagonist and recombinant FSH. The low LH group was defined as LH concentration ≤0.8 mIU/ml at any time during the cycle. Pregnancy results were compared between patients with one episode of low LH or more than two episodes of low LH (study group) and those without low LH (control group). There was no difference in fertilization rates between the two groups (67.5 ± 1.7% versus 68.8 ± 1.0%, respectively). The implantation rates (20.4% versus 25.2%), clinical pregnancy rates (43.9% versus 45.2%) and live-birth rates (LBR) (23.7% versus 30.4%) appeared lower in the study group, but the differences were not significant. In the study group, there were significantly increased early pregnancy loss rates (31.1% versus 16.3%, P = 0.012). The odds of early pregnancy loss increases by 1.55 fold for increased episodes of low serum LH (P = 0.029). Whether the adverse outcome is due to impaired oocyte quality or an endometrial component deserves further investigation.

A pilot proof-of-principle study to compare fresh and vitrified cycle preimplantation genetic screening by chromosome microarray and next generation sequencing.

BACKGROUND: Single embryo transfer (SET) has been utilized as a strategy to reduce the chance of multifetal gestations in in vitro fertilization (IVF) but lower pregnancy rate remains a concern. Recent studies showed that favorable outcome regarding SET can be achieved by selecting embryos with "more normal" genetic components. We explored the use of rapid array comparative genomic hybridization (aCGH) to select blastocysts for fresh SET and compared with the protocols adopting vitrified (ultrarapidly frozen) embryo transfer cycle. Validation of the rapid protocol of aCGH and comparison of the result with the regular protocol of aCGH and next generation sequencing (NGS) are also performed. RESULTS: First-time IVF patients with normal karyotype (n = 21) were enrolled for elective fresh SET cycle (n = 8; designated as fresh SET group) or vitrified embryo transfer cycle (n = 13; designated as vitrified ET group) coupling with comprehensive chromosomal screening by a 9-h rapid aCGH from Day 5 trophectoderm (TE) biopsy. In fresh SET group, 86 blastocysts (10.8 blastocysts/patient) were biopsied and analyzed. Aneuploidy was detected in 53.5 % (46/86) of the biopsied blastocysts. All patients had a single embryo transferred on the following day. The clinical pregnancy rate was 87.5 % (7/8) and the ongoing pregnancy rate was 62.5 % (5/8). In vitrified ET group, 58 blastocysts (4.5 blastocysts/patient) were biopsied and 56 blastocysts were analyzed. Aneuploidy was detected in 39.3 % (22/56) of biopsies. The patients accepted for SET or double embryos transfer (DET) in non-stimulated cycles. The clinical pregnancy rate and the ongoing pregnancy rate was 76.9 % (10/13) and 53.8 % (7/13) respectively. Spontaneous abortions occurred in both of the two patient groups. In the series of fresh SET group, no twin pregnancy was noted and at least one healthy baby had been born at gestational age (GA) 37(+6) weeks when submission. The results of PGS by rapid aCGH, regular aCGH and NGS were comparable in most occasions. CONCLUSION: This study evaluates the use of rapid aCGH to select blastocysts for fresh SET and demonstrates its feasibility in a real clinical IVF program. A successful livebirth is achieved and the favorable outcome is superior to the protocol adopting vitrified ET cycle in our own setting. Additional studies are needed to verify this pilot data and validate its application in large randomized trials.

The effect of androgens on ovarian follicle maturation: Dihydrotestosterone suppress FSH-stimulated granulosa cell proliferation by upregulating PPARγ-dependent PTEN expression.

Intraovarian hyperandrogenism is one of the determining factors of follicular arrest in women with polycystic ovary syndrome (PCOS). Using androgenized rat models, we investigated the effects of androgens on metabolism, as well as on factors involved in follicular arrest and the reduced number of estrus cycles. The dihydrotestosterone (DHT)-treated rats had fewer estrus cycles, higher numbers of large arrested follicles and an increased in body weight gain compared with the dehydroepiandrostenedione (DHEA)- and placebo-treated rats. In cultured rat granulosa cells, DHT suppressed follicle stimulating hormone (FSH)-induced granulosa cell proliferation and increased the accumulation of cells in the G2/M phase. DHT decreased phosphorylated Akt (p-Akt) and cyclin D1 levels through increasing PTEN. DHT-promoted PTEN expression was regulated by peroxisome proliferator-activated receptor gamma (PPARγ) in granulosa cells. Meanwhile, in the large follicles of the DHT-treated rats, the expressions of PPARγ and PTEN were higher, but the expression of p-Akt and proliferating cell nuclear antigen (PCNA) were lower. Conclusively, DHT and DHEA produced differential effects on metabolism in prepubertal female rats like clinical manifestations of women with PCOS. DHT treatment may affect ovarian follicular maturation by altering granulosa cell proliferation through the regulation of enhancing PPARγ dependent PTEN/p-Akt expression in the granulosa cells.

Factors Influencing the Recurrence Potential of Benign Endometrial Polyps after Hysteroscopic Polypectomy.

BACKGROUND: An endometrial polyp is a frequently encountered gynecologic disease with abnormal uterine bleeding and infertility being the two common presenting problems, and hysteroscopic polypectomy is an effective method to remove them. The postoperative polyp recurrence might result in reappearance of abnormal uterine bleeding or infertility, whereas factors influencing the postoperative recurrence potential have limited data. METHODS: This case-series report included 168 premenopausal women who suffered from endometrial polyps and underwent hysteroscopic polypectomy. All of them were awaiting a future pregnancy. Office hysteroscopy was done before and after hysteroscopic polypectomy, in which preoperative hysteroscopy examined the number, type, and location of endometrial polyps, and postoperative hysteroscopy checked the polyp recurrence. Surgical indications, either infertility or the presentation of abnormal uterine bleeding, and follow-up duration were recorded. RESULTS: Seventy-three out of 168 (43%) women had polyp recurrence after hysteroscopic polypectomy. Multivariate logistic regression analysis revealed that more endometrial polyps (P = 0.015) and longer duration of follow-up (P = 0.004) were significantly associated with an increased risk of postoperative polyp recurrence. The type of endometrial polyps was not correlated with polyp recurrence potential, whereas pedunculated type endometrial polyps were closely related to the presentation of abnormal uterine bleeding (P = 0.001). CONCLUSIONS: A higher number of endometrial polyps and longer follow-up duration are associated with a greater potential of polyp recurrence after hysteroscopic polypectomy.

Iron stores and obesity are negatively associated with ovarian volume and anti-Müllerian hormone levels in women with polycystic ovary syndrome.

OBJECTIVE: Obesity and insulin resistance are associated with increased iron stores, but have conflicting effects on ovarian reserve in women with polycystic ovary syndrome (PCOS). Iron-catalyzed oxidative stress might be detrimental to ovarian tissue and granulosa cell function. In this study we determined the association between body iron stores, obesity, and ovarian reserve in women with PCOS. MATERIALS AND METHODS: One hundred and fifty-six women diagnosed with PCOS according to Rotterdam criteria and 30 normoweight healthy control women were enrolled in this cross-sectional study. Ovarian volume, total antral follicle count, and the anti-Müllerian hormone (AMH) level were measured as an indicator of ovarian reserve. RESULTS: Ferritin and transferrin-bound iron levels were significantly higher in women with PCOS than normoweight controls. Obese women with PCOS had higher ferritin levels (p = 0.006), but lower AMH levels (p < 0.0001) than nonobese women with PCOS. Using univariate analysis, the AMH level and mean ovarian volume were inversely related to the ferritin level, homeostasis model assessment of insulin resistance, and body mass index in women with PCOS. Body mass index and ferritin level remained significantly correlated with a lower AMH level and reduced ovarian volume, respectively, after considering other confounding variables. CONCLUSION: An elevated ferritin level and obesity were negatively associated with ovarian volume and the AMH level, respectively, in women with PCOS.

Functional microarray analysis of differentially expressed genes in granulosa cells from women with polycystic ovary syndrome related to MAPK/ERK signaling.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. Although its aetiology and pathogenesis remain unclear, recent studies suggest that the dysfunction of granulosa cells may partly be responsible. This study aimed to use cDNA microarray technology to compare granulosa cell gene expression profiles in women with and without PCOS to identify genes that may be aetiologically implicated in the pathogenesis of PCOS. The study cohort included 12 women undergoing in vitro fertilization, six with PCOS and six without PCOS. Differential gene expression profiles were classified by post-analyses of microarray data, followed by western blot analyses to confirm the microarray data of selected genes. In total, 243 genes were differentially expressed (125 upregulated and 118 downregulated) between the PCOS and non-PCOS granulosa cells. These genes are involved in reproductive system development, amino acid metabolism and cellular development and proliferation. Comparative analysis revealed genes involved in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) signaling pathways. Western blot analyses confirmed that mitogen-activated protein kinase kinase kinase 4 and phospho-ERK1/2 were decreased in PCOS granulosa cells. This study identified candidate genes involved in MAPK/ERK signaling pathways that may influence the function of granulosa cells in PCOS.

Divergent endometrial inflammatory cytokine expression at peri-implantation period and after the stimulation by copper intrauterine device.

Endometrial inflammation has contradictory effects. The one occurring at peri-implantation period is favourable for embryo implantation, whereas the other occurring after the stimulation by copper intrauterine device (Cu-IUD) prevents from embryo implantation. In this study, 8 week female ICR mice were used to investigate the endometrial inflammation, in which they were at proestrus stage (Group 1), at peri-implantation period (Group 2), and had a copper wire implanted into right uterine horn (Group 3). Cytokine array revealed that two cytokines were highly expressed in Group 2 and Group 3 as compared with Group 1, and seven cytokines, including tumour necrosis factor α (TNF-α), had selectively strong expression in Group 3. Immunohistochemistry demonstrated prominent TNF-α staining on the endometrium after Cu-IUD stimulation, and in vitro culture of human endometrial glandular cells with Cu induced TNF-α secretion. The increased TNF-α concentration enhanced in vitro THP-1 cells chemotaxis, and reduced embryo implantation rates. These results suggest that inflammatory cytokine profiles of endometrium are different between those at peri-implantation period and after Cu-IUD stimulation, and TNF-α is the one with selectively strong expression in the latter. It might account for the contradictory biological effects of endometrial inflammation.

Preimplantation genetic screening of blastocysts by multiplex qPCR followed by fresh embryo transfer: validation and verification.

BACKGROUND: Aneuploidy is an important etiology of implantation failure and quantitative real-time polymerase chain reaction (qPCR) seems a promising preimplantation genetic screening (PGS) technology to detect aneuploidies. This verification study aimed at verifying the impact on reproductive outcomes in in vitro fertilization (IVF) cycles using fresh embryo transfer (FET) in which the embryos were selected by blastocyst biopsy with qPCR-based PGS in our settings. RESULTS: A total of 13 infertile couples with more than once failed in vitro fertilization were enrolled during July to October of 2014. PGS was conducted by qPCR with selectively amplified markers to detect common aneuploidies (chromosomes 13, 18, 21, X, and Y). The design of the qPCR molecular markers adopted the locked nucleic acid (LNA) strategy. The blastocyst biopsy was performed on Day 5/6 and the PGS was done on the same day, which enabled FET. A total of 72 blastocysts were biopsied. Successful diagnoses were established in all embryos and the rate of successful diagnosis was 100 %. The aneuploidy rate was 38.9 % (28/72). 28 embryos were transferred. The clinical pregnancy rate was 61.5 % (8/13) per cycle. Early first trimester abortion was encountered in 1 and the ongoing pregnancy rate was 53.8 % (7/13) per cycle. CONCLUSION: This study verified the favorable outcome of adopting PGS with qPCR + FET in our own setting. Expanding the repertoire of aneuploidies being investigated (from a limited set to all 24 chromosomes) is underway and a randomized study by comparing qPCR and other PGS technologies is warranted.

Concurrent exome-targeted next-generation sequencing and single nucleotide polymorphism array to identify the causative genetic aberrations of isolated Mayer-Rokitansky-Küster-Hauser syndrome.

STUDY QUESTION: Can the use of whole-exome sequencing (WES) together with single nucleotide polymorphism (SNP) array help to identify novel causative genes of isolated Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome? SUMMARY ANSWER: OR4M2 (olfactory receptor, family 4, subfamily M, member 2) and PDE11A (phosphodiesterase 11A) gene loss-of-function variants as well as deletions at 15q11.2, 19q13.31, 1p36.21, and 1q44 were identified as possible commonly altered regions in patients with type 1 MRKH. WHAT IS KNOWN ALREADY: The isolated form of Müllerian aplasia is the most common subtype of MRKH syndrome, which invariably leads to difficulties producing offspring in affected women. However, there is little information currently available to allow for genetic testing and counseling to be performed for those affected by this syndrome. STUDY DESIGN, SIZE AND DURATION: This was a case-series genetic study. A total of seven consecutive unrelated women with type 1 MRKH were enrolled. The enrollment and experimental procedures were performed over a 2-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole exome-targeted next-generation sequencing and SNP array (Affymetrix Genome-Wide Human SNP Array 6.0) were performed on the first five unrelated women with type 1 MRKH syndrome. The data were combined, and the '3-hit principal' was applied on a genome-wide scale to search for the common causative genes. Quantitative PCR (qPCR) and Sanger sequencing were used to validate the identified genomic copy number losses and variants. Replication tests using direct Sanger sequencing and qPCR were performed on the remaining two women with type 1 MRKH syndrome to support the credibility of the potential candidate genes and deletions. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 3443 damaging variants based on WES were shown to intersect with 1336 copy number variations (deletions) derived from the SNP array. Four highly recurrent deletions at 15q11.2 (80%), 19q13.31 (40%), 1p36.21 (40%) and 1q44 (40%) were identified in the first five women with type 1 MRKH syndrome and were considered to be novel candidate aberrations. A previously reported 1q21.1 deletion was also recurrent in two of the first five women with type 1 MRKH syndrome. The 1q44 and 19q13.31 deletions were present in at least one of the two additional patients. Damaging variants were detected in HNRNPCL1 (heterogeneous nuclear ribonucleoprotein C-like 1), OR2T2 (olfactory receptor, family 2, subfamily T, member 2), OR4M2, ZNF816 (zinc finger protein 816), and PDE11A in several of the initial five patients. Among these, the damaging variants of OR4M2 (located at 15q11.2) and PDE11A were found in at least one of the two additional patients with type 1 MRKH. LIMITATIONS, REASONS FOR CAUTION: In this study, we only searched for the deletions or damaging variants causing loss-of-function of genes in at least three of the initial five patients (3-hit criteria). Therefore, the study was designed to only detect common causative genes. Genomic duplications and/or rare individual mutations that may have also contributed to MRKH syndrome were not investigated. WIDER IMPLICATIONS OF THE FINDINGS: This study demonstrated the feasibility of the use of combined data from both WES and SNP arrays for the identification of possible common causative genetic aberrations in patients with type 1 MRKH syndrome on a genome-wide scale. Further validation of our found causative genes is required before applying on genetic testing and counseling. STUDY FUNDING/COMPETING INTERESTS: The study was supported by grants from the National Science Council of Taiwan (NSC98-2314-B002-105-MY3 and NSC 100-2314-B002-027-MY3). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.