Advanced glycation end-products and its soluble receptor are not independent predictors of incident dysglycaemia or metabolic syndrome in women with polycystic ovary syndrome: a prospective observational study.

BACKGROUND: To evaluate the association of serum advanced glycation end-products (AGEs) and its soluble receptor of AGE (sRAGE) levels with dysglycaemia and metabolic syndrome in women with polycystic ovary syndrome (PCOS). METHODS: This was an analysis of a cohort of women with PCOS who were prospectively recruited for a longitudinal observational study on their endocrine and metabolic profile between January 2010 and December 2013. The association of serum AGEs and sRAGE levels with dysglycaemia and metabolic syndrome at the second-year visit (the index visit) and the sixth-year visit (the outcome visit) were determined. Comparisons of continuous variables between groups were made using the Mann-Whitney U-test. Spearman test was used for correlation analysis. Multivariate binary logistic regression analysis was employed to identify the factors independently associated with the outcome events. RESULTS: A total of 329 women were analysed at the index visit. Significantly lower serum levels of sRAGE (both p < 0.001), but no significant difference in AGEs, were observed in those with dysglycaemia or metabolic syndrome. At the outcome visit, those with incident metabolic syndrome had a significantly lower initial serum sRAGE levels (p = 0.008). The association of serum sRAGE with dysglycaemia and metabolic syndrome at the index visit was no longer significant in multivariate logistic regression after controlling for body mass index, free androgen index and homeostatic model assessment for insulin resistance (HOMA-IR). sRAGE was also not significantly associated with incident metabolic syndrome at the outcome visit on multivariate logistic regression. CONCLUSIONS: Serum sRAGE levels are significantly lower in women with PCOS who have dysglycaemia or metabolic syndrome, and in those developing incident metabolic syndrome in four years. However, it does not have a significant independent association with these outcome measures after adjusting for body mass index, free androgen index and HOMA-IR.

Independent association of serum vitamin D with anti-Mullerian hormone levels in women with polycystic ovary syndrome.

OBJECTIVE: This study aimed at investigating the association of serum vitamin D (25(OH)D) and anti-Mullerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS) as well as non-PCOS healthy ovulatory women and the possible confounding effects of adiposity and androgen. METHOD: This was a cross-sectional study conducted on serum samples collected from 451 women diagnosed with PCOS as well as 244 age-matched healthy ovulatory women in a tertiary gynaecology out-patient clinic and a family planning clinic. RESULTS: Serum 25(OH)D level was significantly higher in women recruited during summer and autumn than those recruited in winter and spring. Both serum 25(OH)D and AMH levels peaked during summer in women with PCOS. In ovulatory women, only serum 25(OH)D but not AMH level showed such seasonal variation. Serum 25(OH)D level in women with PCOS significantly correlated positively with AMH, AMH/antral follicle count (AFC) ratio, serum total testosterone, sex-hormone-binding globulin and quantitative insulin-sensitivity check index and inversely with body mass index (BMI), insulin, triglycerides and homeostatic model assessment of insulin resistance. After controlling for BMI, 25(OH)D level remained significantly correlated positively with serum AMH, AMH/AFC and total testosterone, and inversely with triglycerides. 25(OH)D level was an independent predictor of serum AMH level after controlling for age, BMI and free androgen index in women with PCOS. CONCLUSION: Serum 25(OH)D level is an independent factor significantly associated with AMH level in women with PCOS but not in ovulatory women.

Effect of ulipristal acetate and mifepristone at emergency contraception dose on the embryo-endometrial attachment using an in vitro human trophoblastic spheroid and endometrial cell co-culture model.

STUDY QUESTION: Do both ulipristal acetate (UPA) and mifepristone inhibit embryo-endometrial attachment at concentrations corresponding to the emergency contraception (EC) dose? SUMMARY ANSWER: Both UPA and mifepristone at concentrations corresponding to the EC dose do not have an inhibitory effect on embryo implantation, although mifepristone at a higher concentration appeared to have such an effect. WHAT IS KNOWN ALREADY: Levonorgestrel is commonly used for EC, but it only acts through inhibition of ovulation. UPA and mifepristone have higher efficacy as EC compared to levonorgestrel; while there is some suggestion that mifepristone may interfere with implantation, whether UPA has post-ovulatory action in inhibiting implantation is yet to be confirmed. STUDY DESIGN, SIZE, DURATION: An in vitro experimental study using trophoblastic spheroids made from JAr cell line as the embryo surrogate, and the Ishikawa cell line and primary human endometrial cells cultured to monolayer as the endometrial surrogate. The primary endometrial cells were collected from nine volunteer women in the mid-luteal phase with consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted in a university gynaecology unit. The JAr and Ishikawa cell lines (or primary endometrial cells) were treated with graded concentrations of UPA (0, 0.04, 0.4 and 4 µM) or mifepristone (0, 0.1, 1 and 10 µM) for 24 h. Embryo-endometrial attachment was studied using an in vitro JAr spheroid-endometrial co-culture model. Expressions of progesterone receptor, ß-catenin and glycogen synthase kinase 3 ß (GSK-3ß) were studied with real-time RT-PCR and Western blotting, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: In the Ishikawa experiments, there was no significant difference in the JAr spheroid attachment rate after treatment with UPA at 0 (93.0%), 0.04 (93.6%), 0.4 (93.4%) and 4 (91.4%) µM concentrations (P > 0.05); the attachment rate was reduced after treatment with mifepristone only at 10 µM (79.8%, P < 0.0001) but not at 0.1 (92.1%) or 1.0 (95.2%) µM concentrations. In the primary endometrial cell experiments, again no significant difference was observed in the JAr spheroid attachment rate after treatment with UPA 4 µM (42.6%) compared to control (46.5%, P > 0.05). Both UPA and mifepristone could significantly up-regulate progesterone receptor expression. There was no significant alteration in expression of ß-catenin and GSK-3ß after treatment with UPA 4 µM or mifepristone 10 µM (P > 0.05). LIMITATIONS, REASONS FOR CAUTION: The co-culture model is only a surrogate which may not fully represent the complicated process of embryo implantation in vivo, although there is no existing perfect model for studying implantation in vitro which fully resembles the latter. WIDER IMPLICATIONS OF THE FINDINGS: The lack of inhibitory effect on embryo implantation by UPA and possibly mifepristone at concentrations corresponding to the EC dose is an important information for contraceptive counseling. STUDY FUNDING/COMPETING INTEREST(S): We had free supply of the UPA compound used in this study from Laboratoire HRA Pharma. This work was supported by a Seed Fund from the Centre of Reproduction, Development and Growth, Faculty of Medicine, The University of Hong Kong, Hong Kong.

Serum adiponectin is independently associated with the metabolic syndrome in Hong Kong, Chinese women with polycystic ovary syndrome.

OBJECTIVE: To evaluate the association of serum adiponectin level with the metabolic syndrome in Chinese women with polycystic ovary syndrome (PCOS). METHODS: This was a cross-sectional study carried out in Hong Kong Chinese women with PCOS at a university-affiliated tertiary hospital between January 2010 and January 2011. Clinical and biochemical parameters of the women were analysed. Prediction of the metabolic syndrome was determined by receiver-operator characteristic (ROC) curves, univariate and multivariate logistic regression analyses. RESULTS: A total of 116 women diagnosed to have PCOS were analysed. The area under the ROC curve of adiponectin for the prediction of metabolic syndrome was 0.820, 95% confidence interval (CI) 0.737-0.886. Univariate binary logistic regression showed that testosterone, sex hormone-binding globulin (SHBG), free androgen index (FAI), waist circumference, body mass index (BMI), quantitative insulin-sensitivity check index (QUICKI), homeostasis model assessment of insulin resistance (HOMA-IR) and adiponectin were significantly associated with the metabolic syndrome. On multivariate logistic regression analysis, adiponectin (p = 0.020), HOMA-IR, age (p = 0.011) and BMI (p = 0.019) were independently associated with the metabolic syndrome, but not FAI (p = 0.256). CONCLUSIONS: Serum adiponectin is independently associated with the metabolic syndrome in Chinese women with PCOS. Further longitudinal follow-up studies are needed to determine whether serum adiponectin adds to the prediction of long-term cardiometabolic morbidity conferred by age, BMI and measures of insulin resistance.

The effect of 7 days of letrozole pretreatment combined with misoprostol on the expression of progesterone receptor and apoptotic factors of placental and decidual tissues from first-trimester abortion: a randomized controlled trial.

OBJECTIVE: To evaluate if letrozole-induced suppression of estradiol reduces progesterone receptor expression and apoptosis in the first-trimester placenta. STUDY DESIGN: We performed a double-blinded, randomized, placebo-controlled trial. We randomized 20 women requesting first-trimester abortion with gestation up to 63 days to receive either letrozole 10 mg daily or placebo pretreatment for 7 days before administrating 400 mcg of vaginal misoprostol followed by suction abortion. We collected the placental and decidual tissues on which we performed immunohistochemical staining for progesterone receptor and apoptotic markers (active caspase 3, caspase 3, Bcl2, CD95, fas ligand) and determined H-scores of each based on the intensities of staining. We performed terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) assay for apoptosis in the samples of four women to confirm the findings from apoptotic markers. RESULTS: We excluded one woman in the letrozole group from the analysis because she had passage of abortus after taking letrozole, leaving 19 women (9 in the letrozole group, 10 in the placebo group) for analysis. There was no significant difference in the H-scorings of progesterone receptor and apoptotic markers, as well as proportion of apoptotic cells on TUNEL assay between the two groups. The H-scores for the progesterone receptor were 8.17 ± 2.67 (mean ± SD) in the letrozole group and 9.01 ± 2.82 in the placebo group (p=0.36). CONCLUSION: We did not detect a difference in the expression of progesterone receptor and apoptotic markers in placental and decidual tissues after letrozole pretreatment for 7 days in first-trimester abortion. IMPLICATIONS: We did not confirm the hypothesis that letrozole reduces progesterone receptor expression and induces apoptosis in the first-trimester placenta. Further studies are required to allow better understanding of the mechanism by which estrogen suppression following the use of letrozole can lead to improved abortion rate in the first trimester.

Sperm fucosyltransferase-5 mediates spermatozoa-oviductal epithelial cell interaction to protect human spermatozoa from oxidative damage.

Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. Interaction between spermatozoa and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. Our previous data showed that oviductal epithelial cell membrane proteins interact with the human spermatozoa and protect them from ROS-induced reduction in sperm motility, membrane integrity and DNA integrity. Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human spermatozoa. In this study, we demonstrate for the first time that sFUT5 is involved in human spermatozoa-oviduct interaction and the beneficial effects of such interaction on the fertilizing ability of human spermatozoa. Anti-sFUT5 antibody-treated spermatozoa had reduced binding to oviductal membrane proteins. It is consistent with the result that affinity-purified sFUT5 is bound to the epithelial lining of human oviduct and to the immortalized human oviductal epithelial cell line, OE-E6/E7. Pretreatment of spermatozoa with anti-sFUT5 antibody and oviductal membrane proteins with sFUT5 suppressed the protective action of oviductal membrane proteins against ROS/cryopreservation-induced oxidative damage in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of oviductal epithelial cell membrane proteins on sperm motility, membrane and DNA integrity. The results enhance our understanding on the protective mechanism of oviduct on sperm functions.

Preimplantation genetic diagnosis using combined strategies on a breast cancer patient with a novel genomic deletion in BRCA2.

PURPOSE: To perform Preimplantation Genetic Diagnosis (PGD) on a paternal Brca2 unknown mutation carrier with early-onset breast cancer, whose paternal grandmother and mother had breast cancer at 60s. METHOD: Elucidating the linkage via single sperm haplotyping on patient's carrier brother, and identifying the genomic deletion via BLAST followed by PCR screening. PGD was subsequently conducted. RESULT: The mutant allele was found by using 4 microsatellite and 2 intragenic SNP markers. Recombination was detected in 8% of sperms. BLAST was utilized to locate putative hairpin structure(s), followed by PCR screening with seven sets of primers. A novel 2,596 bp deletion containing exon 15 ~ 16 was identified. Due to the severity of phenotype and the integrity of exon 11 encoding RAD51 binding domain, and the fact that the patient's mother also had breast cancer at her 60s, we speculate a possible coexistence of maternal breast cancer risk allele(s). Embryo biopsy was performed on day 3. Unaffected morula and blastocyst were replaced on day 5, resulting in a singleton livebirth. A breast lump appeared in the patient after delivery without the presence of malignant cells. CONCLUSION: Concerning the assisted reproductive option for breast cancer patients, the possibility of coexistence of multiple familial risk alleles and the significance of each mutation to the phenotype should be evaluated. To eliminate misdiagnosis resulting from recombination and/or allelic drop-out, both direct mutation detection and linkage analysis approaches may be necessary. BLAST is a very useful and cost-effective tool for identifying large genomic deletion.

Ovarian response and cumulative live birth rate of women undergoing in-vitro fertilisation who had discordant anti-Mullerian hormone and antral follicle count measurements: a retrospective study.

OBJECTIVE: To evaluate ovarian response and cumulative live birth rate of women undergoing in-vitro fertilization (IVF) treatment who had discordant baseline serum anti-Mullerian hormone (AMH) level and antral follicle count (AFC). METHODS: This is a retrospective cohort study on 1,046 women undergoing the first IVF cycle in Queen Mary Hospital, Hong Kong. Subjects receiving standard IVF treatment with the GnRH agonist long protocol were classified according to their quartiles of baseline AMH and AFC measurements after GnRH agonist down-regulation and before commencing ovarian stimulation. The number of retrieved oocytes, ovarian sensitivity index (OSI) and cumulative live-birth rate for each classification category were compared. RESULTS: Among our studied subjects, 32.2% were discordant in their AMH and AFC quartiles. Among them, those having higher AMH within the same AFC quartile had higher number of retrieved oocytes and cumulative live-birth rate. Subjects discordant in AMH and AFC had intermediate OSI which differed significantly compared to those concordant in AMH and AFC on either end. OSI of those discordant in AMH and AFC did not differ significantly whether either AMH or AFC quartile was higher than the other. CONCLUSIONS: When AMH and AFC are discordant, the ovarian responsiveness is intermediate between that when both are concordant on either end. Women having higher AMH within the same AFC quartile had higher number of retrieved oocytes and cumulative live-birth rate.

The effect of endometrial injury on ongoing pregnancy rate in unselected subfertile women undergoing in vitro fertilization: a randomized controlled trial.

STUDY QUESTION: Does endometrial injury in the cycle preceding ovarian stimulation for in vitro fertilization (IVF) improve the ongoing pregnancy rate in unselected subfertile women? SUMMARY ANSWER: Endometrial injury induced by endometrial aspiration in the preceding cycle does not improve the ongoing pregnancy rate in unselected subfertile women undergoing IVF. WHAT IS KNOWN ALREADY: Implantation failure remains one of the major limiting factors for IVF success. Mechanical endometrial injury in the cycle preceding ovarian stimulation of IVF treatment has been shown to improve implantation and pregnancy rates in women with repeated implantation failures. There is limited data on unselected subfertile women, especially those undergoing their first IVF treatment. STUDY DESIGN, SIZE, DURATION: This randomized controlled trial recruited 300 unselected subfertile women scheduled for IVF/ICSI treatment between March 2011 and August 2013. Subjects were randomized into endometrial aspiration (EA) (n = 150) and non-EA (n = 150) groups according to a computer-generated randomization list. PARTICIPANTS/MATERIALS, SETTING, METHODS: Subjects were recruited and randomized in the assisted reproductive unit at the University of Hong Kong. In the preceding cycle, women in the EA group underwent endometrial aspiration using a Pipelle catheter in mid-luteal phase. All women were treated with a cycle of IVF/ICSI. Pregnancy outcomes were compared. MAIN RESULTS AND THE ROLE OF CHANCE: There were no significant differences in baseline or cycle characteristics between the groups. There were 209 subjects (69.7%) who were undergoing their first IVF cycle and 91 (30.3%) subjects who had repeated cycles. There was no significant difference in ongoing pregnancy rates [26.7% (40/150) versus 32.0% (48/150); RR 0.833 (95% CI 0.585-1.187), P = 0.375] in the EA and non-EA groups. The implantation rates [32.8% (67/204) versus 29.7% (68/229); RR 1.080 (95% CI 0.804-1.450), P = 0.120], clinical pregnancy rates [34.0% (51/150) versus 38.0 (57/150); RR 0.895 (95% CI 0.661-1.211), P = 0.548], miscarriage rates [30.3% (17/56) versus 18.6% (11/59), RR 1.628 (95% CI 0.838-3.164), P = 0.150] and multiple pregnancy rates [31.3% (16/51) versus 19.3% (11/57), RR 1.626 (95% CI 0.833-3.172), P = 0.154] were all comparable between the EA and non-EA groups. Subgroup analysis in women having first embryo transfer (n = 209) also demonstrated no significant difference in ongoing pregnancy rates, but for women undergoing repeated cycles (n = 91), the on-going pregnancy rate was significantly lower in the EA group than in the non-EA group. LIMITATIONS, REASONS FOR CAUTION: The study aimed at assessing an unselected population of subfertile women by recruiting consecutive women attending our fertility clinic. However, since the majority of the recruited women (69.7%) were having their first IVF treatments, the results may not be generalizable to all women undergoing IVF. WIDER IMPLICATIONS OF THE FINDINGS: Previous RCTs and meta-analyses have suggested improved pregnancy rates after pretreatment endometrial injury in women with repeated implantation failure. A recent RCT also showed increased pregnancy rates in unselected subfertile women after endometrial injury, although that study was terminated early and thus underpowered. Our study showed with adequate power that no significant improvement in pregnancy rates was observed after endometrial injury in unselected women undergoing IVF treatment. STUDY FUNDING/COMPETING INTERESTS: The study was supported by the Small Project Funding 201309176012 of the Committee on Research and Conference Grants, University of Hong Kong. The authors have nothing to disclose. TRIAL REGISTRATION NUMBER: HKCTR-1646 and NCT 01977976.

Effect of preovulatory progesterone elevation and duration of progesterone elevation on the pregnancy rate of frozen-thawed embryo transfer in natural cycles.

OBJECTIVE: To assess the incidence of P elevation (PE) in natural cycles and evaluate its effect on frozen-thawed embryo transfer cycles performed in natural cycles (FET-NC). STUDY DESIGN: Retrospective analysis. SETTING: A tertiary assisted reproductive unit. PATIENT(S): Subfertile woman who did not conceive in their stimulated IVF cycle and underwent the first FET-NC cycle. INTERVENTION(S): Achieved serum samples were assayed for P concentrations from the day of LH surge up to 3 days before the surge. The cutoff level of PE was defined as 5 nmol/L. MAIN OUTCOME MEASURE(S): Clinical and ongoing pregnancy rates. RESULT(S): The incidence of PE in natural cycles was 173 of 610 (28.4%). There were no significant differences in both clinical and ongoing pregnancy rates (39.0% vs. 37.3% and 32.5% vs. 31.7%) between those with vs. without PE on the day of LH surge. If PE lasted for 2 days or more, there was a significant reduction in the clinical pregnancy rate (39.4% vs. 20.7%). Using multivariate logistic regression, women's age, PE for 2 days or more, and the number of top-quality embryos were the significant factors for clinical pregnancy rates in FET-NC. CONCLUSION(S): The incidence of PE in FET-NC was similar to that in stimulated cycles. Progesterone elevation for 2 days or more before the LH surge impaired the clinical pregnancy rate of FET-NC, whereas PE on the day of LH surge only did not have such an adverse effect.

Cumulative live-birth rate in women with polycystic ovary syndrome or isolated polycystic ovaries undergoing in-vitro fertilisation treatment.

PURPOSE: This retrospective cohort study evaluated the cumulative live birth rate in women with polycystic ovary syndrome (PCOS) and isolated polycystic ovaries (PCO) undergoing in-vitro fertilisation (IVF) treatment. METHODS: We studied 104 women with PCOS, 184 with PCO and 576 age-matched controls undergoing the first IVF treatment cycle between 2002 and 2009. The main outcome measure was cumulative live birth in the fresh plus all the frozen embryo transfers combined after the same stimulation cycle. RESULTS: Women in both the PCOS (n = 104) and isolated PCO groups (n = 184) had higher ovarian response parameters compared to age-matched controls (n = 576), and higher rates of withholding fresh embryo transfer for risk of ovarian hyperstimulation syndrome (OHSS). The actual incidence of moderate to severe OHSS was significantly higher in the PCOS (11.5 %) but not the isolated PCO group (8.2%) compared to controls (4.9%). The live birth rates in the fresh cycle were comparable among the 3 groups, but the PCOS group had a significantly higher miscarriage rate compared to the other 2 groups. Cumulative live birth rate was significantly higher in the isolated PCO group (60.3%), but not the PCOS group (50.0%), compared to controls (47.5%). CONCLUSIONS: Women in the isolated PCO group, but not the PCOS group, had a significantly higher cumulative live birth rate compared to controls. This could be explained by the quantitative effect of the higher number of transferable embryos obtained per stimulation cycle, which is uncompromised by the unfavourable embryo competence otherwise observed in PCOS.

The effect of letrozole with misoprostol for medical termination of pregnancy on the expression of steroid receptors in the placenta.

STUDY QUESTION: What is the effect of letrozole on the expression of steroid receptors in the placentae in cases of termination of pregnancies? SUMMARY ANSWER: The expression of estrogen receptor-α (ERα) and progesterone receptor (PR) transcripts, as well as ERα protein, in placentae was suppressed by letrozole pretreatment in second trimester termination of pregnancy. WHAT IS KNOWN ALREADY: There have been no data in the literature on the effect of letrozole in termination of human pregnancies. STUDY DESIGN, SIZE, DURATION: This study is part of a clinical randomized trial in which 50 subjects were recruited and 44 placentae were collected. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women (n = 50) requesting second trimester abortion between 12 and 20 gestational weeks were randomized to receive either letrozole or placebo pretreatment for 3 days before administration of vaginal misoprostol. Placentae were collected from both groups of women after the abortion. Total RNA from the frozen placenta samples was extracted and subjected to real-time RT-PCR analysis of ERα and estrogen receptor-ß (ERß), PR and glucocorticoid receptor (GR) transcripts. Immunohistochemical studies of ERα, ERß, PR and GR expression, as well as Ki67 and PCNA staining for proliferation, were performed. TUNEL assays were performed to determine the extent of apoptosis. MAIN RESULTS AND THE ROLE OF CHANCE: Real-time RT-PCR demonstrated that the median ERα {3.900 [95% confidence interval (CI): -0.643-8.443] in the letrozole group versus 4.714 (95% CI: 1.776-7.652) in the control group; P = 0.005} and the median PR [0.701 (95% CI: 0.333-1.069) in the letrozole group versus 1.774 (95% CI: 1.07-2.478) in the control group; P = 0.003] were significantly lower in the letrozole group compared with the control group. Furthermore, ERα protein levels, in both syncytiotrophoblasts and cytotrophoblasts but not in villous stromal cells, were significantly reduced [H-score of 113 (95% CI: 103-119) in the letrozole group versus 217 (95% CI: 214-290) in the control group, in syncytiotrophoblasts; 100 (95% CI: 98-105) in the letrozole group versus 210 (95% CI: 200-286) in the control group, in cytotrophoblasts; P = 0.004], while the expression levels of ERß, PR, GR, PCNA, Ki67 and TUNEL were not significantly different between the two groups. LIMITATIONS, REASONS FOR CAUTION: Only the placentae from the second trimester termination of pregnancy were collected in this study. Information from first trimester terminations is still lacking. WIDER IMPLICATIONS OF THE FINDINGS: The results shed some light on the mechanism of action of letrozole pretreatment in termination of pregnancies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the GRF/RGC and CRCG grants of the University of Hong Kong. TRIAL REGISTRATION NUMBER: HKClinicalTrials.com with trial number HKCTR-695.

Cell membrane proteins from oviductal epithelial cell line protect human spermatozoa from oxidative damage.

OBJECTIVE: To study the potential protective action in vitro of oviductal epithelial cell membrane proteins against oxidative damage in human spermatozoa. DESIGN: Prospective in vitro study. SETTING: University research laboratory and infertility clinic. PATIENT(S): Semen from men attending the infertility clinic at the Queen Mary Hospital with normal semen parameters (World Health Organization, 2010). INTERVENTION(S): We studied the effect of oviductal epithelial cell membrane proteins on the sperm functions and endogenous antioxidant enzyme activities. MAIN OUTCOME MEASURE(S): Sperm motility, lipid peroxidation, DNA fragmentation, intracellular reactive oxygen species (ROS) level, superoxide dismutase, and glutathione peroxidase activities. RESULT(S): Oviductal epithelial cell membrane proteins bind to the human spermatozoa and protect them from ROS-induced damages in terms of sperm motility, membrane integrity, DNA integrity, and intracellular ROS level. Spermatozoa-oviduct epithelial cell interaction also enhances the antioxidant defenses in spermatozoa. CONCLUSION(S): Our results demonstrated the protective effects of spermatozoon-oviductal epithelial cell interaction against oxidative stress in human spermatozoa. The results enhance our understanding of the protective mechanism of oviduct on sperm functions.

A pilot study on the use of a 7-day course of letrozole followed by misoprostol for the termination of early pregnancy up to 63 days.

BACKGROUND: Our previous randomized controlled study showed that the complete abortion rate in termination of pregnancy up to 63 days with the combined use of letrozole for 3 days followed by vaginal misoprostol was significantly higher than that of misoprostol alone. A positive correlation was observed between the basal estradiol level and the failure rate. We performed this pilot study to assess if a longer course of letrozole followed by misoprostol would improve the estradiol suppression and the complete abortion rate of pregnancy up to 63 days. STUDY DESIGN: Twenty subjects requesting legal termination of pregnancies up to 63 days were recruited. Medical abortion was offered with letrozole 10 mg daily for 7 days followed by vaginal misoprostol 800 mcg on the 7th day. RESULTS: Median induction-to-abortion interval was 7.5 h (range, 4.75-10.75 h). Overall complete abortion rate was 95%. All subjects with gestation ≤49 days (12/12) as well as 87.5% of subjects with gestation between 50 and 63 days (7/8) had complete abortion. No major adverse event were reported and over 88% of women would like to have medical termination as an option should it be required in the future. CONCLUSION: This pilot study showed that a 7-day course of letrozole followed by vaginal misoprostol was associated with a very high complete abortion rate (95%) which is comparable to the standard regimen with sequential use of mifepristone and misoprostol in medical termination of early pregnancy up to 63 days.

Effect of letrozole on uterine tonus and contractility: a randomized controlled trial.

BACKGROUND: Letrozole inhibits estrogen production. It has been shown to increase efficacy in medical abortion when used with misoprostol. This study investigated if letrozole acts as an abortifacient due to a synergistic effect with misoprostol on uterine contractility. STUDY DESIGN: Sixteen healthy women requesting surgical abortion were randomized to receive either no pretreatment or treatment with letrozole 7.5 mg daily for 3 days prior to the abortion. All women received misoprostol 400 mcg vaginally 3.5 h prior to surgery. Intrauterine pressure was measured for 30 min before and 3.5 h after misoprostol was given using an intrauterine pressure catheter. Main outcome measure was uterine contractility analyzed by repeated-measures analysis of variance. RESULTS: At baseline, uterine contractions were absent and tonus was low. No significant difference was seen between the two groups in tonus (p=.818) or in contractility (p=.423) after misoprostol administration. CONCLUSION: Letrozole does not appear to act as an abortifacient through an effect on uterine contractility or increased sensitivity to misoprostol of the uterine myometrium.

Wnt activation downregulates olfactomedin-1 in Fallopian tubal epithelial cells: a microenvironment predisposed to tubal ectopic pregnancy.

Ectopic pregnancy (EP) occurs when the embryo fails to transit to the uterus and attach to the luminal epithelium of the Fallopian tube (FT). Tubal EP is a common gynecological emergency and more than 95% of EP occurs in the ampullary region of the FT. In humans, Wnt activation and downregulation of olfactomedin-1 (Olfm-1) occur in the receptive endometrium and coincided with embryo implantation in vivo. Whether similar molecular changes happen in the FT leading to EP remains unclear. We hypothesized that activation of Wnt signaling downregulates Olfm-1 expression predisposes to EP. We investigated the spatiotemporal expression of Olfm-1 in FT from non-pregnant women and women with EP, and used a novel trophoblastic spheroid (embryo surrogate)-FT epithelial cell co-culture model (JAr and OE-E6/E7 cells) to study the role of Olfm-1 on spheroid attachment. Olfm-1 mRNA expression in the ampullary region of non-pregnant FT was higher (P<0.05) in the follicular phase than in the luteal phase. Ampullary tubal Olfm-1 expression was lower in FT from women with EP compared to normal controls at the luteal phase (histological scoring (H-SCORE)=1.3±0.2 vs 2.4±0.5; P<0.05). Treatment of OE-E6/E7 with recombinant Olfm-1 (0.2-5 µg/ml) suppressed spheroid attachment to OE-E6/E7 cells, while activation of Wnt-signaling pathway by Wnt3a or LiCl reduced endogenous Olfm-1 expression and increased spheroid attachment. Conversely, suppression of Olfm-1 expression by RNAi increased spheroid attachment to OE-E6/E7 cells. Taken together, Wnt activation suppresses Olfm-1 expression, and this may predispose a favorable microenvironment of the retained embryo in the FT, leading to EP in humans.

Glycodelin-A protein interacts with Siglec-6 protein to suppress trophoblast invasiveness by down-regulating extracellular signal-regulated kinase (ERK)/c-Jun signaling pathway.

During placentation, the cytotrophoblast differentiates into the villous cytotrophoblast and the extravillous cytotrophoblast. The latter invades the decidualized endometrium. Glycodelin-A (GdA) is abundantly synthesized by the decidua but not the trophoblast. Previous data indicate that GdA suppresses the invasion of trophoblast cell lines by down-regulating proteinase expression and activities. This study addresses the signaling pathway involved in the above phenomenon. GdA was found to suppress phosphorylation of ERKs and expression of their downstream effector c-Jun, a component of the transcription factor activator protein-1 (AP-1). The involvement of ERKs and c-Jun in suppressing trophoblast invasion and biosynthesis of proteinases was confirmed by using siRNA knockdown and pharmacological inhibitors. Desialylation reduced binding affinity of GdA toward and invasion suppressive activities on the trophoblast. Co-immunoprecipitation showed that Siglec-6 on the trophoblast was the binding protein of GdA. The binding of GdA to Siglec-6 was sialic acid-dependent. Treatment with anti-Siglec-6 antibody abolished the invasion suppressive activities of GdA. These results show that GdA interacts with Siglec-6 to suppress trophoblast invasiveness by down-regulating the ERK/c-Jun signaling pathway.

Evaluation of serum antimullerian hormone and inhibin B concentrations in the differential diagnosis of secondary oligoamenorrhea.

OBJECTIVE: To evaluate the performance of antimullerian hormone (AMH) and inhibin B as ovarian function markers for differentiating common causes of secondary oligoamenorrhea, namely hypogonadotrophic hypogonadism (HH), polycystic ovary syndrome (PCOS), premature ovarian failure (POF), and hyperprolactinemia (HPRL). DESIGN: Retrospective analysis. SETTING: Two university hospitals. PATIENT(S): A total of 124 women with secondary oligoamenorrhea and 26 women with normal ovulation. INTERVENTION(S): Serum samples from the subjects were analyzed for AMH and inhibin B. MAIN OUTCOME MEASURE(S): Serum AMH and inhibin B concentrations. RESULT(S): Serum AMH concentration was significantly raised in women having World Health Organization group 2 anovulation, either with or without PCOS, and was significantly decreased to very low levels in POF; the diagnostic accuracy in both conditions was excellent, with areas under the receiver operating characteristic curve (AUC) of 0.913 and 0.977, respectively. The discriminatory performance between HH and PCOS was also good, with AUC 0.861. AMH remained unchanged in HH and HPRL compared with ovulatory control subjects. There were large overlap of serum inhibin B levels in the different conditions, and a significant difference from control subjects existed only in the POF group. CONCLUSION(S): Serum AMH, but not inhibin B concentration, serves as a useful diagnostic tool in the differential diagnosis of secondary oligoamenorrhea.

Sequential use of letrozole and gonadotrophin in women with poor ovarian reserve: a randomized controlled trial.

Sequential use of letrozole and human menopausal gonadotrophin (HMG) was compared with HMG only in poor ovarian responders undergoing IVF. Patients (n=53) with less than four oocytes retrieved in previous IVF cycles or less than five antral follicles were randomized to either letrozole for 5days followed by HMG or HMG alone. The letrozole group had lower dosage of HMG (P<0.001), shorter duration of HMG (P<0.001) and fewer oocytes (P=0.001) when compared with controls. Live-birth rate was comparable with a lower miscarriage rate in the letrozole group (P=0.038). Serum FSH concentrations were comparable in both groups except on day 8, while oestradiol concentrations were all lower in the letrozole group from day 4 (all P<0.001). Follicular fluid concentrations of testosterone, androstenedione, FSH and anti-Müllerian hormone were higher in the letrozole group (P=0.009, P=0.001, P=0.046 and P=0.034, respectively). Compared with HMG alone, sequential use of letrozole and HMG in poor responders resulted in significantly lower total dosage and shorter duration of HMG, a comparable live-birth rate, a significantly lower miscarriage rate and a more favourable hormonal environment of follicular fluid. The management of poor ovarian responders or women with poor ovarian reserve in IVF is controversial. The use of letrozole has been studied; however, results are inconsistent. This randomized trial studied the sequential use of letrozole and gonadotrophin compared with gonadotrophin alone in poor responders undergoing IVF. The sequential use of letrozole and gonadotrophin led to a significantly lower dosage and shorter duration of gonadotrophin use, significantly fewer oocytes, comparable live-birth rate, a significantly lower miscarriage rate and a more favourable hormonal environment at a lower cost.

Ovarian stimulation modulates steroid receptor expression and spheroid attachment in peri-implantation endometria: studies on natural and stimulated cycles.

OBJECTIVE: To compare the effect of high serum E(2) levels on endometrial steroid receptors in gonadotropin-stimulated cycles (hCG + 7) and natural cycles (LH + 7), and to study its effect on spheroid attachment. DESIGN: Observational. SETTING: University hospital. PATIENT(S): Infertile patient with normal menstrual cycles undergoing IVF treatment. INTERVENTION(S): Gonadotropin stimulation and endometrial biopsy; trophoblast spheroid (embryo surrogate, Jeg-3)-endometrial cell (Ishikawa) coculture assay. MAIN OUTCOME MEASURE(S): Steroid receptor expression by quantitative polymerase chain reaction and immunohistochemistry; spheroid attachment rate. RESULT(S): Endometrial biopsies from natural (n = 12) and stimulated (n = 23) cycles were obtained. The expression of estrogen receptor α (ERα) but not ERß or progesterone receptor (PR) transcript was significantly reduced in stimulated cycles compared with natural cycles. Glucocorticoid receptor (GR) transcript was significantly increased in the excessive responders of the stimulated cycle. There was no difference in ERα immunoreactivity in endometrial stroma, but a higher immunoreactivity was seen in endometrial glands of stimulated cycles. The endometrium of stimulated cycles had a lower expression of PR protein in glands, but a higher expression in stroma. Although no GR protein was detected in glands, GR protein expression was significantly up-regulated in stroma of the stimulated cycles. Endometrial cells treated with high steroid concentrations had a reduced spheroid attachment rate compared with the controls. CONCLUSION(S): High serum E(2) level affects the expression of steroid receptors in the endometrial cells and suppresses spheroid attachment.