Further delineation of familial polycystic ovary syndrome (PCOS) via whole-exome sequencing: PCOS-related rare FBN3 and FN1 gene variants are identified.

AIM: To identify pathogenic rare coding Mendelian/high-effect size variant(s) by whole-exome sequencing in familial polycystic ovary syndrome (PCOS) patients to elucidate PCOS-related pathways. METHODS: Twenty women and their affected available relatives diagnosed with PCOS according to Rotterdam criteria were recruited. Whole-exome sequencing on germ-line DNA from 31 PCOS probands and their affected relatives was performed. Whole-exome sequencing data were further evaluated by pathway and chemogenomics analyses. In-slico analysis of candidate variants were done by VarCards for functional predictions and VarSite for impact on three-dimensional (3D) structures in the candidate proteins. RESULTS: Two heterozygous rare FBN3 missense variants in three patients, and one FN1 missense variant in one patient from three different PCOS families were identified. CONCLUSION: We identified three novel FBN3 and FN1 variants for the first time in the literature and linked with PCOS. Further functional studies may identify causality of these newly discovered PCOS-related variants, and their role yet remains to be investigated. Our findings may improve our understanding of the biological pathways affected and identify new drug targets.

Comparison of frozen-thawed embryo transfer protocols in patients with previous cycle cancellation due to uterine peristalsis: a pilot study

Background/aim: To investigate the optimal protocol for frozen-thawed embryo transfer (FET) cycles in patients who previously had a cycle cancellation due to uterine peristalsis (UP). Materials and methods: Thirty-four patients with previous embryo transfer (ET) cancellation due to UP during artificial cycle (AC) were included retrospectively. In the proceeding cycle, endometrium was prepared with AC (n: 23) in AC-FET group or with stimulated cycle that contains letrozole (L) (n: 11) in L-FET group. Intravenous bolus dose of 6.75 mg atosiban (Tractocile; Ferring Pharmaceuticals, Switzerland) injection was performed to all patients of AC-FET group due to UP ≥ 4/min on the planned ET day of proceeding cycle. Atosiban was not used in L-FET group. Primary outcome was live birth rate (LBR) per ET. Secondary outcomes were clinical pregnancy rate (CPR) per ET, implantation rate (IR), cycle cancellation rate. Results: The baseline characteristics such as age, body mass index, antral follicle count, duration of infertility, and the number of prior in vitro fertilization attempts of each group were similar. The IR, CPR per ET, LBR per ET, CPR per cycle and LBR per cycle were significantly higher; cycle cancellation rates were significantly lower in L-FET group as compared to the AC-FET group. Conclusion: Endometrial preparation with letrozole significantly improves CPR and LBR in FET cycles of patients with previous cycle cancellations due to UP.

Investigation of oxidative stress status in cumulus cells in patients with in vitro fertilization

Background/aim: The negative impact of oxidative stress on oocytes obtained from in vitro fertilization (IVF) patients is a challenge for the optimization of live birth rates. In this study, it is aimed to investigate whether oxidant/antioxidant parameters have a predictive value in terms of determining the count and quality of oocytes. Materials and methods: Catalase (CAT), glutathione-S-transferase (GST), arylesterase (ARE) enzyme activities, and malondialdehyde (MDA) levels were analysed in cumulus cells of poor responder (n = 28, oocyte count ≤ 4), normo responder (n = 48, 5 ≤ oocyte count ≤ 14), and high responder (n = 26, oocyte count ≥ 15) patient groups continuing IVF treatment. Results: The cumulus cell GST enzyme activity were statistically significantly increased in the high responders group compared to the poor responder and the normo responder's groups (p < 0.001 and p = 0.002, respectively). The cumulus cell MDA levels were significantly decreased in the high responder group compared to the poor responder group (p = 0.008). The cumulus cell CAT (p = 0.175) and ARE (p = 0.124) enzyme activities were examined but no statistically significant difference found between the groups. Conclusion: The significant increase in GST enzyme activity and significant decrease in MDA levels in the high responder group indicate that oxidative stress has an effect oocyte status and quality.

The relationship between H19 and parameters of ovarian reserve.

CONTEXT: The H19 long noncoding RNA (lncRNA) belongs to a highly conserved, imprinted gene cluster involved in embryonic development and growth control. We previously described a novel mechanism whereby the Anti-mullerian hormone (Amh) appears to be regulated by H19. However, the relationship between circulating H19 and markers of ovarian reserve including AMH not been investigated. OBJECTIVE: To determine whether H19 expression is altered in women with decreased ovarian reserve. DESIGN: Experimental study. SETTING: Yale School of Medicine (New Haven, USA) and Gazi University School of Medicine (Ankara, Turkey). PATIENTS OR OTHER PARTICIPANTS: A total of 141 women undergoing infertility evaluation and treatment. INTERVENTION: Collection of discarded blood samples and cumulus cells at the time of baseline infertility evaluation and transvaginal oocyte retrieval, respectively. MAIN OUTCOME MEASURE: Serum and cumulus cell H19 expression. RESULTS: Women with diminished ovarian reserve (as determined by AMH) had significantly lower serum H19 expression levels as compared to controls (p < 0.01). Serum H19 was moderately positively correlated with serum AMH. H19 expression was increased 3.7-fold in cumulus cells of IVF patients who demonstrated a high response to gonadotropins, compared to low responders (p < 0.05). CONCLUSION: In this study, we show that downregulation of H19 in serum and cumulus cells is closely associated with decreased ovarian reserve, as measured by decreased AMH levels and reduced oocyte yield at oocyte retrieval. Further study with expanded sample sizes is necessary to determine whether H19 may be of use as a novel biomarker for diminished ovarian reserve.

A potential novel treatment strategy: inhibition of angiogenesis and inflammation by resveratrol for regression of endometriosis in an experimental rat model.

The aim of our study was to evaluate the effectiveness of resveratrol in experimentally induced endometrial implants in rats through inhibiting angiogenesis and inflammation. Endometrial implants were surgically induced in 24 female Wistar-Albino rats in the first surgery. After confirmation of endometriotic foci in the second surgery, the rats were divided into resveratrol (seven rats), leuprolide acetate (eight rats), and control (seven rats) groups and medicated for 21 d. In the third surgery, the measurements of mean areas and histopathological analysis of endometriotic lesions, VEGF, and MCP-1 measurements in blood and peritoneal fluid samples, and immunohistochemical staining were evaluated. After treatment, significant reductions in mean areas of implants (p < 0.01) and decreased mean histopathological scores of the implants (p < 0.05), mean VEGF-staining scores of endometriotic implants (p = 0.01), and peritoneal fluid levels of VEGF and MCP-1 (p < 0.01, for VEGF and p < 0.01, for MCP-1) were found in the resveratrol and leuprolide acetate groups. Serum VEGF (p = 0.05) and MCP-1 (p = 0.01) levels after treatment were also significantly lower in the resveratrol and leuprolide acetate groups. Resveratrol appears to be a potential novel therapeutic agent in the treatment of endometriosis through inhibiting angiogenesis and inflammation. Further studies are needed to determine the optimum effective dose in humans and to evaluate other effects on reproductive physiology.

Human embryonic poly(A)-binding protein (EPAB) alternative splicing is differentially regulated in human oocytes and embryos.

Oocyte maturation is associated with suppression of transcriptional activity. Consequently, gene expression during oocyte maturation, fertilization and early embryo development, until zygotic genome activation (ZGA) is primarily regulated by translational activation of maternally derived mRNAs. Embryonic poly(A)-binding protein (EPAB) is the predominant poly(A)-binding protein in Xenopus, mouse and human oocytes and early embryos prior to ZGA. EPAB plays a key role in polyadenylation-dependent translational activation of mRNAs by stabilizing polyadenylated mRNAs and by stimulating their translation. Epab-knockout female mice are sterile, fail to generate mature oocytes and display impaired cumulus expansion and ovulation. Consistent with its role during gametogenesis and early embryo development, Xenopus and mouse Epab mRNA is expressed exclusively in oocytes and early embryos, and is undetectable following ZGA or in somatic tissues. Herein, we demonstrate that although EPAB is expressed in human somatic tissues, its transcripts largely consist of an alternatively spliced form lacking the first 58 bp of exon 8, which leads to the formation of a premature stop codon 6 amino acids downstream on exon 8, and omission of the functionally critical poly(A)-binding domain. Moreover, 8-cell and blastocyst stage human embryos also express only the alternatively spliced form of EPAB. On the other hand, the full-length form of EPAB mRNA is exclusively expressed in oocytes. In conclusion, in contrast with the transcriptional regulation in Xenopus and mouse, oocyte- and early embryo-specific expression of EPAB in human is regulated by a post-transcriptional mechanism.

Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH.

Genes critical for fertility are highly conserved in mammals. Interspecies DNA sequence variation, resulting in amino acid substitutions and post-transcriptional modifications, including alternative splicing, are a result of evolution and speciation. The mammalian follicle-stimulating hormone receptor (FSHR) gene encodes distinct species-specific forms by alternative splicing. Skipping of exon 2 of the human FSHR was reported in women of North American origin and correlated with low response to ovarian stimulation with exogenous follicle-stimulating hormone (FSH). To determine whether this variant correlated with low response in women of different genetic backgrounds, we performed a blinded retrospective observational study in a Turkish cohort. Ovarian response was determined as low, intermediate or high according to retrieved oocyte numbers after classifying patients in four age groups (<35, 35-37, 38-40, >40). Cumulus cells collected from 96 women undergoing IVF/ICSI following controlled ovarian hyperstimulation revealed four alternatively spliced FSHR products in seven patients (8%): exon 2 deletion in four patients; exon 3 and exons 2 + 3 deletion in one patient each, and a retention of an intron 1 fragment in one patient. In all others (92%) splicing was intact. Alternative skipping of exons 2, 3 or 2 + 3 were exclusive to low responders and was independent of the use of agonist or antagonist. Interestingly, skipping of exon 3 occurs naturally in the ovaries of domestic cats--a good comparative model for human fertility. We tested the signaling potential of human and cat variants after transfection in HEK293 cells and FSH stimulation. None of the splicing variants initiated cAMP signaling despite high FSH doses, unlike full-length proteins. These data substantiate the occurrence of FSHR exon skipping in a subgroup of low responders and suggest that species-specific regulation of FSHR splicing plays diverse roles in mammalian ovarian function.

The control of oocyte survival by intrinsic and extrinsic factors.

CAPSULE: Mechanisms that control the survival of oocytes and, by extension, the duration of ovarian function have been identified. However, it is still not clear whether oocyte "quality" is related to survival, nor is the role of the granulosa cells of follicles in follicle survival entirely understood. Here, we consider oocyte-intrinsic and oocyte-extrinsic mechanisms of oocyte loss and argue that developing a better understanding of such physiological events is needed to protect fertility, fecundity, and ovarian function in women.The duration that ovaries function is, as is intuitive, controlled by the number of remaining oocytes within follicles. Once the number of follicles drops beneath a threshold number, ovarian function ceases. Thus, understanding mechanisms that control oocyte survival is paramount as we consider strategies to protect or prolong ovarian function in women. It is often assumed that physiological oocyte survival is entirely controlled by "oocyte- intrinsic" factors, such as poor genetic quality or accumulated damage to the oocyte itself. Oocytes that have poor genetic quality due to development or accumulated damage would then die sooner than those of higher "quality." Indeed, new data suggest that oocyte-intrinsic genetic quality as determined by the ability to repair double-stranded DNA breaks is a significant contributor to oocyte survival and the duration of ovarian function. However, the nature of the follicle, where the oocyte and surrounding granulosa cells exist in intimate contact and rely upon each other for survival signals and metabolic function, makes it unlikely that oocyte-intrinsic factors entirely control oocyte survival. We and others are assessing the role of adjacent somatic (granulosa) cells in follicle survival, determining the relative importance of "oocyte-extrinsic" factors.

Protective Effects of Hydrogen-Rich Saline on Experimental Intestinal Volvulus in Rats.

BACKGROUND: Intestinal volvulus can cause morbidity and mortality. Surgical reduction, on the other hand, could result in ischemia-reperfusion (I/R) injury. Hydrogen rich saline solution (HRSS neutralizes free radicals in the body. This study aimed to investigate the effects of HRSS in I/R injury in experimental intestinal volvulus in rats. METHODS: Thirty rats were randomly allocated into 5 groups. All procedures were done under general anesthesia and sterile conditions in each animal. Five ml/kg of saline and HRSS were administered intraperitoneally (ip) in Sham (Group 1) and HRSS (Group 2) groups, respectively. Groups 3, 4, and 5 constituted the study groups in which volvulus was created in a 5-cm- long ileal segment 2 cm proximal to the ileocecal valve. After 2 hours the volvuli were reduced and following 2 hours of reperfusion, these segments were removed. In volvulus-I/R group (Group 3) no additional procedure was done. HRSS was administered shortly before reperfusion (reduction of the volvulus) in Treatment I (Group 4) and 1 h before experimental volvulus in Treatment II (Group 5) groups. Blood and intestinal tissue samples were obtained from all rats at the 4th hour. Both tissue and blood total oxidant (TOS) and antioxidant status (TAS) levels were determined and tissue histomorphologies were studied. Oxidative stress indices (TOS ÷ TAS) (OSI) were calculated. RESULTS: Tissue TOS and OSI levels and histomorphological injury scores were statistically lower in treatment groups than I/R group, whereas blood TOS and OSI levels were similar between the groups. CONCLUSIONS: This study provides biochemical and histomorphological evidence that HRSS prevents intestinal damage in I/R injury caused by volvulus.

Aberrant H19 Expression Disrupts Ovarian Cyp17 and Testosterone Production and Is Associated with Polycystic Ovary Syndrome in Women.

As one of the most common endocrine disorders affecting women, polycystic ovary syndrome (PCOS) is associated with serious conditions including anovulation, endometrial cancer, infertility, hyperandrogenemia, and an increased risk for obesity and metabolic derangements. One contributing etiology to the pathophysiology of hyperandrogenemia associated with PCOS is an intrinsic alteration in ovarian steroidogenesis, leading to enhanced synthesis of androgens including testosterone. Studies have suggested that the increased testosterone synthesis seen in PCOS is driven in part by increased activity of CYP17A1, the rate-limiting enzyme for the formation of androgens in the gonads and adrenal cortex, which represents a critical factor driving enhanced testosterone secretion in PCOS. In this work, we evaluated the hypothesis that dysregulation of the noncoding RNA H19 results in aberrant CYP17 and testosterone production. To achieve this, we measured Cyp17 in ovarian tissues of H19 knockout mice, and quantified serum testosterone levels, in comparison with wild-type controls. We also evaluated circulating and ovarian H19 expression and correlated results with the presence or absence of PCOS in a group of women undergoing evaluation and treatment for infertility. We found that the loss of H19 in a mouse model results in decreased ovarian Cyp17, along with decreased serum testosterone in female mice. Moreover, utilizing serum samples and cumulus cells from women with PCOS, we showed that circulating and ovarian levels of H19 are increased in women with PCOS compared to controls. Findings from our multimodal experimental strategy, involving both a mouse model of dysregulated H19 expression and clinical serum and ovarian cellular samples from women with PCOS, suggest that the loss of H19 may disrupt androgen production via a Cyp17-mediated mechanism. Conversely, excess H19 may play a role in the pathogenesis of PCOS-associated hyperandrogenemia.

The impact of sperm morphology on the outcome of intrauterine insemination cycles with gonadotropins in unexplained and male subfertility.

OBJECTIVE: To determine the impact of sperm morphology on success of intrauterine insemination (IUI) in unexplained and male subfertility. STUDY DESIGN: Basal and post-wash normal percentage of sperm with normal morphology was assessed prospectively in 412 couples with 530 IUI cycles who underwent ovarian hyperstimulation with gonadotropins. The primary outcome was live birth per cycle. Receiver operating characteristics (ROC) curves were used to determine the effectiveness of sperm morphology to predict live birth in IUI cycles. RESULTS: Normal sperm morphology (%) after preparation was higher in patients with live birth (5.4 ± 4.5 vs 4.3 ± 4.1; respectively, p<.05) in study population. In male subfertile group, normal sperm morphology before and after sperm preparation was higher in patients with live birth (1.3 ± 1.4 vs 0.6 ± 0.9; p<0.001 and 4.6 ± 4.4 vs 1.9 ± 2.2; p<0.01, respectively). However, both basal and post-wash normal sperm morphology (%) were similar in patients with and without live birth in unexplained group. The best cut-off value for normal sperm morphology (%) to predict live birth was 4.5% in male subfertile group with a sensitivity of 50.6% and specificity of 61.7%. CONCLUSION: Morphological evaluation is not a reliable parameter alone for predicting pregnancy outcome in unexplained infertility. In male subfertility, post-wash normal sperm morphology percentage higher than 4.5 increases the probability of live birth.

Gastroprotective activity of Nigella sativa L oil and its constituent, thymoquinone against acute alcohol-induced gastric mucosal injury in rats.

AIM: To evaluate the role of reactive oxygen species in the pathogenesis of acute ethanol-induced gastric mucosal lesions and the effect of Nigella sativa L oil (NS) and its constituent thymoquinone (TQ) in an experimental model. METHODS: Male Wistar albino rats were assigned into 4 groups. Control group was given physiologic saline orally (10 mL/kg body weight) as the vehicle (gavage); ethanol group was administrated 1 mL (per rat) absolute alcohol by gavage; the third and fourth groups were given NS (10 mL/kg body weight) and TQ (10 mg/kg body weight p.o) respectively 1 h prior to alcohol intake. One hour after ethanol administration, stomach tissues were excised for macroscopic examination and biochemical analysis. RESULTS: NS and TQ could protect gastric mucosa against the injurious effect of absolute alcohol and promote ulcer healing as evidenced from the ulcer index (UI) values. NS prevented alcohol-induced increase in thiobarbituric acid-reactive substances (TBARS), an index of lipid peroxidation. NS also increased gastric glutathione content (GSH), enzymatic activities of gastric superoxide dismutase (SOD) and glutathione-S-transferase (GST). Likewise, TQ protected against the ulcerating effect of alcohol and mitigated most of the biochemical adverse effects induced by alcohol in gastric mucosa, but to a lesser extent than NS. Neither NS nor TQ affected catalase activity in gastric tissue. CONCLUSION: Both NS and TQ, particularly NS can partly protect gastric mucosa from acute alcohol-induced mucosal injury, and these gastroprotective effects might be induced, at least partly by their radical scavenging activity.

Poor ovarian response in women undergoing in vitro fertilization is associated with altered microRNA expression in cumulus cells.

OBJECTIVE: To analyze the association of micro-ribonucleic acid (miRNA) expression with the number of oocytes retrieved, in women undergoing in vitro fertilization (IVF). DESIGN: Experimental study. SETTING: Academic medical center. PATIENT(S): A total of 189 women undergoing IVF-intracytoplasmic sperm injection (ICSI). INTERVENTION(S): Pooled cumulus cells were collected. MAIN OUTCOME MEASURE(S): Poor responders were identified as patients who produced fewer oocytes than the 25th percentile of their respective age group. MicroRNAs were extracted from cumulus cells, and an miRNA microarray was performed, comparing poor responders (n = 3) to non-poor responders (n = 3). Expression of miR-21-5p (active strand of miR-21) and miR-21-3p was tested in poor responders (n = 21) and non-poor responders (n = 29), using reverse transcription real-time polymerase chain reaction (qRT-PCR). Regulation of miR-21-5p and miR-21-3p, in human granulosa-like tumor (KGN) cells, by estradiol (E2), was tested in vitro. RESULT(S): MicroRNA microarray analysis showed up-regulation of 16 miRNAs and down-regulation of 88 miRNAs in poor responders. Notably, miR-21 was significantly up-regulated 5-fold in poor-responder samples. Analysis using qRT-PCR confirmed that miR-21-5p expression was significantly up-regulated in poor responders, whereas miR-21-3p expression was significantly lower, suggesting that elevated miR-21-5p expression in cumulus cells is not regulated at the pre-miR-21 level in poor responders. Both miR-21-5p and miR-21-3p were increased in KGN cells in response to higher doses of E2; their expression was not affected at lower E2 concentrations. CONCLUSION(S): We found that poor response to IVF is associated with altered miRNA expression in cumulus cells, specifically with elevated expression of miR-21-5p, and that this elevated expression is independent of lower serum E2 levels in poor responders.

Levels of some trace elements and rheumatoid factor in sheep with brucellosis.

Brucellosis is a zoonotic disease that is encountered in sheep rather frequently. In this study, 100 sheep diagnosed with brucellosis that had aborts and 40 healthy sheep were used as materials. Analyses for Cu, Zn, Fe, Cr, Ca, Mg, and K were performed with the atomic absorption spectrophotometric method on blood collected from vena jugularis of all the sheep and rheumatoid factor levels were determined by the nephelometry method. Although it was found that Cu, Ca, and rheumatoid factor values in the sera of the sheep with brucellosis were high when compared to the control group (p<0.001, p<0.05, p<0.001, respectively), their serum Zn values were low (p<0.05). No significant changes in serum Cr, Fe, K, and Mg levels were found.

The effect of captopril on endometriotic implants in a rat model.

OBJECTIVE: To determine the effects of captopril on experimentally induced endometriosis in a rat model. STUDY DESIGN: Twenty-four adult, mature female Wistar-Albino rats in which endometriotic implants were induced by transplanting autologous uterine tissue to ectopic sites on the peritoneum. After the endometriotic implants were formed surgically, the 24 rats were randomly divided into three groups. Group 1 (captopril group, eight rats) were given 50 mg kg(-1)d(-1) of oral captopril for 21 d. Group 2 (leuprolide acetate group, eight rats) were given a single 1 mg kg(-1) subcutaneous injection of leuprolide acetate. Group 3 (control) were given no medication and served as controls (eight rats). The surface area of the endometriotic implants and the score of histologic analysis. Also, VEGF and MCP-1 levels in peritoneal fluids and bloods were analyzed. RESULTS: At the beginning of the medical treatment, the mean surface areas of the endometriotic implants were comparable in all three groups. At the end of the treatment the mean implant surface area in the captopril group and leuprolide acetate group was less than that in the control group. Mean histopathological examination score for the implants post treatment was lower in the captopril and leuprolide acetate groups. Peritoneal fluids VEGF level in the captopril and leuprolide acetate groups was lower than that in the control group. The post-treatment MCP-1 level was also lower in the captopril and leuprolide acetate groups than in the control group. The serum VEGF and MCP-1 levels post treatment were significantly lower in the captopril and leuprolide acetate groups than in the control group. CONCLUSION: Administration of captopril reduced the size and progression of endometriotic lesions in a rat model.

Vitrified human ovaries have fewer primordial follicles and produce less antimüllerian hormone than slow-frozen ovaries.

Slow-freezing and vitrification methods of human ovarian tissue cryopreservation were compared in terms of primordial follicle count and in vitro antimüllerian hormone (AMH) and estradiol production. Compared with fresh and slow-frozen ovaries, vitrified ovaries contained statistically significantly fewer primordial follicles and produced statistically significantly less AMH in vitro. Estradiol production from slow-frozen and vitrified ovaries was similar but statistically significantly lower than from fresh cultured strips.

Protective effects of thymoquinone and methotrexate on the renal injury in collagen-induced arthritis.

The goal of this investigation was to study the protective effects of thymoquinone (TQ) and methotrexate (MTX) on collagen-induced arthritis (CIA) in rats. On day 0 under ether anesthesia, the experimental groups were immunized with 0.5 mg native chick collagen II (CII) solubilized in 0.1 M acetic acid and emulsified in Freund's incomplete adjuvant. Control rats were gavaged with vehicle, whereas CII was administered intradermally. In addition, arthritis treated with TQ group received TQ (10 mg kg(-1) bw by gavage once a week for 3 weeks starting on day 0); and arthritis treated with MTX group received MTX (MTX was suspended in corn oil and administered by gavage at 1 mg kg (-1) bw once a week for 3 weeks starting on day 0). A significant decrease in the incidence and severity of arthritis by clinical and radiographic assessments was found in recipients of therapy, compared with that of controls. The MTX treatment significantly (P<0.01) decreased the elevated serum NO, urea and creatinine in arthritic rats. Likewise, TQ treatment was also able to reduce significantly (P<0.05) serum NO, urea and creatinine levels, but to lesser extent than MTX. The histopathologic abnormalities are consistent with the hydropic epithelial cell degenerations and moderate tubular dilatation in the some proximal and distal tubules. The severity of the degenerative changes in most of the shrunken glomerules and vascular congestion were also observed in arthritic animals. Preventive treatment of TQ and especially MTX significantly inhibited kidney dysfunction and this histopathologic alterations. These studies indicate that TQ can be used similar to MTX as a safe and effective therapy for CIA and may be useful in the treatment of rheumatoid arthritis.