Reduced Endothelin-2 and Hypoxic Signaling Pathways in Granulosa-Lutein Cells of PCOS Women.

Granulosa-lutein cells (GLCs) from PCOS women display reduced HIF-1α and EDN2 levels, suggesting their role in PCOS etiology. Here, we investigated the mechanisms involved in aberrant EDN2 expression in PCOS, and its association with HIF-1α. Various HIF-1α-dependent factors were studied in GLCs from PCOS and compared to normally ovulating women. MicroRNA-210 (miR-210), its target genes (SDHD and GPD1L), and HIF-1α-responsive genes (EDN2 and VEGFA) differed in GLCs from PCOS, compared with those of healthy women. Levels of miR-210-designated hypoxiamiR-and EDN2 were reduced in the PCOS GLCs; concomitantly, GPD1L and SDHD levels were elevated. Cultured GLCs retained low EDN2 expression and had low HIF-1α levels, providing evidence for a disrupted hypoxic response in the PCOS GLCs. However, VEGFA expression was elevated in these cells. Next, miR-210 levels were manipulated. miR-210-mimic stimulated EDN2 twice as much as the miR-NC-transfected cells, whereas miR-210-inhibitor diminished EDN2, emphasizing the importance of hypoxiamiR for EDN2 induction. Intriguingly, VEGFA transcripts were reduced by both miR-210-mimic and -inhibitor, demonstrating that EDN2 and VEGFA are distinctly regulated. Disrupted hypoxic response in the GLCs of periovulatory follicles in PCOS women may play a role in ovulation failure, and in the reduced fertility prevalent in this syndrome.

Sirtuin-1 inhibits endothelin-2 expression in human granulosa-lutein cells via hypoxia inducible factor 1 alpha and epigenetic modifications†.

Endothelin-2 (EDN2) expression in granulosa cells was previously shown to be highly dependent on the hypoxic mediator, hypoxia inducible factor 1 alpha (HIF1A). Here, we investigated whether sirtuin-1 (SIRT1), by deacetylating HIF1A and class III histones, modulates EDN2 in human granulosa-lutein cells (hGLCs). We found that HIF1A was markedly suppressed in the presence of resveratrol or a specific SIRT1 activator, SRT2104. In turn, hypoxia reduced SIRT1 levels, implying a mutually inhibitory interaction between hypoxia (HIF1A) and SIRT1. Consistent with reduced HIF1A transcriptional activity, SIRT1 activators, resveratrol, SRT2104, and metformin, each acting via different mechanisms, significantly inhibited EDN2. In support, knockdown of SIRT1 with siRNA markedly elevated EDN2, whereas adding SRT2104 to SIRT1-silenced cells abolished the stimulatory effect of siSIRT1 on EDN2 levels further demonstrating that EDN2 is negatively correlated with SIRT1. Next, we investigated whether SIRT1 can also mediate the repression of the EDN2 promoter via histone modification. Chromatin immunoprecipitation (ChIP) analysis revealed that SIRT1 is indeed bound to the EDN2 promoter and that elevated SIRT1 induced a 40% decrease in the acetylation of histone H3, suggesting that SIRT1 inhibits EDN2 promoter activity by inducing a repressive histone configuration. Importantly, SIRT1 activation, using SRT2104 or resveratrol, decreased the viable numbers of hGLC, and silencing SIRT1 enhanced hGLC viability. This effect may be mediated by reducing HIF1A and EDN2 levels, shown to promote cell survival. Taken together, these findings propose novel, physiologically relevant roles for SIRT1 in downregulating EDN2 and survival of hGLCs.

Male fertility preservation-Methods, indications and challenges.

Male fertility preservation has been steadily increasing over the past two decades. Significant improvements have been achieved in the treatment modalities of cancer and other severe chronic medical conditions, leading to an increase in patient survivorship and the resulting demand for future parenthood. Recognition and proper patient counselling before commencing therapies with a potential gonadotoxic effect are of paramount importance. Similarly, nonmedically indicated fertility preservation is on the rise. Social sperm banking, gender dysphoria prior to affirmation procedures and posthumous reproduction preservation are becoming more common. When timing and logistics are appropriate, sperm cryopreservation is considered the gold standard for fertility preservation. Testicular tissue and spermatogonial stem cell autotransplantation is considered experimental and represents a promising alternative for pre-pubertal patients. The current paper aims to review the recent trends in male fertility preservation, the common indications for sperm cryopreservation, techniques for sperm retrieval and experimental frontiers.

The cAMP pathway promotes sirtuin-1 expression in human granulosa-lutein cells.

Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, is present in the ovarian granulosa cells (GCs) of various species. This study examined the regulation of SIRT1 expression in human granulosa-lutein cells (hGLCs). Two different, structurally unrelated SIRT1 activators, SRT2104 and resveratrol, dose- and time-dependently enhanced SIRT1 (∼2- and 1.5-fold increase at 50 µmol/L for mRNA and protein levels, respectively), whereas EX-527, an inhibitor of SIRT1 deacetylase activity, significantly suppressed SIRT1 protein induced by these activators. Transfecting cells with SIRT1 siRNA molecules efficiently silenced SIRT1 (∼70 % decrease in 48 h post-transfection). Furthermore, the stimulatory effects of SRT2104 on SIRT1 expression observed in non-transfected or in scrambled siRNA-transfected cells were diminished with SIRT1 silencing. The findings described above imply that SIRT1 autoregulates its own expression. Interestingly, SRT2104 elevated cAMP accumulation (1.4-fold) in the culture media of hGLCs which was further augmented in the presence of hCG (2.2-fold); these effects were evident after 12 h of incubation. This additive effect of hCG and SRT2104 on cAMP accumulation may explain the incremental outcome observed on SIRT1 expression (∼3-fold increase from basal level and ∼1.6-fold stimulation for each compound alone) with these two compounds. SIRT1 knockdown diminished SIRT1 induced by forskolin, providing additional evidence that cAMP promotes SIRT1. These findings imply that by activating adenylyl cyclase (hCG or forskolin) and inhibiting phosphodiesterases (SIRT1 activators), these two signals converge to produce an incremental, positive feedback loop on SIRT1 expression. Such a mechanism highlights the importance of maintaining high SIRT1 levels in human luteinized GCs.

[CLINICAL ASPECTS OF INTRAUTERINE INSEMINATION].

INTRODUCTION: Intrauterine insemination (IUI) is an intervention performed frequently in the treatment of couples suffering from subfertility and women treated using donor sperm. The factors predicting its success include female parameters such as age and ovarian function and male parameters such as sperm quality. However, many details regarding its clinical application such as the methodology of sperm preparation, timing of IUI, the number of inseminations in the same cycle, the recommended number of cycles to be performed and the minimal threshold values of the sperm sample profile required are controversial. The conclusion to be drawn from this literature survey is that there is still lack of sufficient good quality studies performed to allow evidence based recommendations for the performance of IUI.

Male age influences oocyte-donor program results.

BACKGROUND: The purpose of our research was to examine the relationship between male age and semen parameters in a range of ages (from 20s to 60s) in Egg Donation Program (EDP) cycles. EDP provides a pool of high quality oocytes, thus allowing better analysis of the sperm efficacy. DISCUSSION: The retrospective study population consisted of 484 male partners of patients undergoing EDP in in-vitro fertilization (IVF) treatments. A comparison was made of male age and sperm parameters within two groups: cycles resulting in a pregnancy (pregnant group) and cycles which failed to achieve a pregnancy (non-pregnant group). The men involved in the pregnant group were found to be significantly younger 43.2 +/- 8.1 than those of the nonpregnant group 46.81 +/- 7.8 (p = 0.003). Analysis of sperm morphology revealed a significant prevalence of teratozoospermia in males of the non-pregnant group, as compared to the males of the pregnant group (29% vs. 11%, respectively). The results also demonstrate that sperm parameters are apparently not diminished until men reach the age of 40. However, between 40-50 years old semen parameters deteriorate. Male age was found to be related to a reduction in sperm strict criteria: 44.8 in normozoospermia, 47.9 (p = 0.02), 48.4 (p = 0.04) and 51.9 (p = 0.001) years old in mild teratozoospermia, moderate teratozoospermia and severe teratozoospermia, respectively. Additionally, the results showed that the percentage of "healthy" embryos on day 3 of embryo culture was lower in the non-pregnant group (26%), as compared with the pregnant group (34%; p = 0.01). CONCLUSION: Our study confirms that sperm parameters are reduced by age and suggests that this age-dependent effect could be a reason for failures in IVF cycles even in EDP couples.

Cloprostenol, a prostaglandin F(2alpha) analog, induces hypoxia in rat placenta: BOLD contrast MRI.

Blood oxygen level dependent (BOLD) contrast was used to monitor hypoxia induced by cloprostenol, a prostaglandin F(2alpha) (PGF(2alpha)) analog, in the rat embryo-placental unit (EPU). It is shown that administration of cloprostenol (0.025 mg/rat) at mid-gestation (day 16) reduced EPU oxygenation, as detected by BOLD contrast MRI, in correlation with induction of vascular endothelial growth factor (VEGF) gene (Vegfa) expression in the corresponding placenta (r = 0.56, p = 0.03). Elevated VEGF mRNA expression in response to cloprostenol treatment was also observed at early gestation (day 9) in the forming placenta (p = 0.04) and uterus (p = 0.03). Cloprostenol increased the expression levels of endothelin-1 (ET-1) gene (Edn1) (p = 0.03) and its corresponding peptide (p = 0.02) in the forming placenta, as well as the expression of the endothelin receptor type A (ETA) gene (Ednra) in both the forming placenta (p = 0.009) and the uterus (p = 0.01). The levels of the endothelin receptor type B (ETB) gene (Ednrb) were not affected in response to cloprostenol, but a significant elevation in the expression level of this receptor was observed in the uterus at mid- and late gestation (day 22) (p = 0.04 and 0.01 respectively), suggesting a role for ETB in the vasodilatory status of the pregnant uterus. It is suggested that PGF(2alpha) induces uteroplacental vasoconstriction in the rat, and that ET-1 may take part in mediating this effect, probably via activation of ETA receptor. The uteroplacental vasoconstriction induces hypoxia, as manifested by significant changes in BOLD MRI and by upregulation of VEGF.

Human sperm chemotaxis: both the oocyte and its surrounding cumulus cells secrete sperm chemoattractants.

BACKGROUND: Human sperm chemotaxis to pre-ovulatory follicular fluid is well established in vitro. However, it is not known whether the female's oocyte-cumulus complex secretes sperm chemoattractants subsequent to ovulation (for enabling sperm chemotaxis within the Fallopian tube) and, if so, which of these cell types--the oocyte or the cumulus oophorus--is the physiological origin of the secreted chemoattractant. METHODS: By employing a directionality-based chemotaxis assay, we examined whether media conditioned with either individual, mature (metaphase II) human oocytes or the surrounding cumulus cells attract human sperm by chemotaxis. RESULTS: We observed sperm chemotaxis to each of these media, suggesting that both the oocyte and the cumulus cells secrete sperm chemoattractants. CONCLUSIONS: These observations suggest that sperm chemoattractants are secreted not only prior to ovulation within the follicle, as earlier studies have demonstrated, but also after oocyte maturation outside the follicle, and that there are two chemoattractant origins: the mature oocyte and the surrounding cumulus cells.

Involvement of endothelin-1 and its receptors in PGF2alpha-induced luteolysis in the rat.

The possible mediatory role of endothelin-1 (ET-1) in prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis in the rat was examined. The effect of PGF(2alpha) was tested on day 9 of pregnancy either in vivo, by injecting cloprostenol, an analog of PGF(2alpha) or in vitro, in isolated intact corpora lutea incubated with PGF(2alpha). Luteolysis was confirmed by progesterone determination in the peripheral blood serum or in the culture medium, respectively. Administration of cloprostenol (.0025 mg/rat) induced within 1 hr, a significant fall (from 56.8 to 27.6 ng/ml, P < 0.0001) in serum progesterone concentrations that was associated with an increased expression of the mRNA to ET-1 and its protein product in rat luteal tissue. Elevated level of ET-1 were also determined at the spontaneous regression of the CL, upon parturition. Expression of the ET receptors, ETA and ETB was not affected by cloprostenol. On the other hand, this PGF(2alpha) analog induced expression of luteal VEGF mRNA. In vitro experiments demonstrate that the LH (100 ng/ml)-induced increase in luteal progesterone secretion was reduced by PGF(2alpha) (1 microg/ml). The inhibitory effect of PGF(2alpha) was reversed by BQ123 (10(- 7) M), that is a selective ETA receptor antagonist. We conclude that the PGF(2alpha)-induced elevation in luteal expression of ET-1 combined with the reversal of its luteolytic effect by an ETA receptor antagonist suggest that ET-1 may take part in the PGF(2alpha)-induced luteolysis in the rat.