MRAP2 regulates endometrial receptivity and function.

A successful embryo implantation depends on the synchronization between a competent blastocyst and a receptive endometrium. Recently, potential modulators of endometrial receptivity (OVGP1, MRAP2, ZCCHC12, and HAP1) have been reported likely with a functional role during embryo implantation. The aim of this study was to evaluate the gene expression of these genes in the endometrium of infertile women. Eighteen endometrial biopsies, during secretory lutheal phase, were recruited from women with unexplained infertility and women who cannot conceive due to their partners' fertility problems. qRT-PCR was carried out to evaluate MRAP2, OVGP1, ZCCHC12 and HAP1 gene expression. MRAP2 expression was also detected by western blot and it was localized by immunohistochemistry. Morphological analysis was performed by light microscopy. MRAP2 was significantly up-regulated in study vs. control group. Western blot analysis confirmed the observed MRAP2 up-expression. MRAP2 resulted mainly localized in the epithelial cells of uterine glands. Morphological analysis displayed that the epithelium of the uterine glands undergo hypertrophy in women with unexplained infertility in respect to women with male infertility factor. MRAP2 could be considered a mediator of endometrial receptivity likely acting on endometrial stability by binding to MCRs and PKR1.

Clinical experience with an ovarian stimulation protocol for intrauterine insemination adopting a gonadotropin releasing hormone antagonist at low dose.

Studies testing the effectiveness of GnRH antagonists in controlled ovarian stimulation (COS) for intrauterine insemination (IUI) have provided controversial results. The present study was undertaken to evaluate, whether the use of a half of the conventional dose of the GnRH antagonist cetrorelix can be effective in increasing the successful rate of IUI cycles. Patients started COS with human menopausal gonadotropin (hMG) on day three of the menstrual cycle. Cetrorelix was started when at least one follicle of ≥14 mm, was detected at the ultrasound scan, according to the flexible multiple daily dose protocol, and continued until the trigger day with recombinant hCG. Patients adopting GnRH antagonist at low dose had a pregnancy rate (21.7%) that was significantly higher (p < 0.05) in comparison to women receiving hMG only (8.7%). These results suggest that adding a reduced dose of GnRH antagonist to the COS for IUI cycles significantly improves the outcome of the procedure.

Highly purified hMG versus recombinant FSH plus recombinant LH in intrauterine insemination cycles in women ≥35 years: a RCT.

STUDY QUESTION: Is the treatment with recombinant FSH (rFSH) plus recombinant LH (rLH) more effective than highly purified (HP)-hMG in terms of ongoing pregnancy rate (PR) in women ≥35 years of age undergoing intrauterine insemination (IUI) cycles? SUMMARY ANSWER: The ongoing PR was not significantly different in women treated with rFSH plus rLH or with HP-hMG. WHAT IS KNOWN ALREADY: Although previous studies have shown beneficial effects of the addition of LH activity to FSH, in terms of PR in patients aged over 34 years having ovulation induction, no studies have compared two different gonadotrophin preparations containing LH activity in women ≥35 years of age in IUI cycles. STUDY DESIGN, SIZE, DURATION: A single-centre RCT was performed between May 2012 and September 2013 with 579 women ≥35 years of age undergoing IUI cycles. The patients were randomly assigned to one of the two groups, rFSH in combination with rLH group or HP-hMG (Meropur) group, by giving them a code number from a computer generated randomization list, in order of enrolment. The randomization visit took place on the first day of ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Five hundred and seventy-nine patients with unexplained infertility or mild male factor undergoing IUI cycles were recruited in a university hospital setting. All women were enrolled in this study only for one cycle of treatment. Five hundred and seventy-nine cycles were included in the final analysis. Two hundred and ninety patients were treated with rFSH in combination with rLH and 289 patients were treated with HP-hMG. The ovarian stimulation cycle started on the third day of the menstrual cycle and the starting gonadotrophin doses used were 150 IU/day of rFSH plus 150 IU/day of rLH or 150 IU/day of HP-hMG. The drug dose was adjusted according to the individual follicular response. A single IUI per cycle was performed 34-36 h after hCG injection. MAIN RESULTS AND THE ROLE OF CHANCE: The main outcome measures were ongoing PR and number of interrupted cycles for high risk of ovarian hyperstimulation syndrome (OHSS). Ongoing pregnancy rates were 48/290 (17.3%) in the recombinant group versus 35/289 (12.2%) in the HP-hMG group [(odds ratio (OR) 1.50, 95% CI 0.94-2.41, P = 0.09]. The number of interrupted cycles for high risk of OHSS was 13/290 (4.5%) in the rFSH plus rLH group and 2/289 (0.7%) in the HP-hMG group (OR 6.73, 95% CI 1.51-30.12, P = 0.013). LIMITATIONS, REASONS FOR CAUTION: One of the limitations of this study was the early closure and the ongoing PR could be overestimated. Both patient and gynaecologist were informed of the assigned treatment. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrated the lack of differences in terms of ongoing PR between recombinant product and HP-hMG, in women ≥35 years undergoing controlled ovarian stimulation for IUI cycles. HP-hMG was safer than recombinant gonadotrophin concerning the risk of OHSS. STUDY FUNDING/COMPETING INTERESTS: None. TRIAL REGISTRATION NUMBER: NCT01604044.

Endocrine disruptors and human reproductive failure: the in vitro effect of phthalates on human luteal cells.

OBJECTIVE: To evaluate the influence of phthalates on human luteal cell function. DESIGN: Laboratory study. SETTING: University hospital. PATIENT(S): Twenty-three normally menstruating patients in the midluteal phase. INTERVENTION(S): Human luteal cells isolated from corpora lutea for primary cultures. MAIN OUTCOME MEASURE(S): Progesterone (P4) and prostaglandin release assayed by enzyme immunoassay, vascular endothelial growth factor (VEGF) secretion by enzyme-linked immunosorbent assay (ELISA), and VEGF mRNA expression by real-time polymerase chain reaction. RESULT(S): We investigated the effect of di(2-ethylhexyl)phthalate (DEHP), di-n-butyl phthalate (DBP), and butyl benzyl phthalate (BBP) on basal and hCG-induced progesterone (P4) release, as well as DEHP effect on the balance between prostaglandin (PG) E2, vascular endothelial growth factor (VEGF)-luteotrophic factors, and the luteolitic PGF2α in isolated human steroidogenc cells. Phthalates influence on VEGF expression has been also evaluated. DEHP, DBP, and BBP were able to reduce both basal and hCG-stimulated P4 as well as PGE2 release. PGF2α release was reduced after DEHP incubation. VEGF protein release was decreased by the incubation with the tested phthalates. VEGF mRNA expression was not affected by DEHP, DBP, and BBP. As expected, both hCG and cobalt chloride were able to induce P4 release and VEGF release and mRNA expression in human luteal cells respectively. CONCLUSION(S): The results show the ability of phthalates to affect luteal steroidogenesis as well as the balance between luteotrophic and luteolytic factors suggesting an interference of phthalates in human luteal function. These data may contribute to clarify the classically known impaired reproductive health observed after phthalates exposure.

Assessment of insulin resistance in lean women with polycystic ovary syndrome.

OBJECTIVE: To develop and validate a specific simple measure of insulin sensitivity using oral glucose tolerance test (OGTT) values for lean polycystic ovary syndrome (PCOS) women. DESIGN: Retrospective study. SETTING: Gynecologic Outpatient Clinic of University Hospital, affiliated with Unit of Gynecologic Endocrinology. PATIENT(S): Totals of 201 lean and 198 overweight/obese (ov-ob) nondiabetic PCOS patients were retrospectively selected. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): All patients underwent OGTT, euglycemic-hyperinsulinemic clamp, and androgenic and biochemical assays. The predictive performance of each insulin resistance (IR) index was analyzed with the use of receiver operating characteristic (ROC) curves. RESULT(S): Higher correlation coefficients with clamp studies were obtained with the Belfiore Area (RS=0.579) and the homeostasis-model assessment (HOMA)-M120 (RS=-0.576) in lean PCOS patients and with the Sib (RS=0.697) in ov-ob PCOS patients. The best predictive index of IR in lean PCOS was a HOMA-M120 value of ≥12.8 or more (area under the ROC curve [AUC] 92.4%). In the ov-ob PCOS population, the best predictive performance was obtained by a Sib of ≤10.2 or less (AUC 85.7%). CONCLUSION(S): IR should be assessed in all PCOS women, both lean and ov-ob subjects. The HOMA-M120 resulted as a very simple tool, validated specifically for the lean PCOS woman whose cardiometabolic impairment is more frequently misunderstood.

The use of different size-hysteroscope in office hysteroscopy: our experience.

PURPOSE: To evaluate the successful rate and patient acceptance of different-sized hysteroscope in office hysteroscopy. METHODS: We retrospectively evaluated 900 office hysteroscopy performed in ambulatory setting using three different hysteroscopes: 5 mm Hamou II (n = 300), 5 mm Bettocchi (n = 300) and 4 mm Bettocchi (n = 300). Endpoints of our study were the successful rate of hysteroscopy, the eventual side effects/complication and the pain intensity experience from the patients using visual analog scale (VAS). RESULTS: Use of 4 mm Bettocchi leads to a higher rate of successfully performed hysteroscopy (99%, n = 297) and statistically significant when compared to the 5 mm Hamou (95%, n = 285) and to the 5 mm Bettocchi (96%, n = 288) (4 mm Bettocchi vs. 5 mm Bettocchi p < 0.05; 4 mm Bettocchi vs. 5 mm Hamou II p < 0,001; 5 mm Bettocchi vs. 5 mm Hamou II ns). Moreover, the VAS score was higher using 5 mm Hamou II (5.72 ± 1.99) and statistically significant when compared to the 4 mm Bettocchi (3.06 ± 2.14) and to the 5 mm Bettocchi (4.27 ± 1.88) (A vs. B p < 0.05; A vs. C p < 0.001; B vs. C p < 0.001). CONCLUSIONS: Our result suggests that the hysteroscope size plays a pivotal role in the acceptance and for the success of office hysteroscopy.

Endocrine disruptors and human corpus luteum: in vitro effects of phenols on luteal cells function.

Endocrine disruptors are well known to impair fertility. The aim of the present study was to investigate the effects of bisphenol A (BPA) and nonylphenol (p-NP) on human luteal function in vitro. In particular, in luteal cells isolated from 21 human corpora lutea progesterone, prostaglandin (PG) F2α, PGE2 and vascular endothelial growth factor (VEGF) release, as well as VEGF expression were evaluated. BPA and p-NP negatively affected both luteal steroidogenesis and luteotrophic/ luteolytic factors balance, without influencing VEGF mRNA expression. Actually, BPA and p-NP impaired human luteal cells function in vitro, underlining the already suggested correlation between phenols and reproductive failure.

In vitro effect of unacylated ghrelin and obestatin on human luteal cell function.

OBJECTIVE: To evaluate whether unacylated ghrelin and obestatin were able to influence human luteal cell function. The effect of these two ghrelin-related peptides on progesterone (P4), prostaglandin (PG) F(2α), PGE(2), and vascular endothelial growth factor (VEGF) release and on VEGF expression in isolated human steroidogenic cells has been investigated. DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENT(S): Corpora lutea were obtained from 23 normally menstruating patients in the midluteal phase of the menstrual cycle. INTERVENTION(S): Human luteal cells were isolated from corpora lutea, and primary cultures were established. MAIN OUTCOME MEASURE(S): P4 and PGs release was assayed by enzyme immunoassay, VEGF secretion by ELISA, and VEGF mRNA expression by real-time polymerase chain reaction. RESULT(S): P4 and VEGF release were significantly reduced by both unacylated ghrelin and obestatin. Moreover, the highest concentration of obestatin was able to reduce the release of PGE(2) and PGF(2α). VEGF mRNA expression was not affected by the incubation with any of these ghrelin-related peptides. As expected, CoCl(2) was able to induce VEGF release and mRNA expression in luteal cells. CONCLUSION(S): Our results suggest that, similar to ghrelin, both unacylated ghrelin and obestatin might play a role in regulating the luteal cell function that affects both luteal steroidogenesis and luteotrophic/luteolytic imbalance. These results further underline the pivotal correlation between the ghrelin system and reproduction.

Prokineticin 1, homeobox A10, and progesterone receptor messenger ribonucleic acid expression in primary cultures of endometrial stromal cells isolated from endometrium of healthy women and from eutopic endometrium of women with endometriosis.

OBJECTIVE: To examine prokineticin 1 (PROK1), homeobox (HOX) A10, and P receptor (PR) messenger ribonucleic acid (mRNA) expression in primary cultures of endometrial stromal cells (ESC) obtained from eutopic endometrial samples of patients with endometriosis and to clarify whether in vitro steroid hormone dependence of PROK1 gene expression is altered in endometriosis. DESIGN: Prospective laboratory study. SETTING: Tertiary university hospital. PATIENT(S): Twelve normal women (controls) and 12 patients affected by moderate to severe endometriosis in the midsecretory phase of the menstrual cycle. INTERVENTION(S): Endometrial specimens were obtained from control women and from women affected by endometriosis; ESC were isolated from endometrial biopsies, and primary cultures were established. MAIN OUTCOME MEASURE(S): Real-time polymerase chain reaction analysis of PROK1, HOXA10, and PR mRNA expression in ESC after 1-4 days of steroid hormone treatment and after decidual differentiation. RESULT(S): Contrary to ESC from control women, in ESC obtained from women affected by endometriosis PROK1 and PR mRNA expression was not induced by 1-4 days of treatment with steroid hormones. Nevertheless, when ESC from both groups of women were differentiated to decidual phenotype, PROK1 mRNA was up-regulated and PR and HOXA10 mRNA were down-regulated to the same extent. CONCLUSION(S): Our results provide additional evidence for P resistance in endometriosis.

Estrogens and androgens affect human luteal cell function.

OBJECTIVE: To evaluate estrogens (Es)--E2, estrone (E1), and estriol--and androgens--T and androstendione (A)-effect on P, prostaglandin (PG) F2α, PGE2, and vascular endothelial growth factor (VEGF) release and on VEGF expression in human luteal cells. To elucidate whether androgens effects were direct or mediated by their conversion in Es, an aromatase inhibitor was used. Finally, the luteal effect of the non-aromatizable dihydrotestosterone was evaluated. DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENT(S): Corpora lutea (CLs) were obtained from 36 normally menstruating patients in the midluteal phase of the menstrual cycle. INTERVENTION(S): The human luteal cells were isolated from CLs and primary cultures were established. MAIN OUTCOME MEASURE(S): P and PG release were assayed by enzyme immunoassay; VEGF secretion by ELISA; VEGF messenger RNA (mRNA) expression by real-time polymerase chain reaction (PCR). RESULT(S): P and PGF2α secretion were decreased by Es and androgens. The VEGF release was increased by Es and androgens, whereas VEGF mRNA expression was not. The aromatase inhibitor counteracted T and A luteal effects. CONCLUSION(S): Both Es and androgens could participate in the regulation of human luteal function. The effect of T and A seems to be mediated by their conversion to Es, whereas for dihydrotestosterone, both direct androgenic and indirect estrogenic luteal effects could coexist.

Prokineticin 1 mRNA expression in the endometrium of healthy women and in the eutopic endometrium of women with endometriosis.

OBJECTIVE: To examine prokineticin 1 (PROK1) mRNA expression in eutopic endometrial glands obtained from patients with or without endometriosis, to investigate the presence of additional endometrial abnormalities in women with endometriosis. DESIGN: Prospective laboratory study. SETTING: University hospital. PATIENTS: Twelve control women and 12 patients affected by endometriosis in the secretory phase of the menstrual cycle. INTERVENTION(S): Endometrial specimens were obtained from women affected (cases) or not (control group) by endometriosis. Endometrial glands were freshly isolated from endometrial biopsies. MAIN OUTCOME MEASURE(S): PROK1 mRNA expression levels by real-time polymerase chain reaction analysis. RESULTS: PROK1 mRNA was detectable in 4 of 12 (33%) samples obtained from women affected by endometriosis, whereas 10 of 12 (83%) samples obtained from normal women were positive for PROK1 detection by real-time polymerase chain reaction. Moreover, detectable PROK1 mRNA levels were 10 times lower in samples obtained from women with endometriosis than in samples obtained from control women. CONCLUSION(S): PROK1 is a newly discovered angiogenic factor implicated in the vascular function of peri-implantation endometrium and early pregnancy. An altered expression of PROK1 could be one of the several biochemical abnormalities characterizing eutopic endometrium in endometriosis.

Endometriosis and human infertility: a new investigation into the role of eutopic endometrium.

BACKGROUND: Endometriosis is related to infertility even in the absence of mechanical alterations of the reproductive tract. Even though the pathogenesis of this phenomenon is still unclear, an impaired endometrial receptivity has been recently suggested. The aim of the present study was to investigate if endometriotic peritoneal fluids (EPF) could interfere with endometrial stromal cell (ESC) decidualization and if tumor necrosis factor (TNF)-alpha could be involved in the EPF effect. METHODS: Eutopic ESC were isolated from patients with or without endometriosis. ESC were treated with 17beta-estradiol 10(-8) M and 6alpha-methyl-17alpha-hydroxyprogesteroneacetate 2x10(-7) M for 16 days. In vitro decidualization was morphologically and biochemically assessed. We analysed whether ESC decidualization could be affected by EPF or peritoneal fluids from control patients (CPF), with or without soluble TNF-alpha receptor 1 (sTNFR-1). RESULTS: Compared with ESC from control patients, eutopic ESC from patients with endometriosis showed an impaired decidualization. Decidualization of normal ESC was morphologically normal but biochemically abnormal in the presence of EPF, which was able to decrease the secretion of decidualization markers. sTNFR-1 was able to partially counteract this effect. CONCLUSIONS: In endometriosis, the milieu surrounding the uterine cavity may be involved in impaired eutopic ESC decidualization, partially due to increased peritoneal levels of TNF-alpha.

Paracrine regulation of endometriotic tissue.

Endometriosis is a chronic estrogen-dependent gynecological disease, characterized by pelvic pain and infertility, defined as the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites. In the peritoneal cavity endometrial cells adhere, proliferate and induce an inflammatory response. Despite a long history of clinical and experimental research, the pathogenesis of endometriosis is still controversial. Abnormal immunological activation, the endocrine milieu and the peritoneal environment all dramatically affect endometriotic tissue function. Recent studies suggest that the peritoneal fluid of women with endometriosis contains an increased number of activated macrophages and other immune cells that secrete various local products, such as growth factors and cytokines, which exert a paracrine action on endometriotic cells. Since the peculiar biological characteristics of eutopic endometrium from women with endometriosis differ from endometrium of normal subjects, an important role in the pathogenesis of this complex disease has been suggested. All of these factors contribute to enhanced proliferative and angiogenic activity and a number of functional and structural changes, resulting in the particular behavior of this tissue.

Ghrelin affects the release of luteolytic and luteotropic factors in human luteal cells.

CONTEXT: Ghrelin, well-known modulator of food intake and energy balance, is a rather ubiquitous peptide involved in several endocrine and nonendocrine actions. A possible as-yet-unknown role for ghrelin in modulating luteal function has been suggested because both ghrelin and its receptor (GRLN-R) have been immunohistochemically detected in human corpus luteum. OBJECTIVE: We first investigated GRLN-R mRNA expression in midluteal phase human luteal cells. Ghrelin effect on basal and human chorionic gonadotropin (hCG)-stimulated progesterone (P) release was then analyzed. Finally, we investigated whether ghrelin could affect luteal release of vascular endothelial growth factor (VEGF), prostaglandin (PG) E(2), both luteotropic factors, and PGF(2alpha), luteolytic modulator. Ghrelin effect on both basal and hypoxia-stimulated VEGF luteal expression was analyzed. METHODS: Human luteal cells were incubated for 24 h with ghrelin (10(-13) to 10(-7) m) or hCG (100 ng/ml) or CoCl(2) (10 microm), chemical hypoxia, or with hCG or CoCl(2) in combination with ghrelin. Both GRLN-R mRNA and VEGF mRNA were evaluated by real-time RT-PCR. PGs and P release was assayed by RIA, whereas VEGF release by ELISA. RESULTS: GRLN-R mRNA expression was demonstrated in human luteal cells. Both basal and hCG-stimulated P release was significantly decreased by ghrelin, which was able to reduce PGE(2) and increase PGF(2alpha) luteal release. Both basal and hypoxia-stimulated VEGF release was significantly decreased by ghrelin, which did not affect VEGF mRNA luteal expression. CONCLUSIONS: The present in vitro study provides the first evidence of a direct inhibitory influence of ghrelin on human luteal function.

Ghrelin in vitro modulates vasoactive factors in human umbilical vein endothelial cells.

OBJECTIVE: To determine whether Ghrelin could affect prostaglandins (PGs) and nitric oxide synthesis in human umbilical vein endothelial cells (HUVEC). The effect of Ghrelin on endothelial cell proliferation was also evaluated. DESIGN: In vitro research report. SETTING: Third-level referral academic centers, including molecular and cellular biology laboratories. PATIENT(S): Human umbilical cords were obtained from healthy female volunteers at term of uncomplicated pregnancies. INTERVENTION(S): HUVEC were cultured with Ghrelin (from 10(-11) to 10(-7) M). After 24 hours supernatants were collected and HUVEC were treated for total RNA extraction. MAIN OUTCOME MEASURE(S): In the culture medium PGs release was evaluated by RIA. Prostaglandin-endoperoxide synthase 2 (COX2) and both the constitutive and the inducible isoforms of nitric oxide synthases (ECNOS and INOS) mRNA expressions were evaluated by retrotranscriptase polymerase chain reaction. Endothelial cell proliferation was evaluated by bromo-deoxy-uridine incorporation and by cell counting. RESULT(S): Ghrelin negatively affected PGs release as well as COX2, ECNOS, and INOS mRNA expressions in HUVEC. Furthermore, Ghrelin increased bromo-deoxy-uridine incorporation in HUVEC without affecting cell counting. CONCLUSION(S): Our in vitro results allowed to hypothesize that Ghrelin could be involved in the modulation of vascular tone by affecting nitric oxide-related protein synthesis and PGs production in endothelial cells.

Regulation of vascular endothelial growth factor synthesis and release by human luteal cells in vitro.

CONTEXT: Vascular endothelial growth factor (VEGF) is essential for normal luteal development and function, but little is still known about the regulation of its production by human midluteal phase luteal cells. OBJECTIVE: We investigated whether human chorionic gonadotropin (hCG) or local factors, including chemical hypoxia, IGF-I and IGF-II, prostaglandin (PG)E(2), and PGF(2alpha) prevail in modulating VEGF mRNA and protein production in human midluteal phase luteal cells. The effect of progesterone (P) on luteal VEGF mRNA expression and protein secretion was also evaluated. Finally, we investigated whether VEGF could directly affect luteal P secretion. INTERVENTIONS: In human midluteal phase luteal cells, VEGF mRNA expression was evaluated by semiquantitative RT-PCR, whereas VEGF and P release was evaluated by ELISA and RIA, respectively. RESULTS: hCG was unable to significantly affect luteal VEGF mRNA and protein synthesis, which in turn was significantly increased by both chemical hypoxia and IGFs. Conversely, VEGF mRNA and protein production was reduced by PGs and P. Finally, VEGF did not affect P luteal secretion. CONCLUSIONS: Our results suggest that local ovarian factors, rather than hCG, predominate in regulating VEGF mRNA and protein production by human midluteal phase luteal cells. For VEGF, a lack of a direct luteal steroidogenic effect was also demonstrated.