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The changes in endometrial homeostasis found in women with polycystic ovarian syndrome (PCOS) could be associated with alterations in the intracrine metabolism of steroid hormones. The uptake of dehydroepiandrosterone-sulphate (DHEA-S), precursor of the intracrine pathway, is achieved by transporters, such as organic anion transporter polypeptides (OATPs), and molecules with oestrogenic activity, such as androst-5-ene-3beta,17beta-diol (androstenediol), can be generated. We aimed to determine androstenediol generation and the expression of OATPs in human endometria throughout the menstrual cycle and in endometria from PCOS women. Endometrial samples were obtained from control women in the proliferative phase (control endometria (CEp), n=7), secretory phase (CEs, n=7), and from PCOS patients (PCOSEp, n=7). The mRNA levels of OATP-B, OATP-D and OATP-E were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels of OATP-E by immunofluorescence; 3beta-hydroxysteroid dehydrogenase (HSD) by immunohistochemistry/Western blot; the metabolism of DHEA to androstenediol was evaluated by thin layer chromatography-high-performance liquid chromatography (TLC-HPLC). Lower levels of OATP-E transcript were obtained in PCOSEp (p<0.05) compared with CEp, while OATP-E protein levels (p<0.05) and DHEA conversion to androstenediol (p<0.01) were higher in PCOSEp. Lower 3beta-(hydroxysteroid dehydrogenase) HSD protein levels were found in PCOSEp (p<0.05) (Western blot, immunohistochemistry). These results reveal a higher capacity of the endometria from PCOS women to metabolise DHEA to androstenediol, which, coupled with the high oestrogen sensitivity previously found in these endometria, may account for the increase in cell proliferation in PCOSEp already reported., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder highly associated with insulin resistance and compensatory hyperinsulinemia. It is known that the insulin signaling pathway is impaired in endometria from PCOS hyperinsulinemic women, but no information is available about molecules associated with cell surface GLUT4 translocation. We therefore evaluated the protein levels of AS160 target molecules, Rab8A and Rab10, and the WAVE family proteins involved in the cortical-actin remodeling, Neural Wiskott-Aldrich Syndrome Protein (N-WASP) and WASP, in endometria from hyperinsulinemic PCOS women and controls., Methods: Protein levels were assessed by western blot, immunohistochemistry and immunofluorescence in proliferative (PE = 7) and secretory (SE = 7) phase endometria from control women and in endometria from hyperinsulinemic PCOS women (PCOS h-INS = 7)., Results: Similar levels were detected for Rab10 in the three studied groups; however, Rab8A levels decreased in SE (P < 0.05) while higher levels were obtained in PCOSE h-INS compared with PE (P < 0.05). In the normal menstrual cycle, Neural Wiskott-Aldrich syndrome protein (N-WASP) and WASP levels were increased in SE versus PE (P < 0.05), but in PCOSE h-INS, the levels were diminished compared with PE (P < 0.05)., Conclusions: SE is characterized by protein expression changes associated with glucose uptake. In endometria from PCOS women with hyperinsulinemia, reduced levels of WAVE family proteins could compromise the cell surface GLUT4 exposure and the consequent glucose uptake in this tissue.

Fifty to seventy percent of patients with polycystic ovary syndrome (PCOS) present hyperinsulinemia. On the other hand, reports indicate that forkhead box class O 1 (FOXO1) and peroxisome proliferator-activated receptor-gamma (PPARG) are involved in the insulin signaling pathway, regulating the gene expression of SLC2A4 (GLUT4). The negative effect of FOXO1 over PPARG transcription disappears when FOXO1 is phosphorylated (p-FOXO1) and excluded from the nucleus, whereas PPARG can suppress gene expression of SLC2A4. Scarce knowledge is available in endometrium of women with PCOS and hyperinsulinemia (PCOSE h-Ins) about the role of these factors. We aimed to evaluate whether the endocrine and metabolic status of PCOS modify the levels of gene and protein expression of FOXO1, PPARG, and SLC2A4 in the endometria from hyperinsulinemic PCOS women compared with controls. In endometria from control (CE, n=7) or PCOSE h-Ins (n=7), we determined the subcellular location and protein levels of p-FOXO1Ser319 and FOXO1/FOXO4 by immunohistochemistry and western blot respectively; gene and/or protein levels of PPARG and SLC2A4 were evaluated by RT-PCR and/or western blot. Cytoplasm location for FOXO1 and p-FOXO1Ser319 was immunodetected in both groups of endometria, showing significantly higher staining in PCOSE h-Ins for these proteins (P<0.05). In PCOSE h-Ins, gene and protein levels of PPARG were significantly higher than in CE, whereas SLC2A4 mRNA was decreased (P<0.05). In conclusion, the derepression of PPARG transcription by the high levels of p-FOXO1Ser319 could partially account for the lower levels of SLC2A4 found in PCOSE h-Ins, suggesting an alteration of the endometrial function in these patients.

Objective: To examine whether overweight and obesity could lead to increased endometrial proliferation and activation of AKT and ERK1,2 in cycling premenopausal women., Methods: Endometrial and blood samples were obtained from women with normal endometrial histology, and allocated into three groups-normal-weight, overweight and obese-according to the subject's body mass index (BMI). Samples from obese patients with type-I endometrial cancer (EC) were included as a control. Cell proliferation was measured by immunohistochemical detection of Ki67 and phosphorylated histone H3 (p-H3). AKT and ERK1,2 activation was assessed by Western blot. Circulating steroids, leptin and insulin were measured by immunoassays., Results: In endometrial samples with normal histology, epithelial cell proliferation was higher in the overweight and obese groups versus the normal-weight set (P<0.05). Proliferation indexes were positively correlated with the subject's BMI and serum levels of estrogen, leptin and insulin (P<0.05). Increased phosphorylated AKT (pAKT) (1.6-fold) and ERK1,2 (pERK1,2) (8.7-fold) were observed in endometria from obese with respect to normal-weight subjects (P<0.05). Similarly, increased phosphorylation of AKT (0.7-fold) and ERK1,2 (2.3-fold) was detected in endometria from overweight as compared with the normal-weight group (P<0.05). In women with EC, we found a significant increase in endometrial proliferation, and in pAKT and pERK1,2 expression levels when compared to patients with normal endometrial histology., Conclusion: These results show correlation between obesity (and overweight) and increased endometrial cell proliferation, and the activation of AKT and ERK1,2. These features could be related with the higher risk to develop type-I EC in overweight and obese women., (Copyright 2009 Elsevier Inc. All rights reserved.)

Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder associated with insulin resistance and compensatory hyperinsulinemia. Scarce information is available on the expression of molecules involved in the insulin pathway in endometria from women with PCOS. Therefore, we examined the protein levels of insulin-signaling molecules, like insulin receptor, insulin-receptor substrate (IRS)-1, pIRS-1Y612, Akt, AS160, pAS160T642 and GLUT4 in endometria from PCOS women with or without hyperinsulinemia. Protein levels were assessed by Western blot and immunohistochemistry in 21 proliferative-phase endometria from control women (CE = 7), normoinssulinemic PCOS women (PCOSE-NI = 7) and hyperinsulinemic PCOS women (PCOSE-HI = 7). The data show no differences in the expression of insulin receptor between all groups as assessed by Western blot; however, IRS-1 and pIRS-1Y612 were lower in PCOSE-HI than controls and PCOSE-NI (P < 0.05). AS160 was detected in all analyzed tissues with similar expression levels between groups. Importantly, PCOSE-HI exhibited lower levels of pAS160T642 (P < 0.05) and of GLUT4 (P < 0.05) compared with CE. The immunohistochemistry for insulin receptor, IRS-1, Akt, AS160 and GLUT4 showed epithelial and stromal localization; IRS-1 staining was lower in PCOSE-HI (P < 0.05). In conclusion, human endometrium has the machinery for glucose uptake mediated by insulin. The diminished expression of GLUT4, as well as the lower level of pIRS-1Y612 and pAS160T642 exhibited by PCOSE-HI, suggests a disruption in the translocation of vesicles with GLUT4 to the cell surface in these patients.

Prostaglandin E(2) (PGE(2)) is an arachidonic acid metabolite involved in physiological homeostasis and numerous pathophysiological conditions. Furthermore, it has been demonstrated that prostaglandins have a stimulating effect not only on angiogenesis in situ and in vitro but also on chondrocyte proliferation in vitro. Thus, PGE(2) represents an interesting signaling molecule for various tissue engineering strategies. However, under physiological conditions, PGE(2) has a half-life time of only 10 min, which limits its use in biomedical applications. In the present study, we investigated if the incorporation of PGE(2) into biodegradable poly-L-lactide-co-glycolide microspheres results in a prolonged release of this molecule in its active form. PGE(2)-modified microspheres were produced by a cosolvent emulsification method using CHCl(3) and HFIP as organic solvents and PVA as emulsifier. Thirteen identical batches were produced; and to each batch 1.0 mL of serum-free medium was added. The medium was removed at defined time points and then analyzed by gas chromatography tandem mass spectrometry (GC/MS/MS) to measure the residual PGE(2) content. In this study we demonstrated the prolonged release of PGE(2), showing a linear increase over the first 12 h, followed by a plateau and a slow decrease. The microspheres were further characterized by scanning electron microscopy., ((c) 2008 Wiley Periodicals, Inc.)

Objective: To examine whether the abundance, localization, and/or activity of cell cycle regulators CDK2, Cyclin E, p27, and survival proteins AKT and Ras in PCOS-associated endometria (with and without hyperplasia) differ from non-PCOS endometria., Methods: The expression of CDK2, Cyclin E, p27, AKT and Ras was measured by immunohistochemistry and/or Western blot in 9 normal endometria (NE), 12 endometria from PCOS patients without endometrial hyperplasia (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE), and 9 endometria from patients with endometrial hyperplasia (HE). The activity of CDK2 was assessed by an in vitro kinase assay., Results: CDK2, Cyclin E and p27 proteins were expressed mainly in the endometrial epithelial cells of the studied groups. No change in the activity of CDK2 was observed in total extracts obtained from the tissue samples. However, the nuclear expression of CDK2 in epithelial cells was slightly elevated in PCOSE and significantly increased in HPCOSE when compared to NE. Higher expression of p27 was detected in the cytoplasm of epithelial cells of PCOSE and HPCOSE when compared to NE. Also, we found an increment in Ser473-AKT phosphorylation and an over-expression of the Ras oncogene in endometria of patients with PCOS., Conclusion: The PCOS condition is associated with increased Ser473-AKT phosphorylation, elevated expression of Ras, increased cytoplasmic abundance of p27, and increased nuclear abundance of CDK2 in the endometrial epithelial cells. These biological events could potentially provide a chance for endometrial cells from PCOS patients to exit the controlled cell cycle and become hyperplastic at a later stage.

The aim of the present investigation was to study whether the endocrinological status of women bearing polycystic ovarian syndrome (PCOS) affects the endometrial in situ steroid metabolism. For this purpose, we evaluated the mRNA levels (RT-PCR), and the activity of steroid metabolic enzymes: P450 aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in 23 samples of normal endometria (CE), 18 PCOS endometria without treatment (PCOSE), 10 specimens from PCOS women with endometrial hyperplasia (HPCOSE), and 7 endometria from patients with endometrial hyperplasia not associated to PCOS (EH). The data showed lower levels of STS mRNA for PCOSE and HPCOSE (p<0.05, p<0.01, respectively) and of EST for HPCOSE and EH compared to control (p<0.05). However, higher levels for EST mRNA were obtained in PCOSE (p<0.05) versus CE. The mRNA and protein levels for P450 aromatase were undetectable in all analyzed endometria. The relationship between the activities of STS and EST was lower in PCOSE and HPCOSE (p<0.05) versus CE. The ratio between the mRNA from 17beta-HSD type 1/type 2 was higher in PCOSE (p<0.05), whereas, a diminution in the 17beta-HSD type 2 activity was observed in PCOSE (p<0.05). These results indicate that the activity of enzymes related to the steroid metabolism in analyzed PCOSE differ from those found in the CE. Consequently, PCOSE may present an in situ deregulation of the steroid metabolism.

Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic pathology related with infertility and recurrent miscarriage. We have previously shown that the endometrium of these patients can exhibit a potentially higher sensitivity to estrogen action, being estrogens important regulators of the cell cycle and tissue homeostasis. The effect of estrogens on tissues depends on their in situ availability, which is in part regulated by the activity of steroid metabolic enzymes within the tissues. Therefore, the objective of the present study was to analyze if the activity and/or expression of steroid metabolic enzymes in endometria from women with PCOS differ from controls. For this purpose, the activity of the enzymes was determined by using radiometric assays and the mRNA levels measured by semi-quantitative RT-PCR. Both assays were assessed in endometria obtained during mid secretory phase from control (CE, n=12) and PCOS women (PCOSE, n=11). For the statistical analyses, Mann-Whitney and Student's t-tests were used to compare CE and PCOSE, considering a p value <0.05 significantly different. The results showed an increase in the sulfatase activity in PCOS respect to control endometria (200+/-28pmol/mg vs. 115+/-13pmol/mgproth; p<0.05), in agreement with the higher mRNA levels found for the enzyme in PCOSE. In addition, a PCOSE exhibited lower activity of sulfotransferase respect to the control group (50+/-21pmol/mg vs. 124+/-10pmol/mgproth; p<0.05), whereas a higher level of 17beta-hydroxysteroid dehydrogenase type 1mRNA was found in PCOSE compared with the control tissues (p<0.05). The activity of 17beta-hydroxysteroid dehydrogenase type 2 and the mRNA levels of sulfotransferase were similar in both groups; meanwhile, the expression of aromatase was undetectable. These data indicate that the sulfatase pathway could play an important role in the local production of estrogens in PCOSE from secretory phase. This potentially higher bioavailability of estrogens in endometria from PCOS women could influence the deregulation of tissue homeostasis that we have previously reported, and could partially explain the poor reproductive performance observed in this group of patients.

The in vitro cell expansion of autologous chondrocytes is of high interest in regenerative medicine since these cells can be used to treat joint cartilage defects. In order to preserve chondrocyte phenotype, while optimizing adhesion on microspheres, several processing parameters for the microsphere synthesis were varied. In this study three different polylactide-co-glycolides were used with differing lactide-glycolide ratios (85:15 and 50:50) and differing inherent viscosities. An emulsion route was established, where the polymer was dissolved in chloroform and then injected into a stirred polyvinyl alcohol-water solution at different polymer concentrations and different stirring velocities to produce microspheres with varying diameters. The sphere size distribution and morphology was analyzed using image processing software on SEM pictures. Based on previous experiments with commercial microspheres, three optimum samples were selected for further investigations. The degradation of the microspheres was determined in a long-term experiment in culture medium for 3 months. Adherent cells were characterized after 3 and 5 days by FDA+EB vital staining and in SEM.

Objective: To evaluate the protein and messenger RNA expression of sex hormone-binding globulin (SHBG) in endometria from women with polycystic ovary syndrome (PCOS)., Design: Case-control study., Setting: Hospital research unit., Patient(s): Thirty-three women with PCOS, and 17 fertile, healthy women of similar age to those with PCOS., Intervention(s): Endometrial and blood samples were obtained from women with PCOS (PCOSEs) and from control women (CEs) during the proliferative phase of the menstrual cycle., Main Outcome Measure(s): Expression studies for SHBG (immunohistochemistry and reverse transcription-polymerase chain reaction). Hormonal studies for determining sex steroids (T, P, and E(2)) and SHBG concentration. Insulin sensitivity was assessed by composite insulin sensitivity index (ISI(composite))., Result(s): In stroma, the protein expression of SHBG was lower in PCOSEs than in CEs. Epithelial cells had a similar expression of SHBG protein in both groups. Messenger RNA of variant 548 base pairs (wild-type) tended to be lower in PCOSEs compared to CEs. When PCOSEs were classified by insulin resistance, the PCOSEs with normal insulin sensitivity showed an expression of stromal SHBG similar to that observed in CEs., Conclusion(s): The low SHBG expression in the stromal compartment of endometria from women with PCOS with insulin resistance may contribute to generate an abnormal steroid milieu in the endometria of these women.

Objective: To study the proteins involved in endometrial homeostasis in PCOS women., Methods: Protein expression of Ki67, Bcl-2, Bax, Pro-Caspase-3 and Caspase-3 by immunohistochemistry and/or Western blot, and DNA fragmentation using in situ 3'-end labeling of apoptotic cells, was measured in 9 samples of normal endometrium (NE), 12 PCOS endometria without treatment (PCOSE), 7 endometria from PCOS women with endometrial hyperplasia (HPCOSE) and 9 endometria from patients with endometrial hyperplasia (HE)., Results: Cell proliferation was higher in epithelium from PCOSE (P<0.05), HPCOSE and HE vs NE. A higher Bcl-2/Bax relative ratio in PCOSE and HPCOSE was observed, in absence of active Caspase-3 and scarce DNA fragmentation in the four groups of endometria studied., Conclusion: As the apoptosis was scarce in all of the groups studied, endometrial homeostasis deregulation in PCOS could be a result of increased proliferation. Therefore, the onset of endometrial hyperplasia in PCOS endometrium could be linked to inadequate cell proliferation, and concomitantly to inadequate cell survival.

Background: The hormonal alterations observed in women with polycystic ovary syndrome (PCOS) may promote implantation failure as well as disruption of their endometrial homeostasis. To evaluate cell survival of mid-secretory endometrium from untreated women with PCOS, we measured the expression of apoptosis and proliferation-related proteins., Methods: A case-control study of 11 patients with PCOS and 11 fertile women in the Hospital Research Unit was performed. Endometrial samples were obtained from PCOS women (PCOSE) and fertile healthy women (CE) during the mid-secretory phase of the menstrual cycle. Protein expressions for Akt, p-AktSer473 and p-AktThr308, Bad, p-BadSer136, Bcl-2, Bax and pro-caspase-3/caspase-3, were assessed by western blot, and Ki67 and p-histone-3 (p-H3) by immunohistochemistry., Results: In CE and PCOSE, a predominance of p-AktThr308 over p-AktSer473 is observed; p-BadSer136 expression is higher in PCOSE than in CE (P < 0.05). Also, Bcl-2 protein is overexpressed in PCOSE (P < 0.05), with no changes in Bax expression among the two groups, resulting in a significantly higher Bcl-2/Bax ratio in PCOSE than in CE (P < 0.05). No changes in the expression of caspase-3 are obtained between both groups of endometria. Furthermore, cell proliferation detected by the expression of Ki67 and p-H3 proteins is higher in the epithelia than the stroma of PCOSE versus CE (P < 0.05)., Conclusion: The abnormal tissue homeostasis exhibited by the secretory endometrium from PCOS patients with spontaneous ovulation may interfere with their endometrial receptivity.

Objective: To study if the endocrinological status of PCOS women affects the endometrial sensitivity to steroids by evaluating the expression of androgen receptor (AR), estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), co-activators AIB1 and ARA70, and co-repressor NCoR., Methods: Gene and/or protein expression of steroid receptors and co-regulators was measured in 17 samples of normal endometrium (NE), 23 PCOS endometrium without treatment (PCOSE), 11 endometria from PCOS women and with endometrial hyperplasia (HPCOSE), and 10 endometria from patients with endometrial hyperplasia (HE), using RT-PCR and/or immunohistochemistry and Western blot., Results: Gene and protein expression of AR was relatively elevated in PCOSE and HPCOSE compared with NE. A significant increase in ERalpha protein expression was observed in PCOSE, preferentially in the nucleus of endometrial cells, whereas ERbeta gene and protein expression increased gradually from PCOSE to HPCOSE and HE, mainly in the epithelial compartment. Importantly, we found a gradual increase in the ERbeta/ERalpha gene and protein expression ratio in endometria from the four groups of women. AIB1 showed increased nuclear protein expression in PCOSE compared to NE, in the presence of a high expression of ARA70 in all groups. High expression of ARA70 together with a normal expression level of AIB1 was observed in HPCOSE. The cytoplasmic immunostaining of NCoR was similar between the four groups of patients., Conclusion: The PCOS endometrium exhibits a higher sensitivity to steroid action. We can inferred that these alterations could deregulate the transcription of genes involved in the cell cycle, which may lead to the development of endometrial hyperplasia in PCOS women.

Objective: To evaluate gene and protein expression of steroid receptors, nuclear receptor coregulators, and uterine receptivity markers in midsecretory phase endometria from untreated women with polycystic ovary syndrome (PCOS)., Design: Case-control study., Setting: Hospital research unit., Patient(s): Eight patients with PCOS and eight fertile women of similar age to those with PCOS., Intervention(s): Endometrial samples were obtained from women with PCOS (PCOSE) and normal (NE) women during the midsecretory phase of the menstrual cycle., Main Outcome Measure(s): Expression studies (immunohistochemistry, reverse transcription-polymerase chain reaction [RT-PCR] and Western blot)., Result(s): Endometria from PCOS exhibit higher levels of messenger RNA (mRNA) and protein for estrogen receptor alpha and coactivators than NE. Epithelial cells had a greater expression of progesterone receptor in PCOSE, whereas, no differences were observed in gene and protein expression of the nuclear corepressor (NcoR) and the antiadhesion molecule mucin type-1 (MUC-1) between PCOSE and NE. Immunodetection for the coactivator ARA70 was higher in PCOSE than in NE; in contrast, expression of beta3-integrin in epithelia was lower in PCOSE than in control endometria., Conclusion(s): The higher response to steroid hormones of endometria from untreated PCOS-women induces diminished expression of beta3 integrin, which partially explain implantation failure in PCOS patients.

Objective: To assess the effect of clomiphene citrate (CC) on endometrial epithelial integrins and P receptors (PR) during the window of implantation., Design: Controlled, prospective, clinical study., Setting: Teaching hospital and university research laboratory., Patient(s): Thirty-one fertile, normo-ovulatory women participated in this trial. Thirteen women exhibited a CC-stimulated cycle with 50 mg on days 5-9, and 18 women with spontaneous menstrual cycles served as controls., Intervention(s): Endometrial biopsies in the midluteal phase., Main Outcome Measure(s): Immunohistochemical determination and endometrial cellular localization of alpha1, alpha v, beta3, and alpha4 epithelial integrins and PR during the window of implantation. The staining intensity was assessed by a semiquantitative index (HSCORE) and compared by nonparametric Mann-Whitney test., Result(s): Higher plasma levels of P and E2 and delayed histologic dating of the endometrium (38%) were features of CC-treated women. In addition, a low epithelial beta3 integrin expression and persistent PR were observed in glandular epithelial cells of "out-of-phase" endometrial biopsies from CC-treated women. In contrast, in "in-phase" biopsies, neither epithelial PR nor beta3 integrin were different from spontaneous control cycles. There was no difference in the expression of alpha1, alpha v, and alpha4 between the groups studied., Conclusion(s): The administration of clomiphene produces aberrant endometrial beta3 integrin expression in conjunction with a failure in the down-regulation of PR during the window of implantation in a significant number of normo-ovulatory women, notwithstanding the higher plasma P levels. Therefore, CC might affect the expression of endometrial receptivity markers.

Objective: To evaluate the clinical response and endometrial morphology during the implantation window on ovarian hyperstimulation with the aromatase inhibitor letrozole in infertile ovulatory women., Design: Prospective trial in infertile patients., Setting: Tertiary care hospital., Patient(s): Eight ovulatory infertile patient candidates for ovarian superovulation., Intervention(s): Subjects were monitored in one control cycle. In the next cycle, they received letrozole 5.0 mg daily on days 3 through 7 after menses., Main Outcome Measure(s): Number of ovulatory follicles; dominant follicle diameter; endometrial thickness; hormonal profile of FSH, LH, E(2), A, T, and P; endometrial histological dating; and pinopode formation assessed by scanning electron microscopy., Result(s): Cycles stimulated with letrozole resulted in more ovulatory follicles than did natural cycles (mean +/- SD 2.0 +/- 0.9 vs. 1.0 +/- 0.0), which attained a greater preovulatory diameter (mean +/- SD 23.8 +/- 2.7 vs. 19.3 +/- 2.1 mm), with similar endometrial thickness at midcycle compared with spontaneous cycles. Endocrine profile of medicated cycles was characterized on day 7 by increased levels of LH (5.9 +/- 0.8 vs. 3.5 +/- 0.4 IU/mL), reduced E(2) (98.4 +/- 11.4 vs. 161.5 +/- 14.7 pmol/L), and elevated androgens. Preovulatory and midsecretory E(2) were similar to spontaneous cycle, and P levels during midluteal phase were significantly elevated (44.2 +/- 4.6 vs. 27.7 +/- 4.6 pmol/L). Endometrial morphology during the implantation window in letrozole-stimulated cycles was characterized by in-phase histological dating and pinopode expression on scanning electron microscopy., Conclusion(s): Letrozole induces moderate ovarian hyperstimulation in ovulatory infertile patients with E(2) levels similar to spontaneous cycles and higher midluteal P, leading to both a normal endometrial histology and development of pinopodes, considered to be relevant markers of endometrial receptivity.

We report a patient with a nasal hemangiopericytoma associated with an oncogenic hypophosphatemic osteomalacia (OHO). This syndrome results from tumor products that decrease renal tubular phosphate resorption, leading to the osteomalacia. This patient presented with classic bone manifestations of osteomalacia and a nasal tumor. Laboratory studies performed before the first resection of the tumor included normal serum calcium, hypophosphatemia due to decreased tubular reabsorption of phosphate, and an undetectable serum 1,25 dihydroxy vitamin D level. Serum parathormone level was normal. Anterior iliac crest bone biopsy showed characteristic signs of osteomalacia that included increased osteoid and delayed mineralization. A partial resection of the nasal tumor was performed. After the first surgery the patient showed detectable serum level of 1,25 dihydroxy vitamin D, and transient normalization of the tubular reabsorption of phosphate. The patient was also treated with phosphate supplements and vitamin D with transient control of her clinical manifestations and improvement of the radiographic signs of osteomalacia. Three months after surgery, the serum level of 1,25 dihydroxy vitamin D level again became undetectable. After selective embolization of the tumor, followed by an apparent complete tumor resection and postoperative radiation therapy, her hypophosphatemia and decreased phosphate tubular reabsorption persisted. Therefore, biochemical changes associated with hemangiopericytoma induced OHO may persist even after apparent total tumor resection. Clinicians should be aware of the oncogenic basis for some osteomalacia, as seen in this patient.

Objective: To evaluate the expression of steroid hormone receptors, proteins related to apoptosis, and cell proliferation in endometria from women with polycystic ovary syndrome (PCOS)., Design: Case-control study., Setting: Hospital research unit., Patient(s): Eight women with PCOS and 12 fertile healthy women of similar age to those with PCOS., Intervention(s): Endometrial samples were obtained from women with PCOS (PCOSE) and normal (NE) women during the proliferative phase of the menstrual cycle., Main Outcome Measure(s): Expression studies (immunohistochemistry and reverse transcription-polymerase chain reaction) and DNA fragmentation [TdT-mediated dUTP nick end labeling (TUNEL)]., Result(s): In stroma, protein expression of estrogen receptor alpha, Bcl-2, and Bax was higher in PCOSE than in NE; epithelial cells had a greater expression of androgen receptor in the nucleus and a lower expression in the cytoplasm of PCOSE. Cell proliferation was higher in the epithelia of NE, while the expression of caspase-3 and DNA fragmentation was similar in both groups. The bax mRNA expression was higher in PCOSE, and bcl-2 mRNA expression was similar between groups. A higher bcl-2/bax relative ratio in PCOSE was observed., Conclusion(s): An alternating expression of proteins related to cell survival in endometria from PCOSE may potentially be associated with the disruption of their endometrial cell cycle.

The aim of the present study was to investigate the action of insulin-like growth factor I (IGF-I) and insulin-like growth factor-binding protein 3 (IGFBP-3) on steroidogenesis and apoptosis in human corpus luteum during the midluteal phase. Slices from corpora lutea were incubated for 4 h with IGF-I or IGFBP-3. Progesterone, oestradiol, androstenedione and testosterone concentrations were determined by radioimmunoassay; caspase 3 expression was assessed by immunohistochemistry; bcl-2, bax and P(450arom) expression were assessed by RT-PCR; and apoptosis was detected by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. The results showed that addition of IGF-I stimulated progesterone production (150%, P < 0.01), oestradiol production (65%, P < 0.05) and bcl-2 gene expression (approximately 200%, P < 0.05), but decreased apoptosis (P < 0.05). In contrast, IGFBP-3 reduced steroid production and increased bax gene expression and the percentage of apoptotic cells (P < 0.05). Neither IGF-I nor IGFBP-3 had an effect on P(450arom) expression or on the concentrations of its substrates. However, maximum expression of caspase 3 was detected in corpus luteum during the midluteal phase. In conclusion, these results indicate that IGF-I and IGFBP-3 act as regulatory peptides of the function of the human corpus luteum during the midluteal phase. This action may be direct or mediated by steroid production or by bcl-2-bax expression.

Objective: To investigate the role of nitric oxide (NO) and death regulatory genes, bcl-2 and bax, in human endometria apoptosis., Design: Expression of bcl-2, bax, NO synthases (NOS), and the apoptotic effect of L-arginine on endometrial explants in vitro., Setting: Prospective study., Patient(s): Thirty-seven eumenorrheic women., Intervention(s): Endometrial samples were obtained with Pipelle suction curette after women signed institutional informed consent forms., Main Outcome Measure(s): DNA fragmentation (TUNEL), immunohistochemistry, and reverse transcription polymerase chain reaction., Result(s): Apoptosis was detected in mid and late secretory endometria. L-arginine induced an increase in apoptosis in stroma (threefold), glands (eightfold), and surface epithelia (fourfold) in proliferative but not secretory endometria explants. Immunostaining of Bcl-2 was almost absent in the secretory endometria, whereas Bax increased in the stroma at the end of the menstrual cycle, coincident to the decrease in the bcl-2/bax mRNA relative ratio (P<.05) observed in secretory endometria., Conclusion(s): The induction of DNA fragmentation by L-arginine on proliferative endometria suggests that NO may be involved in the endometrial apoptotic process, whose control may be related predominantly to the changes of Bcl-2 expression.

The presence of insulin-like growth factors (IGF), IGF binding proteins (IGFBP) and IGF receptor type 1 (IGF-IR) in the human corpus luteum was investigated by examining the expression and production of related proteins throughout the lifespan of the corpus luteum and the action of nitric oxide upon their production. The expression of proteins in corpora lutea from the early, mid-and late luteal phases was assessed by immunohisto-chemistry, evaluated by a semi-quantitative analysis and the functional study was performed in corpus luteum explants incubated with nitric oxide donors. IGF-I and -II and IGFBP-1 and -3 were measured in the culture media by specific immunoassays. The results showed that IGF-I and -II, IGFBP-1 to -6 and IGF-IR were detected in the human corpus luteum throughout the luteal phase. Moreover, the expression and production of IGF-I and IGFBP-1 increased progressively from corpora lutea from the early to late luteal phases (P < 0.05), whereas the expression and production of IGFBP-2, -4 and -5 were significantly higher in corpora lutea from the mid-luteal phase (P < 0.05). No differences were observed in the expression of IGF-II, IGFBP-3 and -6 and IGF-IR throughout the lifespan of the corpus luteum. However, functional studies showed that nitric oxide donors elicited a stimulatory action on production of IGF-I in corpora lutea from the early luteal phase (80%) and on production of IGFBP-1 in corpora lutea from the late luteal phase (50%) (P < 0.05), whereas production of IGF-II and IGFBP-3 was not affected by nitric oxide. In conclusion, the components of the IGF-IGFBP system are expressed in the human corpus luteum throughout its lifespan. Nitric oxide regulates IGF-I and IGFBP-1 production, indicating that the growth factors may serve, at least in part, as mediators of the action of nitric oxide in the human corpus luteum.

Unlabelled: Determine quantitative expression of endometrial integrins that reflect receptivity during implantation window in fertile women treated with clomiphene citrate (CC), and in intrauterine device users (IUD) as compared to fertile controls. Comparative study of the quantitative expression of a1, a4, av and b3 integrins in epithelial and stromal cells in mid-secretory endometrium of CC treated fertile women, IUD users and controls. All subjects included in this study had regular and ovulatory menstrual cycles., Subjects: Ten women treated with a daily dose of 50 mg of CC. Six women T-Cu device users and nine fertile controls. Age ranges for all groups were similar, 29-41 years old (mean 36.3). Tissue samples were taken at the mid-secretory phase or implantation window. A histological dating of the endometrial biopsies was assessed according to Noyes criteria. Ovulation was assessed by repeated transvaginal ultrasonography. The expression of a1, a4, aV and b3 integrins in dispersions of epithelial (EEC) and stromal (ESC) cells isolated from endometrial biopsies was quantitatively determined by flow cytometry using specific monoclonal antibodies. Immunohistochemistry was also used to detect integrin expression. Biopsies from CC-treated women had a high incidence of out-of-phase endometria. Interestingly, CC-treated women over-expressed a1, aV and b3-ESC integrins and under-expressed b3-EEC subunit (P<0.05). IUD users over-expressed the a1-EEC and under-expressed a4-ESC (P<0.05) at the time of the implantation window. CC treatment in fertile women provokes a high frequency of out-of-phase endometrium and desynchronises the expression of endometrial integrins at the implantation window. The epithelial b3 integrin was under-expressed in all CC-treated patients. The T-Cu intrauterine device alters endometrial receptivity by a different mechanism independent of the expression of the epithelial b3 integrin. However, both CC and IUD use alter the expression of some epithelial and stromal integrins during the implantation window.

The present study aimed to investigate the role of nitric oxide (NO) in regression of the human corpus luteum. We therefore examined the effect of both NO and human chorionic gonadotrophin (HCG) on luteal cell apoptosis, and Bcl-2 production. The effect of NO on oestrogen production during corpus luteum regression was also studied. Slices from corpus luteum collected throughout the luteal phase were incubated for 4 h with the nitric oxide synthase (NOS) substrate, L-arginine (L-Arg, 1 mmol/l), the NOS inhibitor N-monomethyl-L-arginine (L-NMMA) (1 mmol/l), or with HCG (10 IU/ml). Oestradiol concentrations were determined by radioimmunoassay; Bcl-2 concentrations were measured by enzyme-linked immunosorbent assay; apoptosis was detected in-situ by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling; and inducible nitric oxide synthase (iNOS) was assessed by immunohistochemistry. Consistent with our previous findings, L-Arg elicited an inhibitory action on the production of oestradiol (P< 0.05). The number of apoptotic cells increased (P<0.05) from early to late corpus luteum, as did the number of cells positive for the expression of iNOS. The percentage of apoptotic cells in mid and late luteal phase was increased by L-Arg (56% and 310% respectively; P <0.05), and decreased by L-NMMA and HCG. Although no changes were observed in Bcl-2 concentration during the corpus luteum life span, L-Arg inhibited, and HCG augmented, Bcl-2 production (P<0.05) from mid and late corpus luteum cells in vitro. In summary, these results suggest that the opposite actions of L-Arg and HCG on human corpus luteum viability may, in part, be mediated by changes in the level of the anti-apoptotic activities caused by oestradiol and Bcl-2 protein.