Alex Muñoz

The human corpus luteum: life cycle and function in natural cycles.

OBJECTIVE To summarize recent advances in the understanding of the endocrine signaling pathways between the hypothalamus, pituitary, and human corpus luteum (CL); to examine the major paracrine and autocrine mechanisms and the key genes and proteins involved in CL development, function, and regression in natural cycles; to review the endocrine and molecular response of the midluteal phase CL to in vivo administration of human chorionic gonadotropin (hCG); and to describe the ultrasonographic and Doppler evaluation of the ovary and endometrium throughout the luteal phase. DESIGN Published data in the literature, including the basic and clinical research studies of the authors. SETTING University-affiliated hospital and research centers. PATIENT(S) None. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Clinical and molecular analysis of human CL function. RESULT(S) The endocrine function of the subpopulations of luteal cells is critical for the maintenance of CL function, including neovacularization and steroid hormones production. We consider the key genes and proteins that favor development of luteal structure and function throughout the menstrual cycle and in our model of hCG treatment resembling early pregnancy. CONCLUSION(S) These data indicate that the functional lifespan of the CL depends on paracrine and autocrine mechanisms. Therefore, the significance of the key genes and proteins that we analyze in lutein cells during CL development, function, demise, and rescue by hCG is likely to bring new therapeutic applications for the management of fertility defects and the control of fertility.

Recent cigarette smoking and assisted reproductive technologies outcome

OBJECTIVE To assess the association between recent cigarette smoking (CS) in female and male partners and assisted reproduction technology (ART) outcomes. DESIGN Cohort prospective study. SETTING University ART program in Chile. PATIENT(S) One hundred sixty-six couples seeking pregnancy through ART. INTERVENTION(S) Follicular fluid (FF) and serum cotinine concentrations were measured in female partners. Self-reported CS data were collected through personal interviews. MAIN OUTCOME MEASURE(S) The association between female recent smoking, assessed by FF and serum cotinine concentrations, and ART outcomes, such as number of ova retrieved and implantation rates, and the association between self-reported male recent smoking and live birth rates. RESULT(S) A significant age-adjusted association between increased FF cotinine level and decreased number of ova retrieved was found. The male partners smoking habit significantly decreased the live birth rate from 21.1% to 7.8%. Serum cotinine concentrations paralleled those of FF. CONCLUSION(S) The hypothesis of a detrimental effect of recent female smoking over implantation rates is rejected. However, recent male smoking is associated with significantly decreased live birth rates even after adjusting for confounders. Female recent smoking was significantly associated with decreased number of retrieved ova.

Promoter hypermethylation of BRCA1 correlates with absence of expression in hereditary breast cancer tumors.

Germline mutations in BRCA1 account for a low proportion of hereditary cases in diverse populations. Several efforts have been made to find new genes involved in the inheritance of breast cancer with no success until today. The participation of BRCA1 in the development of breast cancer has been proposed in several studies where hypermethylation of its promoter and a decrease in expression has been reported for sporadic cases and one study on familial cases. To explore the participation of BRCA1 in hereditary carcinogenesis through a different mechanism than the inheritance of germline mutations, we studied the methylation status of its promoter in breast tumors, from patients previously screened for BRCA1/BRCA2 germline mutations. We also determined the presence of the BRCA1 protein in these tumors and correlated both events with tumor grade, hormone receptors and ERBB2 presence. Promoter hypermethylation of the BRCA1 gene was detected in 51% of our biopsies, among which 67% did not express the respective protein. This result leads us to suggest that hypermethylation could be considered as an inactivating mechanism for BRCA1 expression, either as a first or second hit. Moreover, a number of biopsies with absence of expression on BRCA1 showed negative detection of estrogen and progesterone receptors, a similar phenotype to BRCA1 mutated breast tumors.

Silencing of tumor suppressor genes RASSF1A, SLIT2, and WIF1 by promoter hypermethylation in hereditary breast cancer

Promoter hypermethylation is gaining strength as one of the main mechanisms through which tumor suppressor genes are silenced during tumor progression. Three tumor suppressor genes are frequently found methylated in their promoter, in concordance with absence of expression, RASSF1A, SLIT2, and WIF1. In addition, a previous array‐CGH analysis from our group showed that these genes are found in deleted genomic regions observed in hereditary breast cancer tumors. In the present work we analyzed the methylation status of these three tumor suppressor gene promoters in 47 hereditary breast cancer tumors. Promoter methylation status analysis of hereditary breast tumors revealed high methylation frequencies for the three genes (67% RASSF1A, 80% SLIT2, and 72% WIF1). Additionally, the presence of methylated PCR products was associated with absence of protein expression for the three genes and statistically significant for RASSF1A and WIF1. Interestingly, methylation of all the three genes was found in 4 out of 6 grade I invasive ductal carcinoma tumors. Association between RASSF1A methylation and DCIS tumors was found. These results suggest that silencing of these tumor suppressor genes is an early event in hereditary breast cancer, and could be a marker for pre‐malignant phenotypes.

Human corpus luteum physiology and the luteal-phase dysfunction associated with ovarian stimulation

The human corpus luteum is a temporary endocrine gland that develops after ovulation from the ruptured follicle during the luteal phase. It is an important contributor of steroid hormones, particularly progesterone, and is critical for the maintenance of early pregnancy. Luteal-phase dysfunction can result in premature regression of the gland, with a subsequent shift to an infertile cycle. Understanding the mechanism of steroidogenesis during corpus luteum growth and regression is crucial for evaluating the normal physiology and pathophysiology of reproductive cycles. The rate-limiting step in corpus luteum steroidogenesis is the transport of cholesterol to the site of steroid production. Steroidogenic acute regulatory protein is a key player in this process and is positively correlated with progesterone concentrations throughout the early and mid-luteal phase. Changes in the endocrine environment brought on by the gonadotrophins used for ovarian stimulation are thought to underlie the corpus luteum dysfunction associated with IVF cycles. While ovarian hyperstimulation syndrome is associated with human chorionic gonadotrophin (HCG), studies suggest that exogenous progesterone provides necessary luteal support in patients undergoing IVF. The current trend towards simple stimulation protocols and the use of single-embryo transfers provide further opportunity to revisit HCG administration as luteal support.

Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells

The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

Decreased anti-Müllerian hormone concentration in follicular fluid of female smokers undergoing artificial reproductive techniques.

BACKGROUND Several reports indicate that women who smoke have an increased risk of failure to conceive compared with their non-smoker counterparts. Here, we assessed the effect of smoking during the Assisted Reproduction Therapy (ART) on a potential marker of ovarian reserve, anti-müllerian hormone (AMH) in the follicular fluid (FF). MATERIALS AND METHODS This was a cohort prospective study to assess the association between cigarette smoking and AMH concentrations in FF in fifty-six women undergoing their first ART cycle. Self-reported smoking status over time was also collected through personal interview. The main outcome measured was the association between current smoking and AMH concentrations in FF. Smoking status was assessed by FF cotinine concentrations. Analysis of covariance was performed to test statistical interaction between the main outcome and confounders. RESULTS The mean concentration of AMH in follicular fluid was significantly decreased among smokers (1.02±0.14 vs. 1.74±0.15, P<0.05). No statistical interaction was found between this difference in AMH concentrations and confounders like age and BMI. Thus, our data support the idea that AMH is decreased in active smokers across the fertile age. CONCLUSIONS The hypothesis of decreased AMH concentration in follicular fluid in female smokers was confirmed. The mechanisms through which cigarette smoking induces this fall in AMH are unknown and additional research is needed to improve our comprehension of the negative impact of smoking on ART outcomes.

Assessment of diagnostic competence of plasmatic androgens on polycystic ovary syndrome based on receiver operator characteristic curves

Objective. This study was designed to assess the diagnostic potency of different androgens in hyperandrogenaemia criterion on polycystic ovary syndrome (PCOS) based on receiver operator characteristic (ROC) curves analysis. Methods. We evaluated 55 PCOS patients and 27 healthy fertile women (control). Androgen evaluation included bio-available testosterone (BAT) by ammonium sulphate precipitation, Free Testosterone Index (FTI), androstenedione (A), total testosterone and dehydroepiandrosterone sulphate (DHEA-S). Results. The androgen tests with the best diagnostic capacities were FTI and BAT. Although T and A had similar diagnostic potencies, A detected 5% of PCOS patients that could not be recognised by FTI, BAT (%), or T. The association of FTI, BAT (%) and A identified 96.36% of the hyperandrogenaemic patients. DHEA-S showed a wide dispersion of values and therefore poor discriminatory competence. Discussion. This study suggests that routine androgen evaluation in PCOS should include FTI, BAT and A to avoid misdiagnosis. ROC curve analysis of these tests on patients with the complete spectrum of PCOS phenotypes is needed to confirm these results.

Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period

OBJECTIVE To study in vivo the progesterone receptor (PR) expression levels in human granulosa cells (GCs) during the periovulatory period and the affect of the protein kinase A (PKA) pathway on PR expression and cathepsin-L expression-activation. DESIGN Experimental study. SETTING University research unit. PATIENT(S) Twenty-five women of reproductive age. INTERVENTION(S) Follicular fluid and GCs obtained from spontaneous cycles before and during the normal luteinizing hormone surge, and samples obtained 36 hours after human chorionic gonadotropin (hCG) administration in patients undergoing in vitro fertilization. MAIN OUTCOME MEASURE(S) To determine PR, cathepsin-L messenger RNA (mRNA) analysis via real-time polymerase chain reaction, and protein of PR, cathepsin-L, and PKA in human GCs. RESULT(S) The Western blot analysis revealed that bands of PR (isoform A) were the most abundant and that mRNA (PR-A and PR-B) have a temporal pattern of expression throughout the periovulatory period. The protein levels of PR and cathepsin-L were up-regulated by hCG. The abundance of PR was diminished in the presence of PKA inhibitor, and cathepsin-L with PR receptor antagonist. CONCLUSION(S) The transient expression of PR in human GCs of the preovulatory follicle suggests that PR and its ligand play a role in the activation of cathepsin-L, which is presumably involved in the degradation of the follicular extracellular matrix during human ovulation.

In-vitro study of gonadotrophin signaling pathways in human granulosa cells in relation to progesterone receptor expression

In humans, data on gonadotrophin-activated (LH, HCG and FSH) progesterone receptor expression and signalling pathways involved in matrix metalloproteinases (MMPs) expression presumably linked to the follicle rupture, are limited. Our hypothesis is LH, HCG and FSH increase progesterone receptor expression in granulosa cells through different signalling pathways, leading to an increased expression of ADAMTS-1 and MMP3/10, which may mediate follicular rupture through the transcription factor, HIF1A. Human granulosa cells were isolated from follicular aspirates obtained from 22 healthy women participating in our IVF programme for male-factor infertility. Progesterone receptor and HIF1A expression was assessed by immunofluorescence, and PKA-PKC-PI3K- ERK1/2, ADAMTS-1 and MMP3/10 expression by Western blot in pre-ovulatory and in cultured granulosa cells. Results show that HCG, LH and FSH regulate progesterone receptor expression and activate PKA, PKC, PI3K and ERK1/2 signalling pathways in granulosa cells but progesterone receptor expression is only mediated by PKA, PKC and ERK pathways. HCG, FSH and LH regulated MMPs expression through progesterone receptors. Moreover, HCG-progesterone-receptor-dependent HIF1A expression stimulated MMP3/10 expression but not that of ADAMTS-1. These results suggest differential downstream progesterone receptor signalling, as progesterone receptor regulates MMP3/10 expression via HIF1A, which is not involved in ADAMTS-1 expression.