Evidence summaries and recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome: assessment and treatment of infertility.

STUDY QUESTION: What is the recommended assessment and management of infertile women with polycystic ovary syndrome (PCOS), based on the best available evidence, clinical expertize and consumer preference? SUMMARY ANSWER: International evidence-based guidelines, including 44 recommendations and practice points, addressed prioritized questions to promote consistent, evidence-based care and improve the experience and health outcomes of infertile women with PCOS. WHAT IS KNOWN ALREADY: Previous guidelines on PCOS lacked rigorous evidence-based processes, failed to engage consumer and multidisciplinary perspectives or were outdated. The assessment and management of infertile women with PCOS are inconsistent. The needs of women with PCOS are not being adequately met and evidence practice gaps persist. PARTICIPANTS/MATERIALS SETTING METHODS: Governance included a six continent international advisory and a project board, a multidisciplinary international guideline development group (GDG), consumer and translation committees. Extensive health professional and consumer engagement informed the guideline scope and priorities. The engaged international society-nominated panel included endocrinology, gynaecology, reproductive endocrinology, obstetrics, public health and other experts, alongside consumers, project management, evidence synthesis and translation experts. Thirty-seven societies and organizations covering 71 countries engaged in the process. Extensive online communication and two face-to-face meetings over 15 months addressed 19 prioritized clinical questions involving nine evidence-based reviews and 10 narrative reviews. Evidence-based recommendations (EBRs) were formulated prior to consensus voting within the guideline panel. STUDY DESIGN SIZE DURATION: International evidence-based guideline development engaged professional societies and consumer organizations with multidisciplinary experts and women with PCOS directly involved at all stages. A (AGREE) II-compliant processes were followed, with extensive evidence synthesis. The Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) framework was applied across evidence quality, desirable and undesirable consequences, feasibility, acceptability, cost, implementation and ultimately recommendation strength. The guideline was peer-reviewed by special interest groups across our partner and collaborating societies and consumer organizations, was independently assessed against AGREE II criteria and underwent methodological review. This guideline was approved by all members of the GDG and has been approved by the NHMRC. MAIN RESULTS AND THE ROLE OF CHANCE: The quality of evidence (QOE) for the EBRs in the assessment and management of infertility in PCOS included very low (n = 1), low (n = 9) and moderate (n = 4) quality with no EBRs based on high-quality evidence. The guideline provides 14 EBRs, 10 clinical consensus recommendations (CCRs) and 20 clinical practice points on the assessment and management of infertility in PCOS. Key changes in this guideline include emphasizing evidence-based fertility therapy, including cheaper and safer fertility management. LIMITATIONS REASONS FOR CAUTION: Overall evidence is generally of low to moderate quality, requiring significantly greater research in this neglected, yet common condition. Regional health systems vary and a process for adaptation of this guideline is provided. WIDER IMPLICATIONS OF THE FINDINGS: The international guideline for the assessment and management of infertility in PCOS provides clinicians with clear advice on best practice based on the best available evidence, expert multidisciplinary input and consumer preferences. Research recommendations have been generated and a comprehensive multifaceted dissemination and translation program supports the guideline with an integrated evaluation program. STUDY FUNDING/COMPETING INTERESTS: The guideline was primarily funded by the Australian National Health and Medical Research Council of Australia (NHMRC) supported by a partnership with ESHRE and the American Society for Reproductive Medicine (ASRM). GDG members did not receive payment. Travel expenses were covered by the sponsoring organizations. Disclosures of conflicts of interest were declared at the outset and updated throughout the guideline process, aligned with NHMRC guideline processes. Dr Costello has declared shares in Virtus Health and past sponsorship from Merck Serono for conference presentations. Prof. Norman has declared a minor shareholder interest in the IVF unit Fertility SA, travel support from Merck and grants from Ferring. Prof. Norman also has scientific advisory board duties for Ferring. The remaining authors have no conflicts of interest to declare.This article was not externally peer-reviewed by Human Reproduction Open.

Feasibility of fluorescence lymph node imaging in colon cancer: FLICC.

BACKGROUND: In colon cancer, appropriate tumour excision and associated lymphadenectomy directly impact recurrence and survival outcomes. Currently, there is no standard for mesenteric lymphadenectomy, with a lymph node yield of 12 acting as a surrogate quality marker. Our goal was to determine the safety and feasibility of indocyanine green (ICG) fluorescence imaging to demonstrate lymphatic drainage in colon cancer in a dose-escalation study. METHODS: A prospective pilot study of colon cancer patients undergoing curative laparoscopic resection was performed. At surgery, peritumoural subserosal ICG injection was done to demonstrate lymphatic drainage of the tumour. A specialized fluorescence system excited the ICG and assessed lymphatics in real time. The primary outcome was the feasibility of ICG fluorescent lymphangiography for lymphatic drainage in colon cancer. Secondary outcomes were the optimal protocol for dose, injection site, and ICG lymphatic mapping timing. RESULTS: Ten consecutive patients were evaluated (six males, mean age 69.5 years). In all, lymphatic channels were seen around the tumour to a varying extent. Eight (80%) had drainage to the sentinel node. In all cases where the lymphatic map was seen, there was no further spread 10 min after injection. In 2 patients (20%), additional lymph nodes located outside of the proposed resection margins were demonstrated. In both cases the resection was extended to include the nodes and in both patients these nodes were positive on histopathology. Factors contributing to reduced lymphatic visualization were inadequate ICG concentrations, excess India ink blocking drainage, and inflammation from tattoo placement. CONCLUSIONS: ICG can be safely injected into the peritumoural subserosal and demonstrate lymphatic drainage in colon cancer. This proof of concept and proposed standards for the procedure can lead to future studies to optimize the application of image-guided precision surgery in colon cancer. Furthermore, this technique may be of value in indicating the need for more extended lymphadenectomy.

Human chorionic gonadotropin (hCG) modulation of TIMP1 secretion by human endometrial stromal cells facilitates extravillous trophoblast invasion in vitro.

STUDY QUESTION: Are secreted extracellular matrix (ECM) remodelling elements, relevant to embryo implantation and placentation, modified by hCG in endometrial stromal cells (ESCs)? SUMMARY ANSWER: hCG decreases tissue inhibitor of metalloproteinase 1 (TIMP-1) secretion in ESCs, thereby facilitating extravillous trophoblast invasion in vitro. WHAT IS KNOWN ALREADY: Successful embryo implantation and placentation depend on the appropriate invasion of the trophoblast into the maternal endometrial stroma. hCG is one of the earliest embryo-derived secreted signals in the endometrium which abundantly expresses hCG receptors. However, there is little data concerning the effects of hCG on endometrial ECM remodelling with respect to embryo implantation. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in an academic research laboratory within a tertiary-care hospital. Samples were collected from 36 women undergoing benign gynaecological surgery during the mid-secretory phase. ESCs were isolated and stimulated with hCG (10 UI/ml) or vehicle. Conditioned media (CM) were analysed to determine changes in the secreted profile of nine matrix metalloproteinases (MMPs) and three tissue-specific inhibitors of MMPs (TIMPs) using an ELISA array. Data were confirmed by gelatine zymography, western blot and ELISA. The HTR8/SVneo cell line served as a model for trophoblast cells. The invasive potential of trophoblast cells was assessed using Transwell invasion assays under CM or co-culture conditions with ECS and the role of regulated molecules was examined by using immunoprecipitation in CM prior to the assessment of invasive potential. MAIN RESULTS AND THE ROLE OF CHANCE: MMP-2 levels increased 30%, whereas TIMP-1 levels decreased 20% in CM from ESCs stimulated with hCG (P < 0.05). Gelatine zymography confirmed an increase in MMP-2 activity (P < 0.05). ELISA and western blotting also confirmed the reduction in TIMP-1 upon hCG treatment (P < 0.05). Invasion assays revealed a ∼50% increase in invading HTR8/SVneo cells in chambers with hCG-stimulated ESCs compared with the control (P < 0.05). Immunodepletion of TIMP-1 from control ESC-CM partially resembled the effect of CM from hCG-stimulated ESCs in the trophoblast invasion assays. LIMITATIONS, REASONS FOR CAUTION: The assays were performed in vitro and ESCs were not decidualized, therefore they reflected the very early stages of embryo implantation or the advanced stages when decidualization fails. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that hCG induces endometrial stromal extracellular remodelling by modulating secreted MMP-2 and TIMP-1. This regulation may be physiologically relevant because it increases the invasive potential of trophoblast-derived cells. At present, few data exist concerning the implications of hCG and endometrial ECM remodelling in embryo implantation. Hence, our results should be confirmed by further in vivo studies. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by FONDECYT 11100443, PBCT-PSD51 (IDIMI) and FONDAP 15010006. None of the authors have any conflicts of interest to declare.

Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

Abnormal pattern of integrin expression at the implantation window in endometrium from fertile women treated with clomiphene citrate and users of intrauterine device.

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.

Expression of steroidogenic acute regulatory protein in the human corpus luteum throughout the luteal phase.

The expression of the steroidogenic acute regulatory protein (StAR) in the human corpus luteum (CL) was examined throughout the luteal phase. The primary 1.6-kb StAR transcript was in greater abundance in early (3.1-fold) and mid (2.2-fold) luteal phase CL compared with late luteal phase CL. The larger StAR transcript (4.4 kb) was found in early and midluteal phase CL, but was not detected in late luteal phase specimens. Mature StAR protein (30 kDa) was present in lower amounts within late CL compared with early and midluteal phase CL. The StAR preprotein (37 kDa) was also detected in greater abundance in early and midluteal CL. Immunohistochemistry revealed that StAR staining was most prominent in thecal-lutein cells throughout the luteal phase. The intensity of the signal for StAR exhibited significant changes throughout the luteal phase, being most intense during the midluteal phase and least during the late luteal phase. Plasma progesterone concentrations were highly correlated (r = 0.73 and r = 0.79) with luteal expression of the preprotein and mature StAR isoforms, respectively, throughout the luteal phase. To examine the LH dependency of StAR expression, the GnRH antagonist, Cetrorelix, was administered during the midluteal phase. Cetrorelix caused a decline in serum LH levels within 2 h, which, in turn, caused a pronounced decline in plasma progesterone within 6 h. The StAR 4.4-kb transcript was not detectable, and the 1.6-kb transcript was reduced by approximately 50% within 24 h of Cetrorelix treatment. The mature 30-kDa StAR protein level declined approximately 30% after Cetrorelix treatment. We conclude that 1) StAR mRNA and protein are highly expressed in early and midluteal phase CL; 2) StAR protein is present in both thecal-lutein and granulosa-lutein cells throughout the luteal phase; 3) StAR protein levels in the CL are highly correlated with plasma progesterone levels; 4) declining StAR mRNA and protein levels are characteristic of late luteal phase CL; and 5) suppression of LH levels during the midluteal phase results in a marked decline in plasma progesterone and a diminished abundance of StAR transcripts in the CL without a corresponding significant decline in StAR protein. Collectively, these data are consistent with the idea that StAR gene expression is a key determinant of luteal progesterone during the normal menstrual cycle. However, the pharmacologically induced withdrawal in the midluteal phase of LH support diminishes luteal progesterone output by mechanisms others than reduced StAR protein levels.

Effects of leptin, interleukin-1alpha, interleukin-6, and transforming growth factor-beta on markers of trophoblast invasive phenotype: integrins and metalloproteinases.

Phenotypic changes of integrin and metalloproteinase secretion of the invasive human cytotrophoblast are regulated by cytokines and growth factors, but how this occurs is not completely understood. We used 24-h cytotrophoblast cultures from first trimester pregnancies to investigate the effects of leptin and cytokines on the expression of the alpha2, alpha5, and alpha6 integrin subunits and on the activity of metalloproteinase-2 (gelatinase A) and metalloproteinase-9 (gelatinase B). The alpha2 subunit was marginally upregulated by leptin and interleukin-1alpha (IL-1alpha). All compounds tested upregulated, in some degree, the alpha5 expression. The a6 integrin subunit was massively upregulated, by leptin, interleukins, and transforming growth factor-beta. None of the factors tested affected metalloproteinase-2 activity, but the activity of metalloproteinase-9 was upregulated by leptin and IL-1alpha. In conclusion, leptin and IL-1alpha actively induce some of the changes that cytotrophoblasts undergo to achieve a more invasive phenotype. A novel role for leptin is proposed during early pregnancy: leptin might be an autocrine/paracrine regulator of cytotrophoblast invasiveness during implantation and placentation.

Resumption of ovarian function during lactational amenorrhoea in breastfeeding women with polycystic ovarian syndrome: endocrine aspects.

BACKGROUND: The aim of this study was to evaluate the changes in gonadotrophin concentrations and the dynamics of the episodic fluctuations of circulating LH during night-time, in fully breastfeeding normal women and in those with polycystic ovarian syndrome (PCOS) during lactational amenorrhoea and after weaning, in order to provide insights into the onset of this syndrome. Additionally, ovarian activity was evaluated by ultrasound examination and steroid concentrations. METHODS: Twelve lactating PCOS (LPCOS) women and six normal lactating (NL) women of similar age were selected. On the 4th and 8th week postpartum (PP) and eight weeks after weaning, blood samples were collected every 10 min (10.00--20.00h). Gonadotrophin concentrations were determined in all samples. Steroid hormones were measured in one fasting sample and ovarian morphology was assessed by ultrasound. RESULTS: On the 8th week PP, LH pulse frequency was higher and FSH concentrations were lower in LPCOS women compared with NL women, and steroid hormone concentrations remained low, except for androstenedione which was higher in LPCOS patients. After weaning, similar differences were observed between both groups. PCOS patients also showed enlarged ovaries with a PCOS pattern in the three study periods. CONCLUSIONS: The enlarged ovaries associated with higher androstenedione concentrations suggest that PCOS is a primary ovarian defect, making it difficult to establish if the abnormal LH pattern observed in these women is primary or secondary to the ovarian dysfunction.

Providing progesterone for pregnancy: control of cholesterol flux to the side-chain cleavage system.

Progesterone, which is required to support human gestation, is derived initially from the corpus luteum and subsequently from the placenta. The rate-limiting step in progesterone synthesis is the delivery of cholesterol to the mitochondrial cholesterol side-chain cleavage system. The steroidogenic acute regulatory protein (StAR) mediates this process in the corpus luteum, whereas in the placenta, which does not express StAR, a StAR homologue, MLN64, may accomplish this function. StAR expression is regulated in the ovary at the transcriptional level by a cAMP-activated signal transduction system and StAR activity is also increased acutely by protein kinase A-mediated phosphorylation. These long-term (transcriptional) and short-term (post-translational, that is, phosphorylation) mechanisms govern luteal steroidogenic activity. The StAR protein has two key functional domains. The StAR C-terminal domain increases cholesterol movement to cytochrome P450scc by promoting sterol desorption from the sterol-rich outer mitochondrial membrane, driving it to the relatively sterol-poor inner membrane. The N-terminal domain mitochondrial targeting sequence directs the StAR protein to the mitochondria.

Endocrine and paracrine-autocrine regulation of the human corpus luteum during the mid-luteal phase.

Human corpora lutea undergo an extremely rapid period of growth, development and regression during the course of non-fertile cycles. The tissue consists of steroidogenic (parenchymal) and non-steroidogenic (stromal) cells. In women and other primates, steroid hormone production by corpora lutea depends on the presence of pituitary-derived LH. Nevertheless, there is also intra-luteal regulation of steroid synthesis. Steroidogenic luteal cells and non-steroidogenic cells interact via endocrine and paracrine pathways, and by contact-dependent pathways (gap junctions). Thus, hormones and locally produced factors including steroids, growth factors, cytokines, reactive oxygen species and nitric oxide may modulate luteal lifespan. The factors regulating regression and rescue of the corpus luteum are not understood completely. This review describes the expression of two representative intragonadal peptides that may influence luteal regression (interleukin 1 beta) and luteal rescue (steroidogenic acute regulatory protein).

Nitric oxide induces apoptosis in the human corpus luteum in vitro.

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.

Leptin and reproduction.

Leptin, the product of the ob gene, is a small peptide molecule synthesized by white adipocytes with an important role in the regulation of body fat and food intake. Leptin and leptin receptor mRNA were first detected in the brain and hypothalamus but now their ubiquitous presence has been demonstrated. Leptin receptor signal transduction involves the activation of signal transducer and activator of transcription (STAT)-3, a member of the transcription family of proteins. Leptin is regulated by hormones and cytokines, interleukin-1, tumour necrosis factor-alpha and transforming growth factor-beta, linking this molecule with the inflammatory response. In addition, emerging evidence has demonstrated that this molecule is related to reproductive function. This small protein is present in the ovary and decidua, in mature oocytes and during embryonic development and trophoblast invasion. Animal models have demonstrated that leptin-deficient ob/ob mice are sterile; however, fertility can be restored by exogenous leptin. In addition, embryos implanted in STAT-3-deficient mice degenerate rapidly and are the target disruption of STAT-3-provoked embryonic lethality. Leptin acts as a novel placental hormone participating in the control of fetal growth and development. Leptin could be a modulator for invasive features of cytotrophoblast cells. We postulate that leptin may have an autocrine/paracrine role in human implantation and placentation.

Leptin and leptin receptor are expressed in the human endometrium and endometrial leptin secretion is regulated by the human blastocyst.

Embryonic implantation is a crucial event for the human reproductive function. Cytokines and paracrine molecules have been proposed as putative local regulators of this process. The leptin or the OB protein has been linked to the reproductive function and inflammatory response. In the present study, we describe for the first time the expression of leptin and leptin receptor (long form) in the secretory endometrium and that endometrial leptin secretion is regulated in vitro by the human blastocyst. Leptin and leptin receptor messenger RNA and protein were identified in secretory endometrium and in cultured endometrial epithelial cells (EECs) by RT-PCR, Western blot, and immunohistochemistry. The concentrations of immunoreactive leptin secreted by human embryos alone or cocultured with EECs were also assessed. We found that human blastocysts secrete significantly higher levels of leptin than arrested embryos. In contrast, leptin concentrations secreted by arrested embryos cocultured with EECs were significantly higher than blastocysts cocultured with EECs. These findings suggest that the human endometrium is a site for local production and a target tissue for circulating leptin. Expression of leptin and its functional receptor in the endometrium and regulation of endometrial leptin secretion by the human embryo suggests that the leptin system may be implicated in the human implantation process.

Insulin and insulin-like growth factor-I and -II modulate human granulosa-lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression.

Insulin and insulin-like growth factors (IGF)-I and -II stimulate granulosa cell steroidogenesis. Since steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroid hormone biosynthesis, the ability of insulin and IGF to modulate StAR protein and mRNA expression was examined in two human granulosa cell culture systems: (i) proliferating granulosa-lutein cells and (ii) luteinized-granulosa cells derived during in-vitro fertilization (IVF). In proliferating granulosa-lutein cells, IGF-I and IGF-II increased StAR protein approximately 4-5-fold, while insulin and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) increased amounts of StAR protein 2.5- and 6-fold respectively. A combination of IGFs/insulin and 8-Br-cAMP increased StAR 7-9-fold. Luteinized granulosa cells also had increased StAR expression after treatment with IGF-I (2. 8-fold), IGF-II (3-fold), insulin (2.5-fold) and 8-Br-cAMP (4. 5-fold). Progesterone production generally followed a pattern similar to StAR protein in both cell systems. In proliferating granulosa-lutein cells, both IGF-I and insulin increased StAR mRNA (3-fold) and 8-Br-cAMP increased StAR mRNA 4-fold, whereas a combination of IGF-I and 8-Br-cAMP had an additive effect on StAR mRNA expression (7-fold). Transient transfection of proliferating granulosa-lutein cells with StAR promoter-luciferase reporter constructs demonstrated that IGF-I, IGF-II, and insulin had no effect on the StAR promoter activity, while 8-Br-cAMP stimulated StAR promoter function. The results indicate that: (i) IGFs and insulin stimulate StAR mRNA and protein expression in human granulosa-lutein cells; (ii) IGF-I and 8-Br-cAMP have an additive effect on StAR gene expression; and (iii) IGF-I increases StAR mRNA and protein by a mechanism that does not involve activation of the proximal StAR gene promoter.

The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking.

Stimulation of steroid-producing cells of the gonads and adrenals with tropic hormone results in a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system. This process of cholesterol translocation is blocked by inhibitors of protein synthesis, suggesting that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory (StAR) protein appears to be the most likely candidate for the "labile" protein, based on the following observations: 1) Expression of StAR in COS-1 cells engineered to contain the cholesterol side-chain cleavage system substantially augments pregnenolone formation; 2) StAR protein is expressed almost exclusively in steroid-producing cells, except the trophoblast of the human placenta, and its presence is correlated with steroid hormone production; 3) StAR mRNA increases in response to cAMP; 4) StAR is a target for serine phosphorylation mediated by protein kinase A, a process that is essential for maximizing StAR activity; and 5) lack of functional StAR causes the autosomal recessive disease, congenital lipoid adrenal hyperplasia, characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. Studies on the mechanism of action of StAR revealed that import into mitochondria is not essential to its steroidogenesis-enhancing activity and more likely represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations established that the C-terminus of the StAR protein contains the functionally important domains. The demonstration of steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence confirmed that StAR import is not essential for its steroidogenic activity and suggested that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. Evidence that StAR functions as a cholesterol transfer protein raises the possibility that StAR acts directly on lipids of the outer mitochondrial membrane, probably stimulating cholesterol desorption from the sterol-rich outer membrane and its movement to the relatively sterol-poor inner membrane.

Interleukin-1beta (IL-1beta) is a modulator of human luteal cell steroidogenesis: localization of the IL type I system in the corpus luteum.

The present investigation examined the effect of interleukin-1beta (IL-1beta) on progesterone production by human luteal cells and the expression and localization of the IL-1 system in the human corpus luteum (CL). Luteal cells were isolated from corpora lutea collected throughout the luteal phase. After dispersion, luteal cells were treated with a panel of monoclonal antibodies directed to leukocyte-specific molecules. The leukocytes were isolated with immunomagnetic beads. Leukocyte-free luteal cells exhibited greater steroidogenic responsiveness to hCG toward the end of the luteal phase. The treatment of mixed luteal cells (total luteal cells) with IL-1beta inhibited by 60% hCG-stimulated progesterone production. Interestingly, the treatment of leukocyte-free luteal cells with IL-1beta did not affect progesterone production. In addition, the treatment of mixed luteal cells with monoclonal antibodies against IL-1 receptor type I (IL-1RtI) resulted in a 2.5-fold increase in the hCG-supported progesterone production. IL-1RtI and IL-1 receptor antagonist were localized by immunohistochemistry in both somatic and immune cells of the CL. Flow cytometric analysis indicated that both nonleukocyte luteal cells and leukocyte-luteal cells exhibited IL-1Rt-I positive cells, representing 56% and 31% of the total luteal cells, respectively. However, 13% of nonleukocyte luteal cells did not express IL-1Rt-I. Northern analysis demonstrated the presence of the 5.1-kb IL-1RtI messenger ribonucleic acid transcript in CL of different ages. RT-PCR indicated that both leukocyte-free luteal cells and luteal leukocytes express IL-1RtI messenger ribonucleic acid. We conclude that 1) luteal leukocytes have an inhibitory effect on hCG-stimulated progesterone production; 2) IL-1beta inhibits hCG-stimulated progesterone production only in mixed luteal cell cultures, indicating that leukocytes mediate the effect; 3) the somatic and immune cells of the CL are sites of action and expression of the IL-1 system; and 4) interaction between the steroidogenic and immune cells of the CL suggests a functional intraovarian role for IL-1beta in CL physiology.

A quantitative evaluation of alpha1, alpha4, alphaV and beta3 endometrial integrins of fertile and unexplained infertile women during the menstrual cycle. A flow cytometric appraisal.

The expression of integrin molecules alpha1beta1, alpha4beta1 and alphaVbeta3 within endometrial tissue has been proposed as a marker of uterine receptivity during the implantation window. The present investigation examines by flow cytometric analysis the concentrations of alpha1, alpha4, alphaV and beta3 integrin subunits in endometrial stromal (ESC) and epithelial cells (EEC) in two groups of women throughout the menstrual cycle: normal fertile women (n = 27) and women with unexplained infertility (n = 26). Integrin concentrations in endometrial cells were calculated in relative fluorescence units against a negative cellular control. The assessment of integrin subunits detected the protein in ESC and EEC from the late proliferative to the late secretory phase. In both groups of women, the alpha1 was the highest integrin expressed in ESC and EEC throughout the menstrual cycle. All women exhibited low concentrations of alpha4-EEC at the time of the implantation window. Infertile women expressed lower concentrations of the alpha4-ESC during the proliferative and early secretory phase while lower concentrations of the alpha1-ESC were seen during the late secretory phase. Interestingly, the infertile women expressed lower concentrations of beta3-EEC in the early, mid-secretory and late secretory phases (P < 0.05). Infertile women also expressed lower concentrations of alpha1-EEC and alphaV-EEC during the late secretory phase (P < 0.05). It can be concluded that the quantitative determination of beta3-EEC by flow cytometry confirmed its potential feature as a marker of endometrial receptivity at the time of the implantation window. In addition, the defective expression of the alpha1-ESC found in the late secretory phase might be associated with the poor fertility outcome of women with unexplained infertility.

Antisteroidogenic action of nitric oxide on human corpus luteum in vitro: mechanism of action.

To analyze the mechanism by which nitric oxide (NO) exerts its antisteroidogenic action, human luteal cells were cultured during 24 and 48 h with L-arginine (L-Arg, 1 mmol/L); 1,2(2-trifluoromethylphenyl)imidazole (TRIM) (50 micromol/L and 1 mmol/L) and cyclic guanosine monophosphate (cGMP) analog (8-Br-cGMP, 1 mmol/L). Estradiol, nitrite, and P450 AROM activity were determined in culture media. Total cGMP concentration was evaluated in the cells and culture media by radioimmunoassay, and NADPH diaphorase was used as a histochemical marker for NO synthase (NOS) activity. During the corpus luteum (CL) life-span, NO affected estradiol secretion in an age-dependent manner, with an inhibition in mid-CL (37%; p < 0.05) in agreement with our previous results, and no significant modification in early and late CL. Basal nitrite concentration in 24 and 48 h of midluteal cell cultures (42 and 93 pmol/10(6) cells, respectively) was increased by L-Arg (53% and 88%) and inhibited by the two TRIM concentrations; also, an intense diaphorase reactivity was observed in endothelial cells and luteal parenchyma. Total cGMP was not detected in cell cultures and 8-Br-cGMP did not modify estradiol secretion, whereas aromatase activity was strongly inhibited by L-Arg (70%, p < .05). These results suggest that both NOS isoforms are active in midluteal cells, and the mechanism of action for NO on in vitro estradiol secretion may be an inhibition of P450 AROM activity.