Effect of anticardiolipin antibodies on prolactin and insulin-like growth factor binding protein-1 production by human decidual cells.

PROBLEM: The effect of anticardiolipin antibodies (ACAs) on basal- and growth factor-stimulated prolactin and insulin-like growth factor (IGF) binding protein (BP)-1 production by cultured human decidual cells was investigated. METHOD OF THE STUDY: Decidual cells were cultured for 24, 48, or 96 hr in medium supplemented with 5% ACA-containing or 5% control serum and increasing concentrations of insulin (1-10 micrograms/mL) or IGF-1 (10-100 ng/mL). RESULTS: No significant increase in prolactin production was observed after addition of increasing doses of insulin and IGF-I in the presence of ACA-containing serum, while a dose-dependent stimulation was seen with control serum. Time-dependent prolactin accumulation was also reduced when cells were cultured in the former conditions. IGF BP-1 release was not affected by insulin and IGF-I in the presence of both sera. However, lower IGF BP-1 levels and a less pronounced time-dependent accumulation were observed in the presence of ACA-positive serum. CONCLUSIONS: Our data suggest that ACAs affect cellular transduction mechanisms regulating critical events, such as decidual cell differentiation. These cellular dysfunctions might be relevant in the induction of some obstetric disorders typical of this syndrome.

Antiphospholipid antibodies inhibit prostaglandin release by decidual cells of early pregnancy: possible involvement of extracellular secretory phospholipase A2.

OBJECTIVE: To investigate the effect of antiphospholipid antibodies on eicosanoid production by human decidual cells and the in vitro interaction between antiphospholipid antibodies and secretory phospholipase A2. DESIGN: Cultures of human decidual cells from early pregnancy. SETTING: All decidual specimens were obtained from the Obstetrics and Gynecology Department of the Catholic University, Rome, Italy. PATIENT(S): Patients were undergoing operative laparoscopy for extrauterine pregnancy, with a period of amenorrhea ranging from 6 to 9 weeks. INTERVENTION(S): Decidual samples were collected at laparoscopy by routine uterine curettage. MAIN OUTCOME MEASURE(S): Decidual cells were incubated with antiphospholipid antibodies, and eicosanoids (prostaglandin [PG] E2, PGF2alpha, and thromboxane B2) were assayed by RIA after 24 hours of culture. In vitro interactions between antiphospholipid antibodies and secretory phospholipase A2 were investigated with use of a modified ELISA for phospholipase A2. RESULT(S): Antiphospholipid antibodies reduced eicosanoid release from decidual cells in a dose-dependent fashion. In vitro assays showed that antiphospholipid antibodies bound secretory phospholipase A2 and that a competition occurred between antiphospholipid antibodies and secretory phospholipase A2 for the common substrate cardiolipin. CONCLUSION(S): In light of the critical role played by eicosanoids in decidual function, we suggest that an interaction between antiphospholipid antibodies and secretory phospholipase A2 occurring in vivo might impair important cellular communications at the decidual level in the antiphospholipid antibody syndrome.

PIP: The presence of antiphospholipid antibodies is often associated with poor obstetric histories, including recurrent abortion, intrauterine growth retardation, and preeclampsia. While it has been suggested that these antibodies can affect the function of vascular endothelial cells at the decidual and placental levels, their cellular target and mode of action remain to be determined. Findings are presented from a study to investigate the effect of antiphospholipid antibodies upon eicosanoid production by human decidual cells and the in vitro interaction between antiphospholipid antibodies and secretory phospholipase A2. Specimens of decidualized endometrium were obtained by routine curettage from patients undergoing operative laparoscopy for extrauterine pregnancy in the Obstetrics and Gynecology Department of the Catholic University in Rome, Italy. Analysis determined that antiphospholipid antibodies reduce eicosanoid release from decidual cells in a dose-dependent manner, while in vitro assays showed that antiphospholipid antibodies bound secretory phospholipase A2 and that competition occurred between antiphospholipid antibodies and secretory phospholipase A2 for the common substrate cardiolipin. An interaction between antiphospholipid antibodies and secretory phospholipase A2 occurring in vivo may impair important cellular communications at the decidual level in the antiphospholipid antibody syndrome.

Further evidence of increased aromatase activity in granulosa luteal cells from polycystic ovary.

This study evaluated the effect of atamestane (a competitive inhibitor of P-450 aromatase) on granulosa luteal cells from polycystic and normal ovaries. Treatment with atamestane (10 micromol/l) determined a strong inhibition of basal aromatase activity in both types of cells; however, its effect was markedly more pronounced in granulosa cells from normal ovary than in granulosa cells from polycystic ovaries (PCO; P < 0.01). Concomitant treatment with insulin (25 microg/ml) and increasing doses of atamestane (0.01-10 micromol/l) caused a dose-dependent inhibition of insulin-stimulated aromatase activity, but again with marked differences between the two types of cells. In granulosa cells from PCO, the minimal effective dose of atamestane was 1 micromol/l and it had an EC50 of 2.23 +/- 0.4 micromol/l and a maximal inhibitory effect of 75%; in granulosa cells from normal ovary, the minimal effective dose of atamestane was 0.01 micromol/l, the EC50 was 0.4 +/- 0.07 micromol/l, and the maximal inhibitory effect was 94%. Significant differences were observed between the different cells at all the studied dose points. Reversibility studies showed that resumption of aromatase activity in granulosa cells from PCO is basally greater and more inducible with insulin treatment. This study provides further evidence of an increased in-vitro function of the aromatase complex in granulosa cells from PCO, that could be induced by an altered cellular autoregulation.

The cellular activity of different sized follicles in cycles treated with gonadotrophin-releasing hormone analogue.

The activity of granulosa cells derived from different sized follicles surrounding oocytes of apparently comparable maturity was evaluated in hyperstimulated ovaries. Granulosa cells were obtained from women undergoing gamete intra-Fallopian transfer procedures who had been treated with gonadotrophin-releasing hormone analogue and gonadotrophins. Only follicles with oocytes of apparently comparable maturity were considered. Granulosa cells from large and small follicles (> or = 18 and < 15 mm diameter respectively) collected from each patient were cultured separately for up to 48 h in the presence or absence of follicle-stimulating hormone (FSH: 50 ng/ml) or insulin (at varying doses, 0.005-25 mg/ml). We found that aromatase activity was elicited by FSH plus insulin, but not by FSH alone, in granulosa cells from both large and small follicles. Progesterone production was maximal in granulosa cells from large follicles, and in these cells was insensitive to further stimuli, in contrast with those collected from small follicles. Prostaglandin oestradiol was secreted in large amounts by granulosa cells from large follicles. Cyclic adenosine monophosphate (cAMP) concentration did not differ between cells from large and small follicles. Our data demonstrate that there are significant differences in granulosa cells derived from different sized follicles with oocytes of apparently comparable maturity.

Effect of follicular fluid on granulosa luteal cells from polycystic ovary.

Recent data suggest that follicular fluid may play an important role in the endocrine balance of polycystic ovary syndrome, probably by acting on the theca-granulosa cell relationship. The aim of this study was to evaluate the effect of steroid-free follicular fluid on steroidal response and cell proliferation of human granulosa luteal cells from polycystic (POGC) and normal ovary (NC). Granulosa cells (from both POGC and NC) were cultured for 48 h with or without increasing dilutions of follicular fluid (FF) obtained from polycystic (FFp) and normo-ovulating (FFc) patients. Both follicular fluids were able to elicit aromatase activity as well as progesterone production and thymidine incorporation. POGC, when incubated with FFp, showed a lower increase of aromatase activity and progesterone production with respect to NC. Furthermore, the proliferation rate was increased by incubation with either follicular fluid, but the increase was less with FFp compared to FFc. Aromatase/[3H]thymidine (A/T) and progesterone/ [3H]thymidine (P/T) ratios could be considered to be representative of the contribution of the single cell unit to steroidogenesis. Using high concentrations of either follicular fluids, POGC showed a higher A/T ratio compared with NC. Moreover, the same treatment strongly decreased P/T ration in POGC, while it was ineffective in NC. Our study show that an abnormal interaction between POGC and their own follicular fluid can be implicated in the pathogenesis of the altered steroidal response in these cells, and that in particular it could affect the proliferation rate.

Increased production and release of prostaglandin-E2 by human granulosa cells from polycystic ovaries.

This study was conducted to compare the levels of prostaglandin E2 (PGE2) released by cultured granulosa cells collected from normally-ovulating women (normal cells, NC) and those with polycystic ovaries (polycystic ovary granulosa cells, POGC). Granulosa cells were collected from 7 normal women and 7 anovulatory women with polycystic ovaries. Both groups underwent laparoscopic oocyte retrieval for gamete intra-fallopian transfer. Cell cultures were carried out under basal conditions and in the presence of various substances known to influence PGE2 biosynthesis. Prostaglandin E2 concentrations in the incubation media were taken as a marker of cyclo-oxygenase activity. Unexpectedly, POGC appeared to release greater amounts of PGE2 compared to the NC. There was no difference between the levels of PGE2 produced by the two types of cells during the first 3 hours after cell explants, whereas a difference (P < 0.01) was observed after 24 and 48 hours of incubation. Interleukin-1 beta enhanced PGE2 secretion (P < 0.01) in both POGC and NC, while lipopolysaccharide increased prostaglandin release only by the NC cells. Indomethacin inhibited PGE2 production to a greater extent in POGC (from -70 to -90% with respect to basal release, P < 0.01) than NC (approximately -50%, P < 0.01). Blockade by indomethacin and the weak inhibitory effect of the glucocorticoid, dexamethasone (P < 0.05 only in NC, and only at 24 hours), provided pharmacological evidence that PG production by granulosa cells in vitro might depend primarily on constitutive cyclo-oxygenase activity.

Growth hormone stimulates androsterone synthesis by rat theca-interstitial cells.

The aim of this study was to assess the possible role of growth hormone (GH) on androsterone synthesis. This effect was analyzed in theca-interstitial cells obtained from immature female rats. The addition of GH to the cultures significantly stimulated androsterone (A) synthesis in a dose- and time-dependent way and this effect was not due to a cellular number increase. When added to the hCG cultures, GH significantly enhanced androgen production even though it did not synergyze with the chorionic gonadotropin. The addition of antibodies anti-IGF-I to the GH cultures did not modify the growth hormone effect suggesting that GH probably does not require IGF-I to achieve its effect on A production. Finally, no effect of GH on cAMP levels were observed in the cultures at the end of the treatment. Our results demonstrate that GH is able to significantly induce A synthesis by rat theca-interstitial cells. Since the presence of GH and its receptors in the ovary is now well established the present data strongly suggest a potential relevance of GH in reproductive biology.

Effectiveness of a somatostatin analogue in lowering luteinizing hormone and insulin-stimulated secretion in hyperinsulinemic women with polycystic ovary disease.

OBJECTIVE: To analyze the action of a long-acting somatostatin analogue on circulating insulin levels and on pituitary sensitivity to GnRH in women affected by polycystic ovary disease (PCOD). DESIGN: Controlled clinical study. SETTING: Normal human volunteers in the Department of Obstetrics and Gynecology, Universita' Cattolica del Sacro Cuore. PATIENTS: Twenty euglycemic women affected by PCOD, aged 19 to 30 years, were studied in their early follicular phase. INTERVENTIONS: A long-acting somatostatin analogue (octreotide) was administered SC for 6 weeks; an oral glucose tolerance test (OGTT), a GnRH test, and plasma hormone determinations were performed at baseline and repeated after 6 weeks of treatment. A glucose load also was performed after 1 week of treatment. MAIN OUTCOME MEASURES: Insulin and glucose serum concentrations were measured in all samples under the oral glucose stimulus, LH and FSH were measured under GnRH test. Plasma levels of PRL, sex hormone-binding globulin, androstenedione (A), T, 17 beta-hydroxyprogesterone, DHEAS, and cortisol were measured. Steroids also were assayed after the octreotide administration. RESULTS: Based on the insulin response to OGTT, subjects were classified as hyperinsulinemic (n = 12) and normoinsulinemic (n = 8). Octreotide administration did not affect the glycemic levels in both groups. Octreotide significantly reduced insulin and LH exaggerated response to GnRH stimulus, as well as the A and T circulating levels, only in the hyperinsulinemic group. CONCLUSIONS: A functional linkage exists between exaggerated insulin and LH-stimulated secretion in a group of polycystic ovary patients. Octreotide may be useful in normalizing these alterations and in reducing ovarian androgen secretion of hyperinsulinemic subjects.

Somatostatin action on rat ovarian steroidogenesis.

To date, very few studies on the effect of somatostatin on female reproductive function have been reported. In our study, we examined the effects of somatostatin on (i) androgen biosynthesis using whole ovarian dispersates, and (ii) aromatase activity and progesterone production using granulosa cells. Whole ovarian dispersates obtained from immature rats were cultured for 96 h in serum-free medium with human chorionic gonadotrophin (HCG; 25 ng/ml) and insulin (10 micrograms/ml) in the presence or absence of an increasing concentration of somatostatin (0.03-3.00 ng/ml). HCG- and insulin-stimulated accumulation of androsterone by these cells was inhibited significantly by somatostatin. Granulosa cells from diethylstilbestrol-treated rats were cultured for 48 h in serum-free medium with follicle-stimulating hormone (FSH; 20 ng/ml) and FSH plus insulin (1 microgram/ml) with or without somatostatin (0.03-3.00 ng/ml). Both aromatase activity and progesterone production stimulated by FSH and FSH plus insulin were significantly inhibited by somatostatin. Somatostatin by itself (1 ng/ml) did not have an effect on any of the evaluated parameters. The action of somatostatin could be immunoneutralized and did not influence the plated viable cell mass. These findings indicate that somatostatin can regulate ovarian steroidogenesis by mediating gonadotrophin and growth factor action on different ovarian cell types.

Effect of gonadotropins, insulin and IGF I on granulosa luteal cells from polycystic ovaries.

The aim of this work is to evaluate the gonadotropin and growth factor effects in vitro on steroidal response in human granulosa luteal cells from polycystic ovaries compared with normal granulosa luteal cells in humans. The granulosa cells from polycystic (polycystic ovarian granulosa cells, POGC) and normo-ovulating women (normal cells, NC) were collected in the preovulatory phase after oocyte retrieval during the GIFT program. The cells were cultured serum-free for 24, 48 and 96 h. Estradiol and progesterone production was determined with or without HCG (1-200 ng/ml), FSH (10-300 ng/ml), insulin (1-50 micrograms/ml) and IGF I (1-50 ng/ml) addition. All treatments significantly induced a 2-3 fold estradiol increase at the 48-h and 96-h time points in POGC. The progesterone production was unaffected by HCG, FSH, insulin and IGF I addition, respectively, in POGC, whereas the NC were responsive at the 48-h and 96-h time points. FSH did not stimulate progesterone production in granulosa cells either from polycystic or normovulating subjects. Our findings indicate that POGC are hypersensitive to all substances in terms of estradiol production, whereas they show a reduced capacity of progesterone production with some treatments.

Cytokine-mediated regulation of ovarian function: interleukin-1 inhibits gonadotropin-induced androgen biosynthesis.

Resident ovarian macrophages have long been recognized as potential in situ regulators of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, interleukin-1 (IL-1) has recently been shown to exert profound regulatory effects at the level of the ovarian granulosa cell. In this report, we examine the possibility that the adjacent theca-interstitial (androgen-producing) cell may also be a site of IL-1 reception and action. The basal accumulation of androsterone, the major androgenic steroid synthesized by whole ovarian dispersates from immature rats, in the presence of insulin (1 microgram/ml), increased 8- to 9-fold after treatment with human CG (1 ng/ml). Although IL-1 alpha or IL-1 beta (10 ng/ml) by themselves were without effect on basal androsterone accumulation, both cytokines (IL-1 beta greater than IL-1 alpha) inhibited human CG hormonal action (in the presence of insulin) in a dose-dependent manner, the maximal inhibitory effect being 75%. Similar results were obtained when using highly purified theca-interstitial cells derived from the same animal model suggesting that IL-1-attenuated androgen biosynthesis is due, at least in part, to IL-1 acting directly at the level of the theca-interstitial cells. The IL-1 effect proved relatively specific since all other known interleukins (IL-1, IL-3, IL-4, IL-5, and IL-6) were without effect. Moreover, IL-1 beta action was effectively immunoneutralized when concurrently applied with anti-IL-1 beta (but not nonimmune) sera. Significantly, the antigonadotropic action of IL-1 could not be accounted for by a decrease in the viable cell mass. Tracer studies with radiolabeled steroid substrates suggested that IL-1-attenuated ovarian androsterone accumulation is due, if only in part, to inhibition of transformations catalyzed by (theca-interstitial) 17 alpha-hydroxylase/17:20 lyase, stimulation of theca-interstitial (or granulosa 20 alpha-hydroxysteroid dehydrogenase-mediated conversions, or both. Taken together, these findings indicate that relatively low concentrations of IL-1, possibly originating from somatic ovarian cells or resident ovarian macrophages, are capable of exerting an inhibitory effect upon gonadotropin-supported androgen production. As such, these and previous observations suggest that intraovarian IL-1 may play a dual regulatory role in the developing ovarian follicle by targeting both the granulosa and theca-interstitial cells as its sites of action.

Cytokine-mediated regulation of ovarian function. Tumor necrosis factor alpha inhibits gonadotropin-supported ovarian androgen biosynthesis.

Resident ovarian macrophages have been implicated in the regulation of ovarian function, presumably through local paracrine secretion of regulatory molecules (i.e. cytokines). One such macrophage product, tumor necrosis factor (TNF) alpha, has been shown to attenuate the gonadotropin-dependent differentiation of the somatic ovarian (estrogen-producing) granulosa cell. This study examines the possibility that TNF alpha may also regulate the adjacent androgen-producing theca interstitial cell. The basal accumulation of androsterone (the major androgenic steroid), synthesized by whole ovarian dispersates from immature rats, remained unchanged following treatment with TNF alpha (30 ng/ml) alone. In contrast, concurrent treatment with increasing concentrations of TNF alpha (0.03-30 ng/ml), yielded dose-dependent inhibition of the human chorionic gonadotropin (1 ng/ml)-stimulated accumulation of androsterone. This reversible and immunoneutralizable effect of TNF alpha was characterized by a minimal effective dose of 0.1 ng/ml, a median inhibitory dose of 0.9 ng/ml, a maximal inhibitory effect of 90%, and a minimal time requirement of less than or equal to 48 h. Comparable results were obtained when using highly purified theca interstitial cells, thereby indicating that TNF alpha is capable of exerting a direct inhibitory effect at the level of the ovarian androgen-producing cell. TNF alpha action was not accounted for by alterations in the plated viable cell mass. Instead, treatment with TNF alpha resulted in significant inhibition of the human chorionic gonadotropin-supported accumulation of cAMP, the putative second messenger of gonadotropin hormonal action. TNF alpha action at sites distal to cAMP generation was associated with profound inhibition of the conversion of the [3H]pregnanolone (3 alpha-hydroxy,5 alpha-pregnane-20-one) and [3H]17 alpha-hydroxypregnanolone (3 alpha, 17 alpha-dihydroxy,5 alpha-pregnane-20-one) substrates to androsterone, suggesting stimulation of 20 alpha-hydroxysteroid dehydrogenase activity, inhibition of 17 alpha-hydroxylase/17:20 lyase activity, or both. Taken together, these findings indicate that TNF alpha, acting at relatively low concentrations, is capable of inhibiting gonadotropin-supported ovarian androgen biosynthesis by selectively modulating the activity of relevant key steroidogenic enzymes. As such, these observations suggest that the theca interstitial cell is a site of TNF alpha reception and action and that TNF alpha, possibly of resident ovarian macrophage origin, may partake in the regulation of ovarian androgen production, an effect due in part to inhibition of the activity of the key steroidogenic enzymes 17 alpha-hydroxylase/17:20 lyase.

Insulin secretion in polycystic ovarian disease: effect of ovarian suppression by GnRH agonist.

Nine obese and ten non-obese women with polycystic ovarian disease (PCO), and seven obese and eight non-obese normal women, had an oral glucose tolerance test (OGTT) before and after treatment with GnRH agonist (buserelin 400 micrograms/day s.c. for 8 weeks) in order to investigate the effect of ovarian suppression on their insulinaemic secretion. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), oestradiol (E2), androstenedione (A), testosterone (T), DHEAS, cortisol and insulin (I) were measured at time 0 of OGTT; in all samples of OGTT, E2, T, A and I were also assayed. PCO patients showed higher basal androgen levels than control patients. All subjects showed a normal glycaemic response to OGTT. The mean fasting and areas under the curve (ISA) of plasma I were significantly greater in the obese PCO women than in non-obese PCO, the normal obese and non-obese women. All PCO patients showed significantly higher fasting I and ISA values in respect to all control patients. Hyperinsulinaemic responses were 89% in PCO obese, 30% in non-obese PCO and 29% in obese control patients. After buserelin treatment, these values did not change significantly in respect to pretreatment in all groups, in spite of a significant decrease of androgen secretion. During OGTT, no variations of steroid plasma concentrations were seen in both normal or hyperinsulinaemic PCO patients. The data of this study show that hyperandrogenism, hyperinsulinism and obesity were associated with different modalities in PCO patients and that a marked decrease of androgen secretion did not restore a normal insulinaemic response to OGTT, suggesting that hyperandrogenism does not produce hyperinsulinism.

Growth hormone and somatomedin-C secretion in patients with polycystic ovarian disease. Their relationships with hyperinsulinism and hyperandrogenism.

Nineteen women with polycystic ovarian disease (PCO; 9 obese) and 15 normal ovulatory women (7 obese) were studied at their follicular phase. All patients had an oral glucose tolerance test (OGTT) before and after treatment with gonadotropin-releasing hormone (GnRH) agonist (Buserelin 400 micrograms/die s.c. for 8 weeks) to investigate the relationship between ovarian steroidogenesis and insulin and growth hormone (GH) and insulin-like growth factor (SmC) secretion. Luteinizing hormone, follicle-stimulating, estradiol, androstenedione, testosterone, dehydroepiandrosterone sulfate, cortisol, insulin, GH and SmC were measured basally at the time of OGTT. PCO patients showed higher androgen basal levels than control patients. All subjects showed a normal glycemic response to OGTT. The mean fasting level and area under the curve of plasma insulin were also significantly greater in PCO than in control patients (p less than 0.05), while GH and SmC plasma concentrations did not differ between the groups. Despite a considerable decrease in androgens and the similar levels in both PCO and control women, buserelin treatment did not determine any significant changes of insulin and GH-SmC secretion. GH and SmC did not correlate with ideal body weight (IBW), insulin or androgens, whereas insulin correlated with both testosterone and androstenedione levels (p less than 0.05) and with IBW (p less than 0.01); after the buserelin regimen only IBW remained related to plasma insulin (p less than 0.01). In conclusion results of this study confirm that hyperinsulinism is a characteristic picture of PCO and is related in an unclear way with hyperandrogenism and obesity.(ABSTRACT TRUNCATED AT 250 WORDS)