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Objective: To evaluate the ability of women affected by functional hypothalamic secondary amenorrhea (FHSA) or polycystic ovary syndrome (PCOS) to adapt to stress. Design: Controlled clinical study. Setting: University hospital. Patient(s): Thirty-one patients affected by FHSA, 29 patients with PCOS, and 30 eumenorrheic women. Intervention(s): The subjects took the Stroop Color Word (Stroop CW) test and underwent blood sampling. Main Outcome Measure(s): Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and serum cortisol levels. Result(s): The healthy controls had better Stroop CW scores than patients with FHSA. Serum cortisol levels significantly increased during Stroop CW with respect to the baseline in patients with FHSA or PCOS but not in the healthy controls. The SBP, DBP, and HR of the controls as well as SBP and DBP of patients with PCOS were significantly higher than those measured in patients with FHSA both at the baseline and during Stroop CW. Conclusion(s): Patients with FHSA do not cope as well as healthy patients, and their autonomic response to stress is worse than both controls and patients with PCOS.

Human placenta is the major source of activin A in maternal circulation. The aim of the present study was to evaluate maternal activin A serum concentration in pregnant women with chronic hypertension (n = 14), pregnancy-induced hypertension (n = 10) or pre-eclampsia (n = 16). In the group of pregnant women with chronic hypertension and of healthy pregnant women (n = 10) activin A was measured in samples collected longitudinally throughout gestation. Using a specific two-site enzyme-linked immunosorbent assay, it has been possible to measure maternal serum activin A concentration. In addition, the effect of recombinant human activin A administration on mean arterial pressure and heart rate in female rats have been also investigated. Mean +/- SEM of maternal serum activin A concentration in pre-eclamptic women (57.4 +/- 28.3 ng/ml), was significantly higher than in women with pregnancy-induced hypertension (14.8 +/- 10.5 ng/ml), chronic hypertension (10.3 +/- 5.4 ng/ml) or healthy control women (9.2 +/- 9.4 ng/ml) (P0.01). Serum activin A levels evaluated 2 weeks after anti-hypertensive treatment were not significantly different in pre-eclamptic women. Moreover, when exogenous recombinant human activin A was administered in female rats arterial pressure or frequency of heart rate did not change. The present study showed that maternal serum activin A concentration is abnormally high in patients with pre-eclampsia. Thus, since the patients with chronic hypertension or pregnancy-induced hypertension have activin A concentration in the normal range of values, activin A may be a prognostic marker of hypertension in pregnancy.

Serum plasma activin-A is measurable in the maternal circulation of healthy pregnant women, increases in specimens collected during the third trimester of gestation, and is highest at parturition. Hormone abnormalities are known to be associated with preterm labor or diabetes in pregnancy. Therefore, in the present study serum activin-A levels in normal controls were compared to those in pregnant women with preterm labor or gestational diabetes. In some cases, values were obtained before and after insulin therapy. In other controls and patients with preterm labor, the activin-A concentration in cord serum was also studied. A newly developed two-site immunotest was used to determine activin-A levels. Subjects included normal controls (n = 7), who were sampled throughout gestation every 5 weeks; pregnant women at term (38-40 weeks) not in labor (n = 22); pregnant women at term in spontaneous labor (< 3.0 cm dilated; n = 42); women in preterm labor (25-35 weeks; n = 38); and women with gestational diabetes (20-39 weeks; n = 9). In control women, serum activin-A levels increased from 4.8 +/- 5.5 micrograms/L (mean +/- SD) at 20 weeks to 25.4 +/- 27.8 micrograms/L at 40 weeks (P < 0.01), and values correlated with gestational age. Pregnant women in preterm labor had serum activin-A concentrations (89.04 +/- 173.31 micrograms/L) higher than those in normal controls (P < 0.01), and no significant correlation to gestational age was found in this group of pregnant women. Healthy women in labor showed serum activin-A concentrations higher than those in women at term but not in labor (P < 0.01). Diabetic patients had serum activin-A concentrations (52.39 +/- 23.32 micrograms/L) significantly higher than those in normal controls. In these patients, maternal serum activin-A concentrations significantly decreased to the range in healthy controls at the same gestational age after insulin therapy (9.48 +/- 3.82 micrograms/L). The present study shows that preterm labor is associated with increased concentrations of activin-A in the maternal circulation and cord serum. Hypersecretion of activin-A is also shown in same patients with gestation diabetes; this reverts to normal after insulin treatment.

In this paper we describe the modification of the galanin (GAL)-like immunostaining in the hypothalamus of rats, which were made hypothyroid at 52 days after birth. On 21st day after the surgical ablation of the thyroid gland, the staining of the GAL-immunoreactive fibers in the median eminence decreased and on the 84th day disappeared almost totally. The GAL-immunoreactive distribution in other areas of the hypothalamus, e.g. the anterior hypothalamus and the dorsomedial nucleus, is only slightly affected by the absence of thyroid hormones, whereas the GAL-staining of medulla oblongata (vagal complex) is equal in both control and hypothyroid rats. In hypothyroid colchicine-treated rats, we were unable to stain GAL-immunoreactive neurons in the paraventricular nucleus (PVN). Oxytocin- and vasopressin-like material was present in the magnocellular neurons and the staining pattern in hypothyroid rats was the same as that of control animals. Our data show a marked reduction in the expression of the GAL-like immunoreactivity of the PVN and median eminence of adult hypothyroid rats. The possible role of this deficit in the pathogenesis of the GH secretion impairment that is observed in hypothyroid rats is discussed.

Objective To investigate a possible correlation between interleukin-12 (IL-12) and IL-13 levels and lymphocyte subsets in the preovulatory follicles of patients with and without polycystic ovarian syndrome (PCOS). Design Controlled clinical study. Setting University hospital. Patient(s) Seventy-eight infertile women undergoing IVF-embryo transfer. Intervention(s) The subjects underwent blood sampling, ovum retrieval, and embryo transfer. Main outcome measure(s) Follicular fluid levels of T, androstenedione (A); IL-12, IL-13, activated T cells, T helper, and T-suppressor lymphocytes. Result(s) The level of IL-12 detected in follicular fluid (FF) was significantly lower in patients with PCOS than in normally ovulating women (mean: 1.47 ± 0.3 pg/mL vs. 2.25 ± 0.7 pg/mL, respectively); in contrast, FF IL-13 concentrations were significantly higher in the patients with PCOS than in the normally ovulating women (mean: 32.5 ± 3.7 pg/mL vs. 19.6 ± 2.5 pg/mL, respectively), as was the total number of activated T lymphocytes (11.5% ± 1.5% vs. 4.8% ± 0.4%). A significant correlation was observed between FF activated T-cell concentrations and FF IL-12, IL-13, T, and A levels. No significant differences were observed when these data were compared with embryological parameters. Conclusion(s) The present study shows significant differences in the correlation between FF IL-12 and IL-13 levels and T lymphocyte numbers in the subset of patients with PCOS as compared to normally ovulating women.

Objective: To evaluate the possible correlation between immunological changes and implantation rates in patients who undergo in vitro fertilization–embryo transfer (IVF-ET). Design: Controlled clinical study. Setting: University hospital. Patient(s): Forty infertile women undergoing IVF-ET. Intervention(s): Stroop Color Word (CW) test, State-Trait Anxiety Inventory (STAI) test, blood sampling. Main Outcome Measure(s): Heart rate and systolic and diastolic blood pressure responses to Stroop CW; circulating T, B, T-helper (CD4), and T-suppressor (CD8) lymphocytes. Result(s): The total number of T lymphocytes increased significantly during superovulation, resulting in significantly higher levels in subjects achieving embryo implantation than in those showing a failure of implantation. An opposite trend was observed for the activated T cells. The number of T-helper lymphocytes and the T-helper/T-suppressor ratio showed a significant increase from baseline to the time of pick-up only in patients with implantation. Conclusion(s): A prolonged condition of stress, which causes a decreased ability to adapt and a transitory anxious state, is associated with high amounts of activated T cells in the peripheral blood. Such a condition, in turn, is associated with a reduced implantation rate in women undergoing IVF-ET.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.

The distribution of neurones expressing POMC mRNA in the cerebral ganglion of the protochordate ascidian, Styela plicata, was investigated using a non-radioactive in situ hybridization technique. Nerve cell bodies of mono and bipolar types expressing POMC mRNA, were observed mainly in the outer layer of the ganglion. Discrete groups of neurones containing POMC mRNA were also localized in the inner portion of the ganglion, and few small monopolar perykaria expressing POMC mRNA were visible at the emergence of the main nerve trunks. POMC mRNA labeling was also found at level of the cytoplasm of previtellogenic and vitellogenic oocytes, and of follicular cells. Our results demonstrate the expression of one or more genes in the cerebral ganglion and ovary, that may be similar to one or more regions of the mammalian POMC gene. Therefore POMC-related molecules seem to be involved in neuromodulatory pathways and regulatory mechanisms of the oogenesis of ascidians.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha MSH) and beta-endorphin were used to localize pro-opiomelanocortin (POMC)-derived peptides in the ovary of the African lungfish Protopterus annectens by immunohistochemistry. Immunoreactivity was observed in the granulosa and the internal theca of the vitellogenic follicles. No immunoreactivity was observed in immature follicles. Using human POMC cDNA as the hybridization probe POMC-like mRNA was identified in situ in cells of the granulosa and internal theca of the vitellogenic follicles. No labeling was observed in primordial follicles. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of the opiate hormones.

Gonadal steroids play an important role in the regulation of neuroendocrine mechanisms involved in the reproductive function. In particular, through a feedback mechanism, they act on the synthesis and release of hypothalamic gonadotropinreleasing hormone (GnRH) and/or pituitary gonadotropic hormones (luteinizing hormone [LH] and follicle stimulating hormone [FSH]. Endogenous opioid peptides (EOP) represent one of the key factors mediating the activity of sex steroids on the hypothalamus-pituitary-gonadal (HPG) axis. Several data, both in animals and humans, indicate that the EOPs play a role in the feedback effect exerted by sex steroids on GnRH secretion. In particular, these peptides control the secretion of LH by inhibiting the hypothalamic GnRH release. Moreover, since EOPs modulate the activity of other neuronal pathways (GABAergic, noradrenergic, dopaminergic, serotoninergic, and cholinergic pathways), they are involved also in sexual behavior, thermoregulation, memory, feeding, pain, and immune response. Therefore a variety of central functions appear to be influenced by gonadal steroids, opioids, and neurotransmitters.

Chronic stress affects the reproductive function by modifying the neuroendocrine homeostasis. The aim of the present study was to clarify the neuroendocrine and the gonadal changes following chronic intermittent stress in male rats and the action of a neuroactive drug, acetyl-l-carnitine (ALC). The effect of two different stressors, cold water swimming or ether, on central beta-endorphin (beta-EP) and GnRH contents, and on plasma testosterone levels was investigated. In addition, the response to an acute stress in chronically stressed rats, treated or untreated with ALC (10 mg/day/rat p.o.), was evaluated. The stressors were applied twice a day for 10 days, and rats were killed before, during and after the last stress session. Mediobasal hypothalamus (MBH) beta-EP and GnRH contents, and plasma testosterone levels were evaluated by radioimmunoassay. The following results were obtained: (1) both chronic swimming and ether stress caused a decrease in hypothalamic beta-EP contents; (2) MBH GnRH contents increased after chronic swimming stress but not after ether stress; (3) chronic swimming stress induced a twofold decrease in plasma testosterone levels, while no changes were observed after ether stress; (4) the treatment with ALC prevented the decrease in plasma testosterone levels after chronic swimming stress, and (5) acute stress in chronically stressed animals caused an increase in MBH-beta-EP. The present data showed that chronic swimming stress reduces the reproductive capacity and impairs the capacity to respond to the acute stress and that ALC modulates the hormonal changes to physical stress and prevents the antireproductive effect of chronic cold swimming.

The aim of the present study was to investigate the possible production, localization, and action of follistatin in human placenta, fetal membranes (amnion, chorion), and maternal decidua. Four different experimental approaches were used: 1) Southern blot analysis following reverse polymerase chain reaction to identify follistatin messenger RNA (mRNA) in tissue homogenates; 2) immunohistochemistry to localize immunoreactive (ir-) follistatin in the various intrauterine tissues; 3) measurement by RIA of ir-follistatin levels in culture medium of placental cells; and 4) possible action of follistatin on human CG (hCG) and progesterone release from cultured placental cells. Placental and decidual cells collected during first trimester or at term gestation express follistatin mRNA; fetal membranes (amnion, chorion) at term also express follistatin mRNA. Immunoreactive follistatin is localized in syncytial cells of placental villi at term as well as in large decidual cells, in amnion epithelium, and in chorionic cells. The placental secretion of follistatin has been confirmed by the evidence of measurable levels of ir-follistatin in the medium of cultured placental cells at term; the release is time dependent and is not modified by the addition of forskolin or progesterone. The addition of increasing doses of recombinant human follistatin does not significantly influence the release of hCG or progesterone from cultured placental cells, whereas the activin A-induced hCG and progesterone release are completely reversed. The present data showed that 1) human placenta, fetal membranes, and decidua express follistatin mRNA; 2) ir-follistatin is localized and released from placental cells at term; and 3) follistatin has a functional role in the local control system regulating placental hormone production.

Immune and neuroendocrine systems interact at various levels. In particular, either cytokines activate the hypothalamus-pituitary-adrenal axis (HPA) or corticotropin-releasing hormone (CRH) induces the release of beta-endorphin from peripheral human mononuclear cells. The aim of the present study was to investigate whether CRH may affect cytokine production and activity in human peripheral blood mononuclear cells (PBMC). Primary cultures of human PBMC and monocytes were used. They were incubated in presence of different doses of synthetic human CRH. Media were collected and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) levels were measured by ELISA, while interferon-gamma (IFN-gamma) levels were measured by bioassay. In addition, phytohemoagglutinin-induced lymphocyte proliferation was evaluated by testing [3H]thymidine incorporation in the presence of various doses of CRH. CRH significantly increased IL-6 release from PBMC (p < 0.01). The addition of CRH to PBMC significantly decreased IFN-gamma levels, in a dose dependent manner (p < 0.01). No significant effect of CRH was observed on lymphocyte proliferation or IL-1 beta production. The present results suggest a role for CRH as a paracrine mediator for human immune cells, increasing the evidence of a clear correlation between immune and neuroendocrine system.

Although supraphysiological levels of 1α, 25 dihydroxyvitamin D3 (VD) have been demonstrated extensively to induce the monomacrophagic differentiation of leukemic myelo- and monoblasts, little is known about the role that physiological levels of this vitamin could play in the regulation of normal hematopoiesis. To clarify this issue, we adopted a liquid-culture model in which cord blood CD34+ hematopoietic progenitors, induced to differentiate in the presence of different combinations of cytokines, were exposed to VD at various concentrations and stimulation modalities. The data obtained show that physiological levels of VD promote a differentiation of CD34+ hematopoietic progenitors characterized by the induction of all the monomacrophagic immunophenotypic and morphological markers. This effect is not only exerted at the terminal maturation but also at the commitment level, as demonstrated by the decrease of highly undifferentiated CD34+CD38− hematopoietic stem cells, the down-regulation of CD34 antigen, and the increase of monocyte-committed progenitors. Molecular analysis suggests that the VD genomic signaling pathway underlies the described differentiation effects.