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STUDY QUESTION Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT is KNOWN ALREADY Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) ±0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors’ ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE of CHANCE hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS of THE FINDINGS This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare. TRIAL REGISTRATION NUMBER N/A.

Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant ( Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 μM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the Ki values were 3.6 ± 0.3 nM (IC50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

Commercial hMG drugs are marketed for the treatment of infertility and consist of highly purified hormones acting on receptors expressed in target gonadal cells. Menopur® and Meriofert® are combined preparation of FSH and hCG and are compared in vitro herein. To this purpose, the molecular composition of the two drugs was analyzed by immunoassay. The formation of FSH receptor and LH/hCG receptor (FSHR; LHCGR) heteromer, intracellular Ca2+ and cAMP activation, β-arrestin 2 recruitment and the synthesis of progesterone and estradiol were evaluated in transfected HEK293 and human primary granulosa lutein cells treated by drugs administered within the pg-mg/ml concentration range. Molecular characterization revealed that Meriofert® has a higher FSH:hCG ratio than Menopur® which, in turn, displays the presence of LH molecules. While both drugs induced similar FSHR-LHCGR heteromeric formations and intracellular Ca2+ increase, Meriofert® had a higher potency than Menopur® in inducing a cAMP increase. Moreover, Meriofert® revealed a higher potency than Menopur® in recruiting β-arrestin 2, likely due to different FSH content modulating the tridimensional structure of FSHR-LHCGR-β-arrestin 2 complexes, as evidenced by a decrease in bioluminescence resonance energy transfer signal. This drug-specific activation of intracellular signaling pathways is consistent with the molecular composition of these preparations and impacts downstream progesterone and estradiol production, with Menopur® more potent than Meriofert® in inducing the synthesis of both the steroids. These findings are suggestive of distinct in-vivo activities of these preparations, but require cautious interpretation and further validation from clinical studies.

To cope physical and/or psychological threats, the human body activates multiple processes, mediated by a close interconnection among brain, endocrine and inflammatory systems. The aim of the study was to assess the hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–thyroid (HPT) axes involvement after an acute stressful event (Emilia Romagna earthquake swarm) with a big data approach. A retrospective, observational trial was performed, collecting all biochemical examinations regarding HPA and HPT axes performed in the same laboratory the year before and the year after the earthquake swarm (20–29 May 2012). Comparing 2576 pre-earthquake to 3021 post-earthquake measurements, a cortisol serum level increase was observed (p

BACKGROUND: The relationship between health status, defined by frailty and comorbidities, and serum T levels has been widely demonstrated in general population, while only one previous retrospective study has explored it in HIV-infected men1. AIM: To investigate the association between frailty and gonadal status by assessing serum total T (TT) with Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) in a cohort of HIV-infected men. METHODS: Prospective, cross-sectional, observational study on HIV-infected men (age 0.21 were considered frail. Statistical analysis: Mann-Whitney U test was used to compare continuous variables. Correlations were performed using linear regression models. RESULTS: 315 consecutive HIV-infected men were enrolled (mean age 45.3±5.3 years; average duration of HIV-infection 16.3±8.8 years). 128 patients (40.5%) were comorbid and 207 (64.9%) were frail. Either cFT (p=0.001) or TT (p CONCLUSIONS: Low cFT and TT levels are associated with multimorbidity and poor health status in HIV infected men. The bidirectional nature of this relationship leads to the figuration of an intriguing vicious circle where T deficiency triggers the onset of comorbidities or, vice versa, poor health status induces hypogonadism. At the same time, notwithstanding the inverse relation between FT and frailty, it seems that other stronger predictive factors, and in particular the duration of infection, are involved in determining the health outcome in this clinical setting. REFERENCES 1Rochira V et al. Low testosterone is associated with poor health status in men with human immunodeficiency virus infection: a retrospective study. Andrology. 2015 Mar;3(2):298-308. 2Guaraldi et al. A frailty index predicts survival and incident multimorbidity independent of markers of HIV disease severity. AIDS. 2015 Aug 24;29(13):1633-41.

Type 5 phosphodiesterase (PDE5) blockade by inhibitors (PDE5i) results in intracellular cyclic guanosine monophosphate (cGMP) increase and smooth muscle relaxation and are used for the treatment of men erectile dysfunction. Although they have high specificity for PDE5, these inhibitors are suspected to cross-interact also with cyclic adenosine monophosphate (cAMP)-specific PDEs, inducing the intracellular accumulation of this cyclic nucleotide and related testosterone increase, positively impacting male reproductive parameters. However, the link between the use of PDE5i and the activation of cAMP-mediated steroidogenesis is still unclear. We have investigated whether three PDE5i, sildenafil, tadalafil and vardenafil, cross-interacts with the high affinity cAMP-specific enzymes type 8A and 8B PDEs (PDE8A and PDE8B), in live, transfected mouse Leydig tumor (mLTC1) and human embryonic kidney (HEK293) cell lines in vitro. The PDE5i-induced production of cAMP-dependent testosterone and its precursor progesterone was evaluated as well. We have developed PDE8A/B biosensors and modified cyclic nucleotides confirming enzyme binding to cAMP, but not to cGMP, in our cell models. cAMP binding to PDE8A/B was displaced upon cell treatment with PDE5i, revealing that sildenafil, tadalafil and vardenafil have similar effectiveness in live cells, in vitro. The cross-interaction between PDE5i and PDE8A/B supports the gonadotropin-enhanced intracellular cAMP increase, occurring together with cGMP increase, as well as steroid synthesis. Indeed, we found that Leydig cell treatment by PDE5i increases progesterone and testosterone production triggered by gonadotropins. We demonstrated that PDE5i may interact with the cAMP-specific PDE8A and PDE8B, possibly inducing intracellular cAMP and sex steroid hormone increase. These findings support clinical data suggesting that PDE5i might increase testosterone levels in men.

Background. The need to re-test adults with negative thyroid ultrasound has not been elucidated yet. The aim of this study was to evaluate the incidence of thyroid disease in patients with previously negative thyroid US and to find any possible correlation with anamnestic or clinical features, and pituitary-thyroid axis function changes. Methods. Subjects who resulted without thyroid nodules in a screening study (1) were re-tested after at least 5 years. Anamnestic (familiarity, radiation therapy, previous pregnancies, smoking habit) and physical data (thyroid palpation, anthropometric measurements, blood pressure) were collected. Neck ultrasound evaluation was performed by an experienced endocrinologist who recorded detailed thyroid and nodules characteristics. When nodules were present, American Thyroid Association classification for prediction of thyroid cancer risk was applied. Serum samples were collected for measurement of TSH, free thyroid hormones, calcitonin and anti-thyroid antibodies. Clinical and anamnestic, physical, sonographic and serological characteristics were analyzed with logistic regression analysis for subjects with nodules versus those without nodules. Results. A total of 111 subjects were enrolled (43 M, 68 F). Half of them presented new thyroid nodules, mostly smaller than 1 cm and not suspicious. The 4% had high levels of anti-thyroid antibodies. Ninety-seven percent were euthyroid. Incidence of thyroid diseases was higher in women, especially in nulliparous. Comparing clinical characteristics of subjects with and without nodules, the only statistically significant difference concerned thyroid volume adjusted for body weight or surface (p

BACKGROUND: HIV-infection is associated to premature decline of serum T1,2. However, prevalence and biochemical characterization of hypogonadism in HIV-infected men are still to be well defined1,2. AIM: To evaluate the gonadal status in HIV-infected men by assessing circulating total T (TT) with either ID-LC-MS/MS or chemiluminescent immunoassay. METHODS: Prospective, cross-sectional, observational study on HIV-infected men with ongoing Highly Active Antiretroviral Therapy (HAART). Serum TT, gonadotropins and sex hormone-binding globulin (SHBG) were measured by chemiluminescent immunoassay. TT was also assessed by the gold standard ID-LC-MS/MS. Free T (FT) was calculated by Vermeulen equation. Hypogonadism was defined as serum TT levels below 320 ng/dL and/or free T levels below 64 pg/ml. Statistical analysis: Categorical variables were compared using Chi-Square test, while correlations were performed using linear regression models. RESULTS: 315 consecutive HIV-infected men were enrolled (mean age 45.56±5.61 years; average duration of HIV-infection 16.57±10.45 years). Considering serum TT levels assessed by LC-MS/MS and immunoassay, 11 patients out of 233 (4.8%) and 10 patients out of 315 (3.2%) had T deficiency, respectively. TT combined with luteinizing hormone (LH) levels was used to classify hypogonadism. No difference was found comparing the two methodologies used for TT measurement (p=0.914). 56 patients (17.8%) showed SHBG above the normal range (>71.4 nmol/L). Considering calculated FT, the incidence of hypogonadism raised to 6.9% using either immunoassay or LC-MS/MS, with no difference between methodologies (p=0.895). Including compensated form of hypogonadism, the prevalence raised to 13% for TT and to 15% for FT. FT showed an inverse relation with age (-0.340,p

Background and aims Sphingosine-1 phosphate (S1P) is a lysosphingolipid present in the ovarian follicular fluid. The role of the lysosphingolipid in gonads of the female is widely unclear. At nanomolar concentrations, S1P binds and activates five specific G protein-coupled receptors (GPCRs), known as S1P1-5, modulating different signaling pathways. S1P1 and S1P3 are highly expressed in human primary granulosa lutein cells (hGLC), as well as in the immortalized human primary granulosa cell line hGL5. In this study, we evaluated the signaling cascade activated by S1P and its synthetic analogues in hGLC and hGL5 cells, exploring the biological relevance of S1PR-stimulation in this context. METHODS AND RESULTS. hGLC and hGL5 cells were treated with a fixed dose (0.1 μM) of S1P, or by S1P1- and S1P3-specific agonists SEW2871 and CYM5541. In granulosa cells, S1P and, at a lesser extent, SEW2871 and CYM5541, potently induced CREB phosphorylation. No cAMP production was detected and pCREB activation occurred even in the presence of the PKA inhibitor H-89. Moreover, S1P-dependent CREB phosphorylation was dampened by the mitogen-activate protein kinase (MEK) inhibitor U0126 and by the L-type Ca2+ channel blocker verapamil. The complete inhibition of CREB phosphorylation occurred by blocking either S1P2 or S1P3 with the specific receptor antagonists JTE-013 and TY52156, or under PLC/PI3K depletion. S1P-dependent CREB phosphorylation induced FOXO1 and the EGF-like epiregulin-encoding gene (EREG), confirming the exclusive role of gonadotropins and interleukins in this process, but did not affect steroidogenesis. However, S1P or agonists did not modulate granulosa cell viability and proliferation in our conditions. Conclusions This study demonstrates for the first time that S1P may induce a cAMP-independent activation of pCREB in granulosa cells, although this is not sufficient to induce intracellular steroidogenic signals and progesterone synthesis. S1P-induced FOXO1 and EREG gene expression suggests that the activation of S1P–S1PR axis may cooperate with gonadotropins in modulating follicle development.

STUDY QUESTION Does the sperm DNA fragmentation index (DFI) improve depending on the FSH receptor (FSHR) genotype as assessed by the nonsynonymous polymorphisms rs6166 (p.N680S) after 3 months of recombinant FSH treatment in men with idiopathic infertility? SUMMARY ANSWER FSH treatment significantly improves sperm DFI only in idiopathic infertile men with the p.N680S homozygous N FSHR. WHAT IS KNOWN ALREADY FSH, fundamental for spermatogenesis, is empirically used to treat male idiopathic infertility and several studies suggest that DFI could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is known that the FSHR single nucleotide polymorphism (SNP) rs6166 (p.N680S) influences ovarian response in women and testicular volume in men. STUDY DESIGN, SIZE AND DURATION A multicenter, longitudinal, prospective, open-label, two-arm clinical trial was performed. Subjects enrolled were idiopathic infertile men who received 150 IU recombinant human FSH s.c. every other day for 12 weeks and were followed-up for a further 12 weeks after FSH withdrawal. Patients were evaluated at baseline, at the end of treatment and at the end of follow-up. PARTICIPANTS/MATERIALS, SETTING, METHODS Eighty-nine men with idiopathic infertility carrier of the FSHR p.N680S homozygous N or S genotype, FSH ≤ 8 IU/l and DFI >15%, were enrolled. A total of 66 patients had DFI analysis completed on at least two visits. DFI was evaluated in one laboratory by TUNEL/PI (propidium iodide) assay coupled to flow cytometry, resolving two different fractions of sperm, namely the 'brighter' and 'dimmer' sperm DFI fractions. MAIN RESULTS AND THE ROLE OF CHANCE Thirty-eight men (57.6%) were carriers of the p.N680S homozygous N and 28 (42.4%) of the homozygous S FSHR. Sperm concentration/number was highly heterogeneous and both groups included men ranging from severe oligozoospermia to normozoospermia. Total DFI was significantly lower at the end of the study in homozygous carriers of the p.N680S N versus p.N680S S allele (P = 0.008). Total DFI decreased significantly from baseline to the end of the study (P = 0.021) only in carriers of the p.N680S homozygous N polymorphism, and this decrease involved the sperm population containing vital sperm (i.e. brighter sperm) (P = 0.008). The dimmer sperm DFI fraction, including only nonvital sperm, was significantly larger in p.N680S S homozygous patients than in homozygous N men (P = 0.018). Total DFI was inversely related to total sperm number (P = 0.020) and progressive sperm motility (P = 0.014). When patients were further stratified according to sperm concentration (normoozospermic versus oligozoospermic) or -211G>T polymorphism in the FSHB gene (rs10835638) (homozygous G versus others), the significant improvement of sperm DFI in FSHR p.N680S homozygous N men was independent of sperm concentration and associated with the homozygous FSHB -211G>T homozygous G genotype. LIMITATIONS, REASONS FOR CAUTION The statistical power of the study is 86.9% with alpha error 0.05. This is the first pharmacogenetic study suggesting that FSH treatment induces a significant improvement of total DFI in men carriers of the p.N680S homozygous N FSHR; however, the results need to be confirmed in larger studies using a personalized FSH dosage and treatment duration. WIDER IMPLICATIONS OF THE FINDINGS The evaluation of sperm DFI as a surrogate marker of sperm quality, and of the FSHR SNP rs6166 (p.N680S), might be useful to predict the response to FSH treatment in men with idiopathic infertility. STUDY FUNDING/COMPETING INTERESTS The study was supported by an unrestricted grant to M.S. and H.M.B. from Merck Serono that provided the drug used in the study. MS received additional grants from Merck Serono and IBSA as well as honoraria from Merck Serono. The remaining authors declare that no conflicts of interest are present. TRIAL REGISTRATION NUMBER EudraCT number 2010-020240-35.

We are grateful to Dr Yves Combarnous (PRC, INRA, CNRS, 37 380, Nouzilly, France) for discussion. We thank Ferring Pharmaceuticals (Saint Prex, Switzerland), the Associazione Scientifica in Endocrinologia, Andrologia e Metabolismo ‘ASEAM’ (Carpi, Italy), and the PhD School in Clinical and Experimental Medicine of the University of Modena and Reggio Emilia (Modena, Italy) for unconditional contributions to this study; International audience; Are four urinary hCG/menotropin (hMG) and one recombinant preparation characterized by different molecular features and do they mediate specific intracellular signaling and steroidogenesis? hCG and hMG preparations have heterogeneous compositions and mediate preparation-specific cell signaling and early steroidogenesis, although similar progesterone plateau levels are achieved in 24 h-treated human primary granulosa cells in vitro. hCG is the pregnancy hormone marketed as a drug for ARTs to induce final oocyte maturation and ovulation, and to support FSH action. Several hCG formulations are commercially available, differing in source, purification methods and biochemical composition. Commercial hCG preparations for ART or research purposes were compared in vitro. The different preparations were quantified by immunoassay with calibration against the hCG standard (Fifth IS; NIBSC 07/364). Immunoreactivity patterns, isoelectric points and oligosaccharide contents of hCGs were evaluated using reducing and non-reducing Western blotting, capillary isoelectric-focusing immunoassay and lectin-ELISA, respectively. Functional studies were performed in order to evaluate intracellular and total cAMP, progesterone production and β-arrestin 2 recruitment by ELISA and BRET, in both human primary granulosa lutein cells (hGLC) and luteinizing hormone (LH)/hCG receptor (LHCGR)-transfected HEK293 cells, stimulated by increasing hormone concentrations. Statistical analysis was performed using two-way ANOVA and Bonferroni post-test or Mann–Whitney's U-test as appropriate. Heterogeneous profiles were found among preparations, revealing specific molecular weight patterns (20–75 KDa range), isoelectric points (4.0–9.0 pI range) and lectin binding (P < 0.05; n = 7–10). These drug-specific compositions were linked to different potencies on cAMP production (EC50 1.0–400.0 ng/ml range) and β-arrestin 2 recruitment (EC50 0.03–2.0 μg/ml) in hGLC and transfected HEK293 cells (P < 0.05; n = 3–5). In hGLC, these differences were reflected by preparation-specific 8-h progesterone production although similar plateau levels of progesterone were acheived by 24-h treatment (P ≥ 0.05; n = 3). N/A. The biological activity of commercial hCG/hMG preparations is provided in International Units (IU) by in-vivo bioassay and calibration against an International Standard, although it is an unsuitable unit of measure for in-vitro studies. The re-calibration against recombinant hCG,quantified in grams, is based on the assumption that all of the isoforms and glycosylation variants have similar immunoreactivity. ShCG/hMG preparation-specific cell responses in vitro may be proposed to ART patients affected by peculiar ovarian response, such as that caused by polycystic ovary syndrome. Otherwise, all the preparations available for ART may provide a similar clinical outcome in healthy women.

Background Human luteinizing hormone (LH) and chorionic gonadotropin (hCG) are glycoprotein hormones regulating development and reproductive functions by acting on the same receptor (LHCGR). We compared the LH and hCG activity in gonadal cells from male mouse in vitro, i.e. primary Leydig cells, which is a common tool used for gonadotropin bioassay. Murine Leydig cells are naturally expressing the murine LH receptor (mLhr), which binds human LH/hCG. Methods Cultured Leydig cells were treated by increasing doses of recombinant LH and hCG, and cell signaling, gene expression and steroid synthesis were evaluated. Results We found that hCG is about 10-fold more potent than LH in cAMP recruitment, and slightly but significantly more potent on cAMP-dependent Erk1/2 phosphorylation. However, no significant differences occur between LH and hCG treatments, measured as activation of downstream signals, such as Creb phosphorylation, Stard1 gene expression and testosterone synthesis. Conclusions These data demonstrate that the responses to human LH/hCG are only quantitatively and not qualitatively different in murine cells, at least in terms of cAMP and Erk1/2 activation, and equal in activating downstream steroidogenic events. This is at odds with what we previously described in human primary granulosa cells, where LHCGR mediates a different pattern of signaling cascades, depending on the natural ligand. This finding is relevant for gonadotropin quantification used in the official pharmacopoeia, which are based on murine, in vivo bioassay and rely on the evaluation of long-term, testosterone-dependent effects mediated by rodent receptor. Electronic supplementary material The online version of this article (doi:10.1186/s12958-016-0224-3) contains supplementary material, which is available to authorized users.

Luteinizing hormone (LH) and choriogonadotropin (hCG) are glycoprotein hormones regulating ovarian function and pregnancy, respectively. Since these molecules act on the same receptor (LHCGR), they were traditionally assumed as equivalent in assisted reproduction techniques (ART), although differences between LH and hCG were demonstrated at molecular and physiological level. In this study, we demonstrated for the first time that co-treatment with a follicle-stimulating hormone (FSH) dose in the ART therapeutic range potentiates different LH- and hCG-dependent responses in vitro, measured in terms of cAMP, phospho-CREB, -ERK1/2 and -AKT activation, gene expression, progesterone and estradiol production in human granulosa-lutein cells (hGLC). We show that in the presence of FSH, hCG biopotency is about 5-fold increased, in the presence of FSH, in terms of cAMP activation. Accordingly, CREB phosphorylation and steroid production is increased under hCG and FSH co-treatment. LH effects, evaluated as steroidogenic cAMP/PKA pathway activation, do not change in the presence of FSH, which, however, increases LH-dependent ERK1/2 and AKT, but not CREB phosphorylation, resulting in anti-apoptotic effects. The different modulatory activity of FSH on LH and hCG action in vitro corresponds to their different physiological functions, reflecting proliferative effects exerted by LH during the follicular phase and before trophoblast development, and the high steroidogenic potential of hCG requested to sustain pregnancy from the luteal phase onwards.

Introduction Chronic hepatitis C is the main cause of death in patients with end-stage liver disease. Prognosis depends on the increase of fibrosis, whose progression is twice as rapid in men as in women. Aim of the study was to evaluate the effects of reproductive stage on fibrosis severity in women and to compare these findings with age-matched men. Materials and Methods A retrospective study of 710 consecutive patients with biopsy-proven chronic hepatitis C was conducted, using data from a clinical database of two tertiary Italian care centers. Four age-matched groups of men served as controls. Data about demographics, biochemistry, liver biopsy and ultrasonography were analyzed. Contributing factors were assessed by multivariate logistic regression analysis. Results Liver fibrosis was more advanced in the early menopausal than in the fully reproductive (P

A volume-controlled hemorrhagic shock was produced in anesthetized rats by intermittent bleeding from an iliac vein over a period of 20–30 min, until the carotid mean arterial pressure (MAP) stabilized around 20–24 mmHg. In this condition, which caused the death of all saline-treated animals within 25–30 min, the intravenous (i.v.) bolus injection of the adrenocorticotropin fragment 1–24 (ACTH(1–24)) at a dose of 160 μg/kg promptly restored MAP, as well as pulse pressure, heart rate and respiratory function, and greatly prolonged the survival time. Capsaicin (125 mg/kg cumulatively, s.c., 1 week before) completely prevented the anti-shock effect of ACTH(1–24), which, on the other hand, was shared by i.v. [Nle 11 ]-substance P (SP) (200–300 μg/kg). Finally the SP-antagonist [ d -Arg 1 , d -Pro 2 , d -Trp 7.9 ,Leu 11 ]-SP prevented the effect of ACTH(1–24). These results suggest that SP-containing nerve fibers are required for the effect of ACTH in hemorrhagic shock.

In a rat model of hemorrhagic shock which caused the death of all control rats within 30 min, i.v. injection of the ganglion-stimulating drug dimethylphenylpiperazinium (DMPP) caused a dose-dependent reversal of the shock condition--without the need for reinfusion of the shed blood--starting from the dose of 4 ng/kg i.v. Shock reversal was associated with the mobilization of residual blood and improvement in blood flow, particularly at the carotid level. These results could influence our thinking on pathophysiology and first-aid management of shock.

In anesthetized rats, step-wise bleeding to a severe condition of hemorrhagic shock causes a decrease in arterial and venous pH and in venous P O 2 and S O 2 and an increase in arterial P O 2 and in venous P CO 2 and lactic acid. The intravenous bolus injection of ACTH-(1–24)(160 μg/kg)—which causes a rapid and sustained reversal of the shock condition — produces a gradual and almost complete recovery (within 60 min) of venous P O 2 , P CO 2 and S O 2 ; on the other hand, the normalization of blood pH and lactate is preceded by a further worsening during the first minutes after treatment. On the whole, these data are compatible with the ACTH-(1–24)-induced mobilization of the residual blood — which is pooled in poorly oxygenated tissues — and with the improved circulatory and respiratory functions.

Summary In a model of haemorrhagic shock causing the death of all saline-treated rats within 25.8 ± 2.7 min after treatment, the intravenous injection of thyrotropin-releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induces a prompt and sustained increase of arterial pressure and pulse amplitude, with survival of all rats. Bilateral vagotomy, atropine sulphate (2 mg/kg intraperitoneally) and hemicholinium-3 (20 μ g/rat intracerebroventricularly) partially prevent the TRH-T-induced shock reversal, whereas atropine methylbromide has no effect. These data indicate that afferent vagal fibres, brain cholinergic neurons and central muscarinic receptors play a role in the mechanism of the anti-shock effect of TRH-T.

The ability of ACTH-(1-24) to prolong survival and to extend the deadline for effective blood reinfusion has been studied in a model of lethal hypovolemic shock in the rat. Anesthetized rats were bled to a mean arterial pressure of 18 to 25 mm Hg and then subjected to one of the following iv treatments: a) saline; b) ACTH-(1-24), 160 micrograms/kg; c) blood reinfusion; d) ACTH-(1-24), 160 micrograms/kg; c) blood reinfusion; d) ACTH-(1-24), with saline 5 min after bleeding died within 0.05 h. On the other hand, the treatment with ACTH-(1-24) induced an almost complete and sustained recovery of cardiovascular and respiratory functions associated with a survival time of 44 +/- 18 h, while four of six rats reinfused with the withdrawn blood were still alive 15 days later. The time-lapse between bleeding and treatment was of crucial importance, and neither ACTH-(1-24) injection nor blood reinfusion had any effect if performed 25 min after bleeding. However, treatment with ACTH-(1-24) shortly after bleeding (5 min) greatly improved the effect of a later blood reinfusion. These data indicate that ACTH-(1-24) can prolong survival and permit the time-lapse between blood loss and blood reinfusion to be extended.

In haemorrhage-shocked rats, the recovery of mean arterial pressure (MAP), pulse pressure (PP) and respiratory rate (RR), as well as the improvement of survival rate, induced by the i.v. administration of centrally acting cholinergic drugs (physostigmine, oxotremorine) are not affected by morphine at the dose of 2·5 mg/kg i.v., and only partially (MAP, PP, survival rate) or not at all (RR) prevented by a dose of morphine of 5 mg/kg i.v. These results indicate that the anti-shock effect of cholinergic drugs is largely independent of the opioid tone, this possibly being of practical relevance.

In rats bled to hypovolemic shock, the intracerebroventricular injection of hemicholinium-3 (20 micrograms/rat) completely prevented the shock reversal induced by the intravenous injection of ACTH (1-24) (160 micrograms/kg), but had no influence on the shock reversal induced by the intravenous injection of physostigmine (70 micrograms/kg). These data indicate that brain cholinergic neurons are involved in the anti-shock effect of ACTH-peptides, but not in that of centrally acting cholinergic drugs.

Cholinergic mechanisms are currently thought to play an essential role in blood pressure homeostasis. Here we show that, in urethane-anaesthetized rats bled to severe hemorrhagic shock, the i.v. administration of nicotine 0.2-50 micrograms/kg causes a prompt, sustained and dose-dependent improvement in cardiovascular and respiratory functions, the animals' survival rate being significantly higher than that of animals treated with saline. These effects are prevented by bilateral cervical vagotomy and by concurrent local anaesthesia of the carotid bodies, which suggests that stimulation of visceral afferents is the main mechanism of action of nicotine in hemorrhagic shock.

Summary Putrescine, intraperitoneally injected either into intact or into hypophysectomized rats, caused a reduction in urine volume at doses of 200–300 mg/kg. At doses of 100 mg/kg or more, there was also a significant loss of potassium. The highest dose (300 mg/kg) caused haemoglobinuria, proteinuria, increased natriuresis, increased urinary osmolarity, reduced aldosteronaemia, ectasis of glomerular capillaries and tubular damage. The underlying mechanisms(s) are probably mostly linked to the strong cationic charge of putrescine and to its binding to fixed anions of tubular-cell membrane.

The influence of putrescine on cardiac arrhythmias induced by either permanent ligature of the left anterior coronary artery or heart reperfusion following a 5-min coronary occlusion was studied in anesthetized rats. Reperfusion-induced arrhythmias were significantly prevented by the i.v. injection of 150-200 mg/kg of putrescine, the survival rate being 100% in treated animals and 40% in controls. At a dose level of 200-300 mg/kg i.v., putrescine also significantly reduced the duration of ventricular tachycardia induced by permanent coronary occlusion. These findings show that putrescine significantly reduces the consequences of cardiac ischemia and reperfusion, probably as a consequence of its multiple stabilizing effects at the membrane level.

In a rat model of volume-controlled hemorrhagic shock causing the death of all saline-treated animals within 30 min of treatment, the intravenous bolus injection of thyrotropin- releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induced the prompt and sustained disappearance of the ECG and EEG signs of heart and brain ischemia, along with the reversal of hypotension and respiratory depression and with 100% survival rate at the end of the 2 h observation period. These data confirm that, in a pre-terminal condition induced by massive hemorrhage, timely treatment with TRH-T will restore heart and brain perfusion to levels compatible with survival and with functional recovery from ischemia and maintain it at those levels for some hours.

In an experimental model of bleeding-induced hemorrhagic shock causing the death of all saline-treated rats within 30 min, the intravenous injection of ACTH-(1-24) at the dose of 160 micrograms/kg induced a sustained reversal of the shock condition, with almost complete recovery of blood pressure, pulse amplitude, respiratory rate, heart rate, and 100% survival, at least for the 2 h of observation. This effect of ACTH-(1-24) was prevented by the intracerebroventricular injection of 4-DAMP (a highly selective antagonist for M1 and M3 muscarinic receptors), but unaffected by the intracerebroventricular injection of pirenzepine (a highly selective antagonist for M1 muscarinic receptors). These data indicate that an essential step in the complex mechanism of the ACTH-induced shock reversal may be the activation of brain M3 muscarinic receptors.

In a rat model of severe hypotension and respiratory depression induced by step-wise bleeding, protoveratrines cause a prompt and sustained improvement of cardiovascular and respiratory functions, both in anesthetized and in conscious animals, seemingly through a magnification of the reflex response originated by the chemoreceptors of aortic and carotid bodies. The restoration of cardiovascular function is attributable to an increase both in total peripheral resistance and cardiac output. The finding could provide the basis for a new approach to the first-aid management of massive blood losses.

Intraperitoneal injection of putrescine induced dose-related hypothermia in rats. The effect was more pronounced at room temperature (22 degrees C) than in a warm environment (30 degrees C), the maximum hypothermia (-2.64 +/- 0.29 degrees C, 30 min. after treatment) being obtained with the dose of 300 mg/Kg and remaining significant throughout 3 hr of observation. Putrescine also had antipyretic activity, as it significantly reduced pyrogen-induced fever at a dose level (100 mg/Kg i.p.) ineffective in causing hypothermia in normal rats. The hypothermic and antipyretic effects of putrescine were not associated with any obvious sign of toxicity.