LH increases the response to FSH in granulosa-lutein cells from sub/poor-responder patients in vitro.

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). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (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.

Membrane Estrogen Receptor (GPER) and Follicle-Stimulating Hormone Receptor (FSHR) Heteromeric Complexes Promote Human Ovarian Follicle Survival.

Classically, follicle-stimulating hormone receptor (FSHR)-driven cAMP-mediated signaling boosts human ovarian follicle growth and oocyte maturation. However, contradicting in vitro data suggest a different view on physiological significance of FSHR-mediated cAMP signaling. We found that the G-protein-coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with preferential Gαs protein/cAMP-pathway coupling and FSH responsiveness of patients undergoing controlled ovarian stimulation. In contrast, FSHR/GPER heteromers triggered anti-apoptotic/proliferative FSH signaling delivered via the Gßγ dimer, whereas impairment of heteromer formation or GPER knockdown enhanced the FSH-dependent cell death and steroidogenesis. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation.

Inferring biallelism of two FSH receptor mutations associated with spontaneous ovarian hyperstimulation syndrome by evaluating FSH, LH and HCG cross-activity.

RESEARCH QUESTION: What is the cumulative effect of two follicle-stimulating hormone receptor (FSHR) mutations in spontaneous ovarian hyperstimulation syndrome (sOHSS) pathogenesis? Are these mutations in the mono- or biallelic state? DESIGN: Two FSHR mutations were found in a pregnant patient affected by sOHSS with no predisposing conditions. While the p.Asn106His mutation is novel, the p.Ser128Tyr mutation has been associated with sOHSS previously. The patient's FSHR gene was analysed by Sanger sequencing, and FSHR cDNAs carrying a single or both point mutations were created by mutagenesis in vitro. cAMP activation by recombinant FSH, luteinizing hormone (LH), human chorionic gonadotropin (HCG) and thyroid-stimulating hormone (TSH) was evaluated in transfected HEK293 cells by bioluminescence resonance energy transfer. RESULTS: All mutations decreased the 50% effective concentration of FSH calculated for cAMP (P < 0.05, n = 6), resulting in two- to 10-fold lower ligand potency. TSH failed to induce an FSHR-mediated increase in intracellular cAMP, while LH was approximately four-fold more potent than HCG in p.Ser128Tyr FSHR-expressing HEK293 cells despite lower cAMP plateau levels (P < 0.05, n = 5). The p.Ser128Tyr FSHR mutation was found to be responsible for an LH-/HCG-induced increase in cAMP when it was in the biallelic heterozygous state with p.Asn106His, but no increase in cAMP was induced in the monoallelic state. CONCLUSION: In-vitro data support that, in pregnant patients with sOHSS, the two FSHR mutations have an opposing effect on the pathogenesis of sOHSS and are in the biallelic heterozygous form, allowing HCG to induce a p.Ser128Tyr FSHR-mediated increase in cAMP.

Telomere and telomerase in chronic liver disease and hepatocarcinoma.

The pathogenesis of liver cirrhosis is not completely elucidated. Although in the majority of patients, the risk factors may be identified in B and C viral hepatitis, alcohol intake, drugs or fatty liver disease, there is a small percentage of patients with no apparent risk factors. In addition, the evolution of chronic liver disease is highly heterogeneous from one patient to another. Among patient with identical risk factors, some rapidly progress to cirrhosis and hepatocellular carcinoma (HCC) whereas others have a benign course. Therefore, a genetic predisposition may contribute to the development of cirrhosis and HCC. Evidence supporting the role of genetic factors as a risk for cirrhosis has been accumulating during the past years. In addition to the results from epidemiological studies, polymorphisms studies and data on twins, the concept of telomere shortening as a genetic risk factor for chronic liver disease and HCC has been proposed. Here we review the literature on telomerase mutations, telomere shortening and liver disease including hepatocellular carcinoma.

Age-associated alterations in cholesterol homeostasis: evidence from a cross-sectional study in a Northern Italy population.

BACKGROUND: The modifications of cholesterol metabolism associated with aging are ill-defined. The objective of this study was to define age-associated alterations of the different metabolic pathways controlling cholesterol homeostasis by analyzing circulating sterols. METHODS: We analyzed serum samples collected from 201 adult (75 male, 126 female) subjects within the epidemiological MICOL study (Multicentrica Italiana Colelitiasi). The age range was 38-79 years; 103 had evidence of gallstones. The concentrations of the different sterols, recognized as markers of the main pathways of cholesterol homeostasis, were analyzed by gas chromatography-mass spectrometry, including lathosterol (synthesis), campesterol and sitosterol (absorption), and 7α-hydroxy-4-cholesten-3-one (degradation to bile acids). RESULTS: A significant direct correlation was detected between age and cholesterol levels (r =0.34, P<0.01). The lathosterol/cholesterol ratio was lower in older age quartiles (P<0.05 by analysis of variance), with an inverse correlation between the lathosterol/cholesterol ratio and age (r=-0.32, P<0.01). Such correlation was particularly evident in females. The campesterol/cholesterol and sitosterol/cholesterol ratios were inversely correlated with aging in control, but not in gallstone patients. The levels of 7α-hydroxy-4-cholesten-3-one were not correlated with age. CONCLUSION: These data show a reduction of cholesterol synthesis with aging which is associated with increased circulating cholesterol levels. The finding might be related to a reduced metabolic need for cholesterol in advancing age, leading to a downregulation of the main mechanisms of cholesterol intake in the liver. A different age-related behavior was observed in gallstone-free versus gallstone patients regarding cholesterol absorption. The possible implications in terms of the pharmacological management of hypercholesterolemia in the elderly remain to be defined.

In vivo degradation of cholesterol to bile acids is reduced in patients receiving parenteral nutrition.

BACKGROUND: Artificial nutrition is frequently associated with hepatobiliary complications, probably due to the inherent derangement of the gastrointestinal tract physiology. Alterations of hepatic lipid metabolism are likely to be involved. The aim of the present study was to investigate the effect of artificial nutrition on bile acid production, a key event in cholesterol homeostasis, in humans. PATIENTS AND METHODS: Eleven patients receiving artificial nutrition, either parenteral nutrition (PN; n = 6) or enteral nutrition (EN; n = 5) with no previous history of liver disease, underwent analysis of cholesterol 7α-hydroxylation rates in vivo, a measure of bile acid formation, by isotope release analysis after intravenous injection of [7α-(3)H]cholesterol. The results were compared with those obtained in a population of 16 age-matched control subjects. RESULTS: Hydroxylation rates were lower in patients with artificial nutrition (PN: 94 ± 13 mg/d; EN: 230 ± 39 mg/d, mean ± SEM) when compared with controls (385 ± 47 mg/d) (P < .01, 1-way analysis of variance). In a patient receiving EN, hydroxylation rates increased 3.5-fold after treatment with the cholecystokinin analogue ceruletide (20 µg bid for 2 weeks intramuscularly). Serum lathosterol-to-cholesterol ratio, a marker of cholesterol synthesis, was also significantly reduced in artificial nutrition, whereas serum levels of fibroblast growth factor 19 (FGF19) were increased. CONCLUSION: In vivo 7α-hydroxylation is suppressed in artificial nutrition, particularly in PN. The finding associates with reduced cholesterol production, possibly as a metabolic consequence. The data suggest a regulatory role of gastrointestinal hormones and FGF19 on bile acid production and might suggest a pathophysiological basis for some common complications of artificial nutrition, such as gallstone disease and cholestasis.

ABCB4 and ABCB11 mutations in intrahepatic cholestasis of pregnancy in an Italian population.

BACKGROUND: Genetic alterations in the ATP-binding cassette subfamily B member 4 (ABCB4) and ATP-binding cassette subfamily B member 11 (ABCB11) have been associated to the onset of intrahepatic cholestasis of pregnancy (ICP) in predisposed women. AIMS: To identify new and/or frequent ABCB4 and ABCB11 genes variants in a cohort of Italian patients with ICP and to evaluate the possible pathogenetic role for the novel mutations identified. METHODS: DNA of 33 unrelated Italian women with obstetric cholestasis were screened for mutations in the entire coding sequence of ABCB4 and ABCB11 genes. Polymerase chain reaction and automated sequencing was performed on the 27 coding exons of both genes. RESULTS: Genotyping revealed 11 mutations, 5 of whom were novel variants: 2 localized on ABCB4 (p.I587DfsX603, p.I738LfsX744) and 3 on ABCB11 (p.V284D, p.Q558H, p.P731S). The most severe phenotypes were associated with the variants p.I587DfsX603, p.I738LfsX744 and p.V284D. Moreover, the already described mutation p.N510S found in ABCB4 seems to be strictly involved in the onset of ICP in that particular patient. CONCLUSIONS: Our data support the hypothesis of a significant involvement of ABCB4 mutations in the onset of ICP, but also confirm an important role for ABCB11 mutations in increasing the susceptibility to cholestasis of pregnancy.

Effects of bile duct ligation and cholic acid treatment on fatty liver in two rat models of non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease, one of the most prevalent liver disorders in Western countries, is characterized by hepatic accumulation of triglycerides. Bile acids have long been known to affect triglyceride homeostasis through a not completely understood mechanism. AIM: To analyse the effects of two different manipulations of bile acid circulation on non-alcoholic fatty liver disease. METHODS: Two animal models of non-alcoholic fatty liver disease were developed by either feeding rats with a choline deficient or with a high fat diet. After 4 weeks, rats were randomized to undergo either bile duct ligation, sham operation or cholic acid administration. RESULTS: During cholestasis there was an increased CYP7A1 expression, the rate limiting enzyme in bile acid synthesis, and a reduction of hepatic concentration of oxysterols, ligands of the liver X receptors. Target genes of the liver X receptors, involved in fatty acid and triglyceride synthesis, were down-regulated in association with decreased hepatic triglyceride content and improvement of fatty liver. Administration of cholic acid, ligand of farnesoid X receptor, also had a beneficial effect on fatty liver in rats on choline deficient diet. CONCLUSION: These results indicate that pharmacological approaches increasing the expression of CYP7A1 or stimulating farnesoid X receptor pathway could represent a promising treatment for non-alcoholic fatty liver disease.

Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes.

BACKGROUND AND AIM: Studies have shown monounsaturated oleic acid to be less toxic than palmitic acid and to prevent/attenuate palmitic acid hepatocites toxicity in steatosis models in vitro. However, to what degree these effects are mediated by steatosis extent is unknown. METHODS: We evaluated whether steatosis per se is associated with hepatocytes apoptosis and determined the role of oleic and palmitic acid, the most abundant fatty acids in western diets, on triglyceride accumulation and apoptosis in an in vitro model of steatosis induced in three hepatocytic cell lines (HepG2, HuH7, WRL68). The impact of incubation for 24 h with oleic (0.66 and 1.32 mM) and palmitic acid (0.33 and 0.66 mM), alone or combined (molar ratio 2 : 1) on steatosis, apoptosis, and insulin signalling, was evaluated. RESULTS: Concurrent with PPARgamma and SREBP-1 gene activation, steatosis extent was larger when cells were treated with oleic than with palmitic acid; the latter fatty acid was associated with increased PPARalpha expression. Cell apoptosis was inversely proportional to steatosis deposition. Moreover, palmitic, but not oleic acid, impaired insulin signalling. Despite the higher amount of fat resulting from incubation of the two fatty acids combined, the apoptosis rate and impaired insulin signalling were lower than in cells treated with palmitic acid alone, indicating a protective effect of oleic acid. CONCLUSIONS: Oleic acid is more steatogenic but less apoptotic than palmitic acid in hepatocityc cell cultures. These data may provide a biological basis for clinical findings on dietary patterns and pathogenetic models of nonalcoholic fatty liver disease.

Correlation between plasma levels of 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic patients.

BACKGROUND/AIM: Hepatic bile acid synthesis is the main mechanism whereby the organism can degrade cholesterol. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one have been reported to reflect bile acid synthesis and the expression or activity of the limiting enzyme of the main biosynthetic pathway, cholesterol 7alpha-hydroxylase. Aim of this study was to correlate the levels of this metabolite with the rates of cholesterol 7alpha-hydroxylation in vivo, a direct measurement of bile acid synthesis, in hyperlipidemic patients. DESIGN: Concentrations of 7alpha-hydroxy-4-cholesten-3-one were assayed by gas-liquid chromatography: mass spectrometry in plasma samples obtained in 18 patients with primary hyperlipoproteinemia who previously underwent determination of cholesterol 7alpha-hydroxylation rates in vivo by tritium release analysis. Both determinations were performed in basal conditions and after treatment with hypolipidemic drugs (the fibric acid derivatives gemfibrozil and bezafibrate, cholestyramine alone or associated with simvastatin). RESULTS: Changes in plasma 7alpha-hydroxy-4-cholesten-3-one profile closely reflected in vivo cholesterol 7alpha-hydroxylation rates during treatment with fibrates, cholestyramine and cholestyramine plus simvastatin. When plotting determinations from all studies (n=40), a very strict correlation was disclosed between plasma 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates (r=0.81, P<0.001). CONCLUSIONS: Plasma 7alpha-hydroxy-4-cholesten-3-one closely mirrors measurements of cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic subjects and therefore stands as a reliable marker of global bile acid synthesis. In view of the correlation observed, these data may help to interpret changes of plasma levels of this metabolite in terms of cholesterol balance quantification.

17 Beta-estradiol prevents cytotoxicity from hydrophobic bile acids in HepG2 and WRL-68 cell cultures.

BACKGROUND: Epidemiological and clinical studies suggest the possibility that estrogens might have a cytoprotective effect on the liver. The aim of the present study was to test the hypothesis that 17beta-estradiol (E2) prevents hepatocellular damage induced by deoxycholic acid (DCA), a hydrophobic bile acid. METHODS: HepG2 cells were exposed for 24 h to DCA (350 micromol/L). Cell viability, aspartate aminotransferase and lactate dehydrogenase activity and apoptosis were measured as indices of cell toxicity. The effect of DCA was compared to that observed using either a hydrophilic bile acid, ursodeoxycholic acid (UDCA; 100 micromol/L), or E2 at different concentrations (1 nmol/L, 10 nmol/L, 50 nmol/L and 50 micromol/L) or mixtures of E2/DCA or UDCA/DCA. The same experiments were performed using WRL-68 cells that, at variance with HepG2, express a higher level of nuclear estrogen receptor. RESULTS: High concentrations of E2 and UDCA prevented DCA-induced decrease in cell viability, increase in enzyme activity and apoptosis evaluated both by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labeling (TUNEL) assays. In addition, DCA-related apoptosis, assessed by caspase activity, was also prevented by E2 (P < 0.01) in physiological (1-10 nmol/L) doses. The cytoprotective effects of E2 and UDCA was also observed in the WRL-68 cell line. CONCLUSIONS: 17Beta-Estradiol prevents DCA-induced cell damage in HepG2 and WRL-68 cell lines to an extent comparable to UDCA. The hypothesis that the protective effect of E2 may be mediated by a mechanism that is nuclear estrogen receptor independent, deserves further verification.

ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum (PXE).

Pseudoxanthoma elasticum (PXE) is a genetic disorder, characterized by cutaneous, ocular and cardiovascular clinical symptoms, caused by mutations in a gene (ABCC6) that encodes for MRP6 (Multidrug Resistance associated Protein 6), an ATP-binding cassette membrane transporter. The ABCC6 gene was sequenced in 38 unrelated PXE Italian families. The mutation detection rate was 82.9%. Mutant alleles occurred in homozygous, compound heterozygous and heterozygous forms, however the great majority of patients were compound heterozygotes. Twenty-three different mutations were identified, among which 11 were new. Fourteen were missense (61%); five were nonsense (22%); two were frameshift (8.5%) and two were putative splice site mutations (8.5%). The great majority of mutations were located from exon 24 to 30, exon 24 being the most affected. Among the others, exons 9 and 12 were particularly involved. Almost all mutations were located in the intracellular site of MRP6. A positive correlation was observed between patient's age and severity of the disorder, especially for eye alterations. The relevant heterogeneity in clinical manifestations between patients with identical ABCC6 mutations, even within the same family, seems to indicate that, apart from PXE causative mutations, other genes and/or metabolic pathways might influence the clinical expression of the disorder.