Ghrelin misbalance affects mice embryo implantation and pregnancy success by uterine immune dysregulation and nitrosative stress.

Introduction: In a previous study we found that ghrelin (Ghrl) misbalance during the peri-implantation period significantly impaired fetus development. In this study we aimed to evaluate the putative mechanisms underlying these effects, including embryo implantation success, uterine nitric oxide synthase (NOS) activity, nitric oxide synthesis and the inflammatory/immune uterine profile. Methods: Ghrelin misbalance was induced by injecting 4nmol/animal/day of Ghrl (hyperghrelinemia) or 6nmol/animal/day of a Ghrl antagonist (Ant: (D-Lys3)GHRP-6) from day 3 to 8 of pregnancy. Control animals (C) were injected with de vehicle. Females were euthanized at pregnancy day 8 and their uteri excised in order to evaluate: the percentage of reabsorbed embryos (microscopically), eNOS, iNOS and nytrotirosine expression (by immunohistochemistry), nitrite synthesis (by Griess technique), VEGF, IL-10, IL-17, IL-6, MMP9 and GM-CSF expression (by qPCR) and leukocyte infiltration by flow cytometry (evaluating T cells, NK cells, granulocytes, dendritic cells and macrophages). Results: Ant-treatment significantly increased the percentage of reabsorbed embryos and the uterine expression of eNOS, iNOS and nytrotirosine. (D-Lys3)GHRP-6-treatment increased also the expression of the inflammatory cytokines IL-6, IL-17 and MMP9, and decreased that of IL-10 (anti-inflammatory). Moreover, Ant-treatment increased also the NK cells population and that of CD11b+ dendritic cells; and decreased T cells percentages. Similarly, hyperghrelinemia showed a significant increase vs. C on eNOS, iNOS and nytrotirosineuterine expression and a decrease in T cells percentages. Conclusion: Ghrl misbalance during the peri-implantation period induces pro-inflammatory changes and nitrosative stress in the gravid uterus, impairing significantly embryo implantation and/or development.

Role of the hypothalamus in ghrelin effects on reproduction: sperm function and sexual behavior in male mice.

In brief: Ghrelin signals to the hypothalamus inhibit reproduction during times of food scarcity. In this study, we demonstrate that ghrelin impairs sperm quality in male mice. Abstract: Ghrelin (GHRL) is an orexigenic peptide that has been investigated as one of the signals responsible for the reproductive performance of mammals under fluctuating metabolic conditions. Central GHRL administration impairs spermatogenesis in mice by regulating the hypothalamic-pituitary-gonadal axis function. In the present study, the hypothalamus role as a mediator of GHRL effects on sperm fertilizing capacity and male sexual behavior was evaluated. After 42 days of hypothalamic GHRL infusion or artificial cerebrospinal fluid, in vitro and in vivo sperm fertilizing capacity, testicular α-tubulin, speriolin gene expression and spermatic α-tubulin protein were evaluated. Hypothalamic expression of genes Kiss1, Gpr54 and Gnrh was also studied. The second group of animals was infused with one time only GHRL or artificial cerebrospinal fluid into the hypothalamus to evaluate the effects on sexual behavior. Results demonstrated that chronic GHRL administration to male mice significantly increased the percentages of pre-implantation embryo loss and the number of post-implantation embryo loss. In relation to the gene expression, our results show a relative decrease of Kiss1, Gpr54 and Spatc1. Although no significant differences were observed in the quantitative expression of α-tubulin protein, qualitative changes in its expression pattern were observed. In addition, a dual effect on sexual behavior was observed: 40% of the treated animals showed a significant reduction in the number of mounts and intromissions, while a 60% showed a significant decrease in ejaculation latency vs control animals. In conclusion, our results provide evidence that central GHRL administration possibly induces failure in embryo development and/or implantation in the females mated with treated males, possibly because of a negative effect in the α-tubulin pattern.

Inter- and trans-generational effects of gestational ghrelin imbalance on development and reproduction in the mouse.

CONTEXT AND AIMS: We have demonstrated that ghrelin (Ghrl) participates in fetal programming, since intragestational hyperghrelinaemia increased pup's growth and a Ghrl-receptor antagonist accelerated offspring's sexual maturation and impaired their adult reproductive function. Now, we aim to analyse if these phenotypic changes (found in F1) also occurred in F2 and/or F3 generations. METHODS: We treated mice dams (F0), with 4nmol/animal/day of Ghrl or 6nmol/animal/day of an antagonist [Ant:(d -Lys3)GHRP6] from day 1 of pregnancy until delivery. When F1 female pups reached adulthood, they were paired to obtain F2, and subsequently, F2 females were paired to obtain F3. Parameters evaluated in F2 and F3 pups were: growth, physical development, neurobiological maturation, puberty onset and in adulthood, reproductive function. KEY RESULTS: The F2 and F3 Ant groups showed a significant increase in litter size. Although no differences were detected in the weight of these pups at birth, in adulthood, they were heavier. At F3, pups from the Ant group showed advanced incisors eruption and eye opening compared to controls. Furthermore, F3 male pups from the Ant group showed earlier testis descent, although in adulthood, these males exhibited reduced sperm concentration in comparison to Ghrl. No differences were detected in F2 or F3 females regarding puberty onset or reproduction. CONCLUSIONS AND IMPLICATIONS: Some fetal programming effects of Ghrl seen in F1, also appeared transgenerationally. Since many women at reproductive age suffer from conditions with reduced Ghrl levels (i.e. obesity or polycystic ovarian syndrome), these results could be relevant to the health of their descendants.

Do aging, drinking, and having unhealthy weight have a synergistic impact on semen quality?

PURPOSE: To evaluate if age, alcohol consumption, and body mass index (BMI) have synergistic effects on seminal quality, and to rank these factors based on their impact on semen. METHODS: Retrospective study of 9464 patients attending an andrology laboratory. Data on patients' age and daily alcohol intake were provided by the patients. BMI was recorded in the laboratory. Seminal parameters evaluated were volume, sperm concentration and total count, motility, morphology, viability, nuclear maturity, and membrane functional integrity. RESULTS: All the seminal parameters evaluated were affected by the synergistic interaction Age x BMI, suggesting that this combination is more potent in affecting semen quality. The variables sperm morphology and nuclear maturity seemed to be especially susceptible since they were affected by the three synergistic interactions. In the logistic regression analysis, age was the most powerful factor since it impacted first on five of the nine parameters, impacting mainly on sperm motility, viability, and morphology, with no effects on sperm count. On the contrary, BMI impacted first in sperm concentration and total sperm count; which was confirmed also by the logistic predictions analysis. Alcohol consumption impacted first on membrane functional integrity and nuclear maturity. A J-shaped association between BMI or alcohol consumption with semen quality was found in the multivariate analysis. CONCLUSION: The factors considered in this study showed a synergistic negative impact on semen quality, being age and unhealthy weight the most important ones. Reducing the exposure to lifestyle risk factors may be promising for improving sperm quality in infertile patients.

Fetal Programming Effects of a Mild Food Restriction During Pregnancy in Mice: How Does It Compare to Intragestational Ghrelin Administration?

To explore in mice if a 15% food restriction protocol during pregnancy programs the offspring postnatal development, with emphasis on reproductive function, and to assess if ghrelin (Ghrl) administration to mouse dams exerts effects that mimic those obtained under mild caloric restriction. Mice were 15% food-restricted, injected with 4 nmol/animal/day of Ghrl, or injected with the vehicle (control) thorough pregnancy. After birth, the pups did not receive further treatment. Pups born from food-restricted dams (FR pups) were lighter than Ghrl pups at birth, but reached normal weight at adulthood. Ghrl pups were heavier at birth and gained more weight than control pups (C pups). This effect was not associated with plasma IGF-1. FR pups showed a delay in pinna detachment and eye opening, while an advance was observed in Ghrl pups. FR pups showed also impairment in the surface-righting reflex. In both female FR and Ghrl pups, there was an advance in vaginal opening and, in adulthood, FR pups showed a significant decrease in their own litter size and plasma progesterone, and an increase in embryo loss. A delay in testicular descent was evident in male Ghrl pups. Changes in puberty onset were not associated with differences in the expression of Kiss1 in hypothalamic nuclei. Finally, in adulthood, FR pups showed a significant decrease in sperm quality. In conclusion, a mild food restriction thorough gestation exerted programming effects on the offspring, affecting also their reproductive function in adulthood. These effects were not similar to those of intragestational Ghrl administration.

Male adiposity, sperm parameters and reproductive hormones: An updated systematic review and collaborative meta-analysis.

The present updated systematic review and meta-analysis aims to summarize the evidence from published studies with low risk for any important bias (based on methodological quality assessment) investigating the potential associations of adiposity with sperm quality and reproductive hormones. We conducted a systematic search of the literature published in MEDLINE-PubMed and EMBASE through June 2019. Based on the criteria in our review, 169 eligible publications were used for data abstraction. Finally, 60 articles were included in the qualitative analysis and 28 in the quantitative analysis. Our systematic review results indicated that overweight and/or obesity were associated with low semen quality parameters (i.e., semen volume, sperm count and concentration, sperm vitality and normal morphology) and some specific reproductive hormones (e.g., inhibin B, total testosterone and sex hormone-binding globulin). Overweight and/or obesity were also positively associated with high estradiol concentrations. Meta-analysis indicated that overweight and/or obesity categories were associated with lower sperm quality (i.e., semen volume, sperm count and concentration, sperm vitality, total motility and normal morphology), and underweight category was likewise associated with low sperm normal morphology. In conclusion, our results suggest that maintaining a healthy body weight is important for increasing sperm quality parameters and potentially male fertility.

Recategorisation of body mass index to achieve andrological predictive power: a study in more than 20 000 patients.

The aim of this study was to recategorise body mass index (BMI) in order to classify patients according to their risk of semen abnormalities. Patients (n=20563) presenting at an andrology laboratory were classified into five groups according to BMI: underweight (BMI <20kg m-2), normal weight (BMI 20-24.9kg m-2), overweight (BMI 25-29.9kg m-2), obese (BMI 30-39.9kg m-2) and morbidly obese (BMI >40kg m-2). Semen quality was evaluated to determine: (1) differences between groups using analysis of variance (ANOVA); (2) the chances of semen abnormalities (using generalised linear models, Chi-squared tests and odds ratios); (3) reference BMI values with andrological predictive power (multivariate conglomerate analyses and multivariate analysis of variance (MANOVA)); and (4) expected values of abnormalities for each new group resulting from BMI recategorisation. Morbidly obese and underweight patients exhibited the highest decrease in semen quality and had higher chances of semen abnormalities. The smallest number of sperm abnormalities was found at a BMI of 27kg m-2. Four reference values were identified, recategorising BMI into four groups according to their risk of semen abnormalities (from lowest to highest risk): Group1,BMI between 20 and 32kg m-2; Group2, BMI <20 and BMI >32-37kg m-2; Group3, BMI >37-42kg m-2; and Group4, BMI >42kg m-2. A BMI <20 or >32kg m-2 is negatively associated with semen quality; these negative associations on semen quality increase from a BMI >37kg m-2 and increase even further for BMI >42kg m-2. The BMI recategorisation in this study has andrological predictive power.

Modulatory effects of ghrelin on sperm quality alterations induced by a fructose-enriched diet.

The objectives of this study were: 1) to evaluate the effects of a fructose enriched diet (FED) on rat sperm quality, epididymal function (i.e. oxidative stress and alpha-glucosidase expression) and testosterone concentrations; 2) to determine if the administration of ghrelin (Ghrl), reverses the effects induced by FED. After validating the protocol as an inductor of metabolic syndrome like-symptoms, adult male rats were assigned to one of the following treatments for 8 weeks: FED = 10% fructose enriched in water (v/v); FED + Ghrl = fructose enriched diet plus Ghrl (6 nmol/animal/day, s.c.) from week 6-8; or C = water without fructose (n = 5-10 animals/group). FED significantly decreased sperm concentration and motile sperm count/ml vs C (FED: 19.0 ± 1.6 × 106sperm/ml and 834.6 ± 137.0, respectively vs C: 25.8 ± 2.8 × 106 and 1300.4 ± 202.4, respectively; p < 0.05); ghrelin injection reversed this negative effect (23.5 ± 1.6 × 106sperm/ml and 1381.7 ± 71.3 respectively). FED resulted in hypogonadism, but Ghrl could not normalize testosterone concentrations (C: 1.4 ± 0.1 ng/ml vs FED: 0.8 ± 0.2 ng/ml and FED + Ghrl: 0.6 ± 0.2 ng/ml; p < 0.05). Ghrelin did not reverse metabolic abnormalities secondary to FED. FED did not alter epididymal expression of antioxidants enzymes (superoxido-dismutase, catalase and glutathione peroxidases -Gpx-). Nevertheless, FED + Ghrl significantly increased the expression of Gpx3 (FED + Ghrl: 3.47 ± 0.48 vs FED: 0.69 ± 0.28 and C: 1.00 ± 0.14; p < 0.05). The expression of neutral alpha-glucosidase, which is a marker of epididymal function, did not differ between treatments. In conclusion, the administration of Ghrl modulated the negative effects of FED on sperm quality, possibly by an epididymal increase in Gpx3 expression. However, Ghrl could not neither normalize the metabolism of FED animals, nor reverse hypogonadism.

Reproductive performance of male mice after hypothalamic ghrelin administration.

It has been demonstrated that food intake and reproductive physiology are both simultaneously modulated to optimize reproductive success under fluctuating metabolic conditions. Ghrelin (GHRL) is an orexigenic peptide identified as the endogenous ligand of the growth hormone secretagogue receptor that is being investigated for its potential role on reproduction. Considering that data available so far are still limited and characterization of GHRL action mechanism on the reproductive system has not been fully elucidated, we studied the participation of hypothalamus in GHRL effects on sperm functional activity, plasma levels of gonadotropins and histological morphology in mice testes after hypothalamic infusion of 0.3 or 3.0 nmol/day GHRL or artificial cerebrospinal fluid (ACSF) at different treatment periods. We found that GHRL 3.0 nmol/day administration for 42 days significantly reduced sperm concentration (GHRL 3.0 nmol/day = 14.05 ± 2.44 × 106/mL vs ACSF = 20.33 ± 1.35 × 106/mL, P < 0.05) and motility (GHRL 3.0 nmol/day = 59.40 ± 4.20% vs ACSF = 75.80 ± 1.40%, P < 0.05). In addition, histological studies showed a significant decrease percentage of spermatogonia (GHRL 3.0 nmol/day = 6.76 ± 0.68% vs ACSF = 9.56 ± 0.41%, P < 0.05) and sperm (GHRL 3.0 nmol/day = 24.24 ± 1.92% vs ACSF = 31.20 ± 3.06%, P < 0.05). These results were associated with a significant reduction in luteinizing hormone and testosterone plasma levels (P < 0.05). As GHRL is an orexigenic peptide, body weight and food intake were measured. Results showed that GHRL increases both parameters; however, the effect did not last beyond the first week of treatment. Results presented in this work confirm that central GHRL administration impairs spermatogenesis and suggest that this effect is mediated by inhibition of hypothalamic-pituitary-gonadal axis.

Role of ghrelin in fertilization, early embryo development, and implantation periods.

In order to clarify the physiological role of ghrelin in gestation, we evaluated the effects of administration of exogenous ghrelin (2 or 4 nmol/animal per day) or its antagonist (6 nmol/animal per day of (d-Lys3)GHRP6) on fertilization, early embryo development, and implantation periods in mice. Three experiments were performed, treating female mice with ghrelin or its antagonist: i) starting from 1 week before copulation to 12 h after copulation, mice were killed at day 18 of gestation; ii) since ovulation induction until 80 h later, when we retrieved the embryos from oviducts/uterus, and iii) starting from days 3 to 7 of gestation (peri-implantation), mice were killed at day 18. In experiments 1 and 3, the antagonist and/or the highest dose of ghrelin significantly increased the percentage of atrophied fetuses and that of females exhibiting this finding or a higher amount of corpora lutea compared with fetuses (nCL/nF) (experiment 3: higher nCL/nF-atrophied fetuses: ghrelin 4, 71.4-71.4% and antagonist, 75.0-62.5% vs ghrelin 2, 46.2-15.4% and control, 10-0.0%; n=7-13 females/group; P<0.01). In experiment 2, the antagonist diminished the fertilization rate, and both, ghrelin and the antagonist, delayed embryo development (blastocysts: ghrelin 2, 62.5%; ghrelin 4, 50.6%; and antagonist, 61.0% vs control 78.4%; n=82-102 embryos/treatment; P<0.0001). In experiment 3, additionally, ghrelin (4 nmol/day) and the antagonist significantly diminished the weight gain of fetuses and dams during pregnancy. Our results indicate that not only hyperghrelinemia but also the inhibition of the endogenous ghrelin effects exerts negative effects on the fertilization, implantation, and embryo/fetal development periods, supporting the hypothesis that ghrelin (in 'adequate' concentrations) has a physiological role in early gestational events.