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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 4 nmol/animal/day of Ghrl or 6 nmol/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.

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.

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.

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.

The purpose of this study was to evaluate the intragestational role of ghrelin in offspring development and reproductive programming in a mouse model of ghrelin imbalance during pregnancy. Female mice were injected with ghrelin (supraphysiological levels: 4 nmol/animal/day), antagonist (endogenous ghrelin inhibition with (D-Lys3)GHRP-6, 6 nmol/animal/day) or vehicle (control = normal ghrelin levels) throughout the pregnancy. Parameters evaluated in litters were growth, physical, neurobiological and sexual development and, at adulthood, reproductive function. Litter size and initial weight did not vary between treatments. Male pups from dams treated with ghrelin showed higher body weight increase until adulthood (31.7 ± 0.8 vs control = 29.7 ± 0.7, n = 11-14 litters/treatment; P < 0.05). Postnatal physical and neurobiological development was not modified by treatments. The antagonist accelerated male puberty onset, evidenced as earlier testis descent and increased relative testicular weight (antagonist = 0.5 ± 0.0% vs ghrelin = 0.4 ± 0.0% and control = 0.4 ± 0.0%, n = 5-10 litters/treatment; P < 0.05). At adulthood, these males exhibited lower relative testicular weight and reduced sperm motility (63.9 ± 3.6% vs control = 70.9 ± 3.3 and ghrelin = 75.6 ± 3.0, n = 13-15 animals; P < 0.05), without changes in plasma testosterone or fertility. Female pups intragestationally exposed to the antagonist showed earlier vaginal opening (statistically significant only at Day 25) and higher ovarian volume (antagonist = 1085.7 ± 64.0 mm3 vs ghrelin = 663.3 ± 102.8 mm3 and control = 512.3 ± 116.4 mm3; n = 4-6 animals/treatment; P < 0.05), indicating earlier sexual maturation. At adulthood, these females and those exposed to ghrelin showed a tendency to higher percentages of embryo loss and/or foetal atrophy. In conclusion, ghrelin participates in reproductive foetal programming: Alterations in ghrelin activity during pregnancy modified body weight increase and anticipated puberty onset, exerting (or tending to) negative effects on adult reproductive function. Fil: Torres, Pedro Javier. Universidad Nacional de Córdoba. Secretaria de Ciencia y Tecnología; Argentina Fil: Luque, Eugenia Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Ponzio, Marina Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Cantarelli, Verónica Inés. Ministerio de Ciencia, Tecnología e Innovación Productiva. Agencia Nacional de Promoción Científica y Tecnológica. Fondo para la Investigación Científica y Tecnológica; Argentina Fil: Diez, Marcela. Universidad Nacional de Córdoba. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Figueroa, S.. Universidad Nacional de Córdoba. Facultad de Medicina; Argentina Fil: Vincenti, Laura María. Universidad Nacional de Córdoba. Facultad de Medicina; Argentina Fil: Carlini, Valeria Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina Fil: Martini, Ana Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina

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 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 32–37kg m−2; Group3, BMI >37–42kg m−2; and Group4, BMI >42kg m−2. A BMI 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.

High-fat diets (HFDs) are an acknowledged risk factor for male subfertility, but the underlying mechanisms remain unclear. In the present study we compared the effects of two HFDs with different ω6 : ω3 ratios, one enriched with soy oil (SOD; ω6 : ω3 = 9.62) and another enriched with sunflower oil (SFOD; ω6 : ω3 = 51.55), with those of a commercial diet (CD; ω6 : ω3 = 19.87), supplied from pregnancy to adulthood, on morphometric parameters and reproductive performance in adult male mice (recommended ω6 : ω3 for rodents = 1–6). Bodyweight was significantly higher in the SFOD than CD group, and relative testicular weight was significantly lower in the SFOD than the other two groups. SFOD altered sperm performance: it reduced sperm viability (mean ± s.e.m.; 76.00 ± 1.35% vs 82.50 ± 1.45% and 80.63 ± 1.00% in the SFOD vs CD and SOD groups respectively; P

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, 46.2K15.4% and control, 10–0.0%; nZ7–13 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%; nZ82–102 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. Fil: Luque, Eugenia Mercedes. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Cordoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: Torres, Pedro Javier. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: de Loredo, Nicolás. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: Vincenti, Laura María. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: Stutz, Graciela. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: Santillán, María Emilia. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: Ruiz, Ruben Daniel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Cordoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: Fiol, Marta Haydee. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Cordoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina Fil: Martini, Ana Carolina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Cordoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; Argentina

Ghrelin (Ghr) is a gut/hypothalamus peptide with inhibitory actions on reproductive physiology; however, there are no previous reports of its role on estrous behavior. Under the hypothesis that the increase of plasma Ghr during food restriction (FR) is responsible for receptivity reduction, we intended to evaluate the receptivity percentage of female mice subjected to: exp. 1) acute and chronic FR and Ghr administration (3 nmol/animal/day, s. c.) and exp. 2) the co-administration of a ghrelin antagonist [ant=(d-Lys3)-GHRP-6; 6 nmol/animal/day s. c.]. All females were ovariectomized, primed with steroids, trained, and randomly subjected every week to each one of several protocols, followed by a behavioral test. Experiment 1 (n=8): basal, no treatment; acute FR (aFR), 24-h fasting; chronic FR (cFR), 50% FR for 5 days; acute ghrelin (aGhr), Ghr 30 min before test and chronic ghrelin (cGhr), Ghr for 5 days. Except for cGhr, all treatments significantly decreased the percentage of receptivity (mean±SEM): basal 61.9±6.0, aFR 33.1±8.1, cFR 18.8±7.7, aGhr 45.6±10.6, p

Hexarelin (HEXr), a synthetic ghrelin analogue, has been associated with modifications of reproductive physiology. In previous studies of adult mice, we detected that HEXr induced significantly reduced ovulation rate and significant correlation coefficients between sexual maturation and corporal weight in offspring. In this study, we investigated the effects of chronic HEXr administration on sperm concentration and functional activity, oestrous cyclicity andpregnancyindex,inadditiontothenumberoffetusesanditscorrelationwiththenumberofcorporalutea.AdultAlbino swiss mice were injected (sc) daily with HEXr: 100mgkg � 1 day � 1 (HEXr D1) or 200mgkg � 1 day � 1 (HEXr D2) for 53 daysinmalesand30daysinfemales.Wedetectedasignificantlydecreasedratiointhenumberoffetusespercorporalutea in females treated with HEXr D2 for 30 days before mating and during the first 6 days of pregnancy, in addition to a downwardtrendinthepregnancyindexandpercentageoffemalesimpregnatedbyeachmaletreatedwithbothdosesofthe analogue. Although we did not find any significant effect on additional parameters evaluated in both genders, we propose certaineffectsofHEXrontheimplantationprocessand/orearlydevelopmentofembryosandovertheinvivoreproductive capability of males. Additional keywords: ghrelin, growth hormone secretagogues, reproduction. Received 5 January 2011, accepted 6 July 2011, published online 11 November 2011

Ghrelin (Ghr) has been associated with reproductive physiology and pre- and postnatal development. The objectives of the present study were to evaluate the effects of hexarelin (HEX; 100 or 200 µg kg−1 day−1), a therapeutic Ghr analogue, on: (1) embryo development 60 h post ovulation, induced pharmacologically, in pregnant mice; (2) the physical, neurobiological and sexual development of offspring of female mice injected with HEX during the first, second or third week of pregnancy or throughout the entire pregnancy; and (3) adult memory acquisition in these offspring. We also evaluated the effects of chronic HEX administration on memory acquisition in adult mice. Treatment of non-pregnant female mice with HEX decreased ovulation rate. However, treatment of pregnant mice with HEX at any time during pregnancy tended to accelerate offspring maturation, regardless of bodyweight. This effect was only significant on neurobiological parameters following treatment during the first week. HEX treatment during the first week and/or throughout the entire pregnancy resulted in impaired memory acquisition in the offspring, with female mice being more susceptible to these effects. Similar results were observed for the effects of chronic HEX treatment on memory acquisition in adult mice. In conclusion, HEX seems to exert differential effects depending on when it is administered. Because HEX has started to be used therapeutically, its deleterious effects on ovulation and memory acquisition must be further evaluated.