Phd in ENDOCRINOLOGICAL SCIENCES

Thesis title: Obesità maschile, riproduzione ed epigenetica: effetti della dieta chetogenica e impatto delle alterazioni metaboliche in PMA

Obesity is a complex metabolic disorder arising from the interplay of hereditary, environmental, and behavioural factors, and is frequently associated with severe comorbidities, including hypogonadism and infertility. It is linked to endocrine dysfunction, impaired spermatogenesis, heightened inflammatory responses, and oxidative stress. Moreover, obesity may exert transgenerational effects, influencing the susceptibility to chronic diseases in the offspring through epigenetic mechanisms. In light of these observations, weight loss achieved through appropriate nutritional interventions represents a promising strategy to counteract the adverse effects of obesity.Among dietary approaches, the ketogenic diet (KD), characterised by a low carbohydrate intake, high fat content, and adequate protein levels, has proven effective in reducing body weight, serum lipids, and glycaemia, as well as improving inflammatory status. Furthermore, ketone bodies generated under this regimen may serve as an energy source supporting sperm motility. Nevertheless, evidence regarding the impact of KD on male reproductive health remains limited. This research was conducted within the PNRR Flagship 4 programme, which promotes health and well-being through scientific research and technological innovation. By exploring the molecular and epigenetic mechanisms underlying the relationship between obesity and male fertility, the project contributes to a deeper understanding of the biological processes linking obesity, gametogenesis, and embryonic development, with the aim of identifying potential biomarkers for the diagnosis and treatment of male infertility. The study addresses an issue of major clinical and social relevance, combining prevention, through the promotion of a healthy lifestyle, with methodological innovation, employing advanced analytical techniques for miRNA profiling, DNA methylation assessment, and chromatin integrity evaluation. The findings provide novel insights that may inform the development of diagnostic and therapeutic strategies aimed at improving reproductive health and preventing non-communicable diseases. The project comprises three main studies: Study 1 – Cytological, molecular, and epigenetic parameters were compared between obese (BMI > 30 kg/m²) and normal-weight men (BMI < 25 kg/m²). Specifically, sperm DNA fragmentation (SDF%, TUNEL), expression of miR-34c-5p, miR-449b-5p, and miR-19b-3p (Digital PCR), and methylation levels of H19, IGF2, and PEG1/MEST (pyrosequencing) were evaluated in semen samples from 24 obese patients and 19 normal-weight controls. Normal-weight men exhibited significantly higher testosterone levels (p < 0.001), ejaculate volume (p = 0.058), and sperm concentration (p = 0.012). Obese subjects showed increased SDF% (p < 0.001) and reduced miR-34c-5p and miR-449b-5p expression (p < 0.001 and p = 0.015, respectively). Differential methylation patterns were observed across imprinted genes. In H19, CpG1 negatively correlated with miR-19b-3p expression (ρ = –0.354; p = 0.076), while CpG2 correlated positively with miR-449b-5p (ρ = 0.413; p = 0.036). In IGF2, total methylation correlated positively with sperm concentration (ρ = 0.379; p = 0.051), and CpG2 correlated negatively with ejaculate volume (ρ = –0.369; p = 0.058). PEG1/MEST CpG1 methylation was significantly higher in obese men (p = 0.018), and CpG3 showed positive correlations with BMI (ρ = 0.353; p = 0.071) and SDF% (ρ = 0.426; p = 0.027). CpG4 correlated positively with progressive motility (ρ = 0.480; p = 0.011), vitality (ρ = 0.404; p = 0.037), and showed a trend with sperm concentration (ρ = 0.364; p = 0.062). Study 2 – The effects of a three-month KD using commercial products specifically standardized and formulated for this dietary regimen, were evaluated in 8 obese men (BMI > 30 kg/m²) to determine its impact on male fertility. Blood ketone levels confirmed adherence to the diet. Following intervention, testosterone levels significantly increased (p = 0.019), SDF% markedly decreased (p < 0.0019), and miR-34c-5p expression significantly rose (p = 0.007), indicating improvements in both sperm quality and hormonal balance. Study 3 – 61 men from infertile couples attending the ART Centre of Policlinico Umberto I were stratified by BMI into normal-weight (< 25 kg/m²), overweight (25–29.9 kg/m²), and obese (> 30 kg/m²) groups. Overweight and obese subjects exhibited a higher percentage of abnormal sperm forms (p = 0.026) and increased SDF% (p = 0.023), along with a trend towards reduced miR-449b-5p expression. Moreover, miR-34c-5p expression was inversely correlated with embryo quality, suggesting a regulatory role of these miRNAs in early developmental processes. Despite substantial advances in reproductive medicine, male infertility remains a major contributor to couple infertility. The findings of this project demonstrate that even in normal conventional semen parameters, alterations in SDF%, miRNA expression, and DNA methylation can impair gamete competence and early embryonic development. These results underscore the need for an integrated approach to infertility management that includes evaluation and optimisation of the male partner’s metabolic and nutritional status, both prior to natural conception and within ART protocols. Enhancing male metabolic health thus emerges as a key strategy for preserving fertility, supporting optimal embryonic development, and safeguarding the well-being of future generations.