Paternal nutrition and reproductive fitness

Obesity, defined in human subjects as a BMI >30 kg/m², is associated with adverse metabolic effects and higher CVD risk^{(Reference Lavie, Milani and Ventura23)}. Obesity has also been linked to reproductive dysfunction and an association with male infertility^{(Reference Cabler, Agarwal and Flint24)}. However, while conflicting effects of obesity on semen quality and fertility have been reported in men, multiple studies in animal (mostly rodent) models have shown negative effects of obesity of a high-fat diet (HFD) on male reproductive fitness. Hammoud et al. demonstrated that with increasing BMI in men, the incidence of oligozoospermia increased also, with obese men having a 15⋅62 % incidence compared to 5⋅32 % for normal-weight men^{(Reference Hammoud, Wilde and Gibson25)}. Disturbances in endocrine homoeostasis underpinning spermatogenesis indicate one mechanism by which obesity-mediated alterations in hormonal profiles may alter male fertility. For example, obese men have been found to have decreased testosterone, inhibin B and increased oestrogen levels, all associated with impaired spermatogenesis^{(Reference Du Plessis, Cabler and McAlister26)}. Furthermore, alterations in the balance of gonadotropin-releasing hormone and luteinising hormone/follicle-stimulating hormone (GnRH-LH/FSH) may further contribute to an obesity-related impairment of spermatogenesis by disrupting Leydig and Sertoli cell function in bulls^{(Reference Thundathil, Dance and Kastelic27)}. Additionally, fat accumulation in male obesity can increase the scrotal temperature which impairs spermatogenesis, contributing to decreased sperm quality^{(Reference Kasturi, Tannir and Brannigan28)}. In men, obesity has also been associated with an increased risk of sperm DNA damage^{(Reference Dupont, Faure and Sermondade29)}, while in mice, increased reactive oxygen species (ROS) production and higher levels of DNA damage in sperm of HFD fed males have been reported^{(Reference Bakos, Mitchell and Setchell30)}. Elevated levels of testicular ROS, and the subsequent increase in oxidative stress, have been shown to have detrimental effects on mammalian sperm integrity through the impairment of the sperm plasma membrane^{(Reference Aitken, Smith and Jobling31)}. Furthermore, the elevation of ROS in men and the resultant increase in sperm cytotoxicity and DNA fragmentation^{(Reference Mahfouz, Sharma and Thiyagarajan32)}, have been shown to reduce sperm vitality and motility^{(Reference Shi, Chen and Huang33)}, lower levels of sperm capacitation^{(Reference Morielli and O'Flaherty34)} and diminish sperm-oocyte binding capacity^{(Reference Aitken, Gordon and Harkiss35)}.

The impact of paternal nutrition has on a male's reproductive health extends beyond the pre-conception period and into the peri-conception period. An association between elevated paternal BMI and impaired embryo development has been identified in men and animal models. In men undergoing ART interventions, an increasing BMI was significantly associated with a decreased rate of embryo blastulation on day 5 of culture^{(Reference Bakos, Henshaw and Mitchell36)}. Furthermore, the same study reported rates of pregnancy, embryo implantation and live birth decreased from 41⋅3 % for men of a BMI <25 kg/m² to 22⋅6 % for obese men^{(Reference Bakos, Henshaw and Mitchell36)}. In rats, similar observations were reported for embryos derived from obese males, with HFD-induced obesity reducing the cleavage rates of preimplantation embryos. Furthermore, these embryos demonstrated an impaired ability to achieve developmental milestones in vitro and ultimately failed to achieve blastocyst expansion at an appropriate time-point^{(Reference Mitchell, Bakos and Lane37)}. Early embryo cleavage dynamics have been associated with rates of ongoing development and live birth within clinical ART settings^{(Reference Bartolacci, Dal Canto and Guglielmo38)}. Recently, the association between being overweight in men and a reduction in fertility has been supported further through a large-scale meta-analysis of 115 158 study participants, revealing obese men had an increased likelihood of infertility^{(Reference Campbell, Lane and Owens39)}. Similar to the metabolic insult of high fat and obesogenic diets, paternal undernutrition and nutrient-deficient diets have also demonstrated an impact on sperm quality and early embryo development. In mice, pre-implantation embryos from low-protein fed stud males were found to have a reduction in genes associated with metabolic homoeostasis, particularly a decreased expression of genes involved in the AMPK pathway^{(Reference Watkins, Sirovica and Stokes40)}. Separately, paternal global dietary restriction found undernutrition in male mice resulted in a faster cleavage time in preimplantation embryos, yet reduced rates of blastocyst expansion were observed^{(Reference McPherson, Fullston and Kang41)}.

Paternal nutrition and offspring health

The influence of paternal nutritional status extends beyond alterations in early embryonic development and a number of studies in animal models have highlighted the deleterious effects paternal over- and under-nutrition have on the health of a male's offspring. In mice, perturbed patterns of fetal growth and skeletal formation have been reported in response to both paternal low protein and low folate diets^{(Reference Watkins, Sirovica and Stokes40,Reference Lambrot, Xu and Saint-Phar42)} . Interestingly, in both studies, altered placental development was highlighted as one central regulator of the changes in fetal growth, mirroring observations from many maternal programming studies^{(Reference Sferruzzi-Perri and Camm43)}. Changes in expression of several hepatic genes for lipid metabolism have also been observed in response to paternal low protein diet (LPD)^{(Reference Carone, Fauquier and Habib44)}. These differential growths and metabolic profiles seen during fetal development are then mirrored in postnatal life. In mice, a paternal 70 % caloric restriction model, designed to reflect the nutritional availability in of developing countries, increased levels of adiposity in male offspring adiposity as well as inducing dyslipidaemia^{(Reference McPherson, Fullston and Kang41)}. Similarly, a paternal reduction in caloric intake (reduced by 25 %) in the rat resulted in an increase in displays of anxiety-such as behaviours in the adult offspring in addition to inducing a reduction in food intake, weight gain and serum leptin levels^{(Reference Govic, Penman and Tammer45)}. Data from the Overkalix epidemiological data sets also connect patterns of paternal and grand-paternal nutrition with significant changes in offspring development^{(Reference Bygren, Kaati and Edvinsson46)}. Here, periods of low availability of food between the ages of 9 and 12 in males, defined as a ‘slow growth period’ decreased the mortality risk from CVD in their offspring^{(Reference Kaati, Bygren and Edvinsson47,Reference Pembrey, Bygren and Kaati48)} . In contrast, paternal and grand-paternal over-nutrition during this same period was associated with an increased predisposition to diabetes-related mortality^{(Reference Kaati, Bygren and Edvinsson47,Reference Pembrey, Bygren and Kaati48)} . Intergenerational paternal programming has also been reported within experimental animal models. Paternal HFD-induced obesity in male rats results in increased adiposity, impaired glucose tolerance and insulin sensitivity within a second (F2) generation^{(Reference Fullston, Ohlsson Teague and Palmer49)}. Underlying these changes was a significant decrease in DNA methylation in the paternal testicular germ cells associated with the differential expression of 414 genes and 11 miRNAs^{(Reference Fullston, Ohlsson Teague and Palmer49)}. More recently, we have shown that offspring cardiovascular dysfunction and impaired renin-angiotensin system homoeostasis were programmed into a second generation in response to a paternal LPD in mice^{(Reference Morgan, Paganopoulou and Akhtar50)}.

Identifying the mechanisms of paternal programming

The observation that paternal programming can operate over multiple generations implicates epigenetic transmission of paternal traits as one potential mediator. Epigenetic alterations to the sperm (DNA and histone modifications, RNA populations) have been proposed as one mechanism for transmission of paternal programming effects in the offspring. In mice, global sperm DNA hypomethylation, coupled with a reduced testicular expression of the key regulatory methyltransferase genes Dnmt1 and Dnmt3L, have been reported in response to a paternal LPD^{(Reference Watkins, Dias and Tsuro51)}. Aberrant patterns of sperm DNA methylation have also been observed in response to caloric restriction^{(Reference McPherson, Fullston and Kang41)} and dietary insufficiency of key vitamins and minerals such as folate^{(Reference Lambrot, Xu and Saint-Phar42)}. Similarly, obesity has been shown to induce alterations to DNA methylation profiles as well as miRNA populations in sperm of male mice^{(Reference Fullston, Ohlsson Teague and Palmer49)}. In men, differential sperm DNA methylation profiles and ncRNA profiles have also been observed between obese and lean men^{(Reference Donkin, Versteyhe and Ingerslev52)}. With regard to paternal obesity in men, studies have shown hypomethylation of the IGF2 differentially methylated region in offspring leucocytes at birth^{(Reference Soubry, Murphy and Wang53)}. Furthermore, significant hypomethylation of other imprinted genes including MEST, PEG3 and NNAT were found in offspring of obese fathers^{(Reference Soubry, Murphy and Wang53)}. Due to the involvement of these genes in growth and metabolic regulation, their differential DNA methylation could be one mechanism linking paternal obesity with altered offspring growth and metabolism. Separately, infertility has been associated with differential sperm DNA methylation^{(Reference Lujan, Caroppo and Niederberger54)}, histone distribution^{(Reference Wang, Gao and Li55)} and RNA content^{(Reference Bianchi, Boekelheide and Sigman56)}. Interestingly, mature sperm contain several different populations of RNA, both within the nucleus and the mitochondria^{(Reference Miller and Ostermeier57)}. Sperm RNAs are detectible within the fertilised oocyte and can contribute to early embryonic development^{(Reference Krawetz58)}. The significance of sperm RNAs in programming offspring development is exemplified through the observation that the injection of sperm tsRNA fragments isolated from dietary-induced obese mice into control zygotes is able to programme the long-term metabolic ill health in the offspring^{(Reference Chen, Yan and Cao59)}.

Changes to a dietary status not only induce change to the composition of the sperm, it can also impact the non-sperm fraction of the semen, the seminal plasma. The role of the seminal plasma in modulating the maternal inflammatory and immune status during the periconception period has been reviewed in detail recently^{(Reference Morgan and Watkins22,Reference Schjenken and Robertson60)} . However, a relatively unexplored connection is between the seminal plasma microbiome and paternal reproductive fitness. The mammalian microbiome consists of anywhere between 10 and 100 trillion microorganisms and functions in a symbiotic relationship with its host^{(Reference Turnbaugh, Ley and Hamady61)}. Until recently, our view of our microbes has centred on their role in pathogenic processes. However, it is now widely accepted that our body's microbiota is central in many developmental, physiological, metabolic and even psychological areas of everyday life. Our bodies possess many different and diverse bacterial populations including our skin, gut and oral microbiomes. Due to modern advances in sequencing capabilities, we are beginning to understand the association between our microbiota and complex conditions such as inflammatory bowel disease^{(Reference Frank, St Amand and Feldman62)} and even obesity^{(Reference Ley, Turnbaugh and Klein63)}. The interplay between our microbiota and reproductive health has come to the forefront over the past few years with the discussion regarding the sterility of the intra-uterine environment^{(Reference Stinson, Boyce and Payne64)}. Gaining a better insight into the parental interplay between the maternal reproductive tract and the developing fetus is critical for developing new biomarkers for gestational well-being and both maternal and offspring long-term health. Initial studies into the seminal microbiome focused on the detection of pathogenic bacterial species, using comparatively simple techniques such as microscopy and RT-qPCR. In some of the earlier studies, negative associations between the levels of Anaerococcus and semen quality were reported^{(Reference Hou, Zhou and Zhong65)}. In a separate study, semen samples identified as ‘normal’ within a clinical setting were populated predominantly with Lactobacillus, while samples of ‘low quality’ displayed a predominance of Prevotella ^{(Reference Weng, Chiu and Lin66)}. One influence of bacteria on male reproduction stems from the toxic effects of inflammatory cytokines or ROS produced by them within the male reproductive tract^{(Reference Alahmar67)}. In addition, bacteria may also bind directly to the sperm, influencing motility or inducing apoptosis^{(Reference Calogero, Duca and Condorelli68)}. Prebiotics supplementation in both human and animal models has been shown to influence seminal plasma composition and sperm quality. In obese mice, supplementation with Lactobacillus rhamnosus PB01 (DSM 14870) improved sperm kinetics^{(Reference Dardmeh, Alipour and Gazerani69)}. The authors observed increased testosterone levels and sperm with higher velocity and motility in supplemented obese males than non-supplemented obese males^{(Reference Dardmeh, Alipour and Gazerani69)}. In men, increased sperm motility, reduced sperm DNA fragmentation and intracellular H₂0₂ levels have also been reported following L. rhamnosus CECT8361 and Bifidobacterium longum CECT7347 supplementation in asthenozoospermic males^{(Reference Valcarce, Genoves and Riesco70)}.

Not only can the seminal microbiome influence male reproductive health, unprotected sexual intercourse can result in the exchange of microbes between partners, suggesting that each partner's reproductive microbiota can affect that of the other. Factors such as frequency of sexual intercourse and number of partners can all be related to the vaginal microbiota and incidences of bacterial vaginosis^{(Reference Brotman, Ravel and Cone71,Reference Plummer, Vodstrcil and Danielewski72)} . Therefore, it is conceivable that the male's metabolic status at the time of conception could influence his seminal microbiome, which in turn influence the female reproductive microbiota. As the female reproductive microbiota is directly related to that of the neonate^{(Reference Mueller, Bakacs and Combellick20)}, this offers a novel mode of paternal programming of offspring metabolic health. However, such direct demonstration of seminal microbiota paternal programming has yet to be demonstrated.