Biosolids and Male Reproduction

Abstract

Over the past 50 years there has been a concerning decline in male reproductive health and increase in male infertility which is now recognised as a major health concern globally. While male infertility can be linked to some genetic and lifestyle factors, these do not fully explain the rate of declining male reproductive health. Increasing evidence from human and animal studies suggests that exposure to chemicals found ubiquitously in the environment may in part play a role. Many studies on chemical exposure however, have assessed the effects of exposure to individual environmental chemicals (ECs), usually at levels not relevant to everyday human exposure. There is a need for study models which reflect the “real-life” nature of EC exposure. One such model is the biosolids treated pasture (BTP) sheep model which utilises biosolids application to agricultural land to examine the effects of exposure to low level mixtures of chemicals. Biosolids are the byproduct of the treatment of wastewater from industrial and domestic sources and so their composition is reflective of the ECs to which humans are exposed. Over the last 20 years the BTP sheep model has published multiple effects on offspring physiology including consistent effects on the male reproductive system in fetal, neonatal, juvenile, and adult offspring. This review focuses on the evidence from these studies which strongly suggests that low-level EC exposure during gestation can alter several components of the male reproductive system and highlights the BTP model as a more relevant model to study real-life EC exposure effects.

Introduction

The last 50 years has witnessed a concerning decline in male reproductive health, including a significant increase in the incidence of congenital genital malformations such as hypospadias and cryptorchidism as well as testicular germ cell carcinoma (Giwercman et al, 1993; Toppari et al, 2001). Several studies published in recent years have also indicated a concerning decline in sperm quality and concentration over the same period (Cannarella et al, 2021; Carlsen et al, 1992; Levine et al, 2017). Male infertility is responsible for 20–30% of infertility cases worldwide (Agarwal et al, 2015) which has resulted in an increase in the number of couples relying on assisted reproductive techniques. Taken together, male reproductive health/infertility is now recognised globally as a major health issue. While male infertility has been linked to numerous genetic and lifestyle factors, approximately 30% of cases are idiopathic (Sinclair, 2000). Given that the rate at which male fertility is declining is too quick to be explained by genetic drift, and the decline in male reproductive health coincides with the increase in industrialisation, anthropogenic activities and significantly increased pollution, it has been speculated that environmental factors may play a role in the deterioration in male reproductive health (Skakkebæk et al, 2022). In the last 50 years over 140,000 new chemicals have been produced and released into the environment. Some of these chemicals, known as endocrine disrupting chemicals (EDCs), are of particular concern and are recognised as a global problem for environmental and human health. EDCs are defined as exogenous chemicals or chemical mixtures which, once they enter the body, have the ability to interfere (e.g. mimic or block) the release or action(s) of endogenous hormones, altering normal function of the endocrine system (Zoeller et al, 2012). Most EDCs are lipophilic and can accumulate in adipose tissue, where they can be stored and thus have a long half-life in the body. During periods of increased energy demand, these stored EDCs can be then mobilised leading to delayed and or concentrated exposure.

With regard to effects on male reproduction, many EDCs have estrogen-like or anti-androgenic effects. The negative effects of EDCs on spermatogenesis and male fertility, have been widely investigated and demonstrated in many clinical experiments and epidemiological studies (Gore et al, 2015). EDCs come from a range of anthropogenic sources and are found ubiquitously in the environment. They include bisphenol A (BPA) found in food packaging, phthalates found in plastic packaging, lubricating oils, personal care products, polychlorinated biphenyls (PCBs) from industrial manufacturing, polybrominated diphenyl ethers (PBDEs) used in flame retardants, organophosphates (OPs) in pesticides and heavy metals (Cannarella et al, 2023) to name a few. Studies that have examined the effects of adult exposure to some of these chemicals on semen parameters have yielded a range of outcomes. For example, BPA exposure has been negatively associated with sperm concentration, sperm motility and total sperm count (Adoamnei et al, 2018; Hu et al, 2017; Radwan et al, 2018). There is moderate evidence of an inverse relationship between exposure to di(2-ethylhexyl)phthalate (DEHP) and dibutyl phthalate (DBP) anogenital distance (AGD) and (Radke et al, 2018) and a positive association between mono-butylphthalate (MBP) exposure and anopenile distance (Arbuckle et al, 2018). However, other studies have shown no association between semen quality and urinary phthalate metabolite concentrations (Albert et al, 2018), and in another cohort study, urinary phthalate metabolites were negatively associated with sperm concentration and sperm motility but positively associated with semen volume and progressive motility (Chen et al, 2017). Serum concentrations of polychlorinated biphenyl (PCB) congeners PCB-118 and PCB-77 have been shown to have both negative and positive associations with semen volume and progressive motility (Paul et al, 2017). Concentrations of PBDEs in hair show no association with any semen parameters (Albert et al, 2018) whereas PBDE concentrations in house dust have been found to be negatively associated with sperm concentration, total sperm count and progressive motility and viability (Yu et al, 2018). While these studies indicate that the effects of adult exposure to EDCs are equivocal, and may be dependent on the specific chemicals, the observed variability of effects may also, in part, be explained by the timing of exposure.

It is well recognised that the timing of exposure to EDCs is important in terms of effects. The normal development of the male reproductive system and external genitalia in-utero is critically dependent on the orchestrated release and action of androgens (e.g. testosterone) and other growth factors (e.g. insulin-like growth factor 3, INSL3). It is evident from both human and animal studies that this is a sensitive period during which time adverse events that alter the hormonal environment required for normal male hypothalamic-pituitary-gonadal development have long lasting consequences on adult reproductive health and fertility. Evidence from animal studies has shown that exposure to anti-androgens during this sensitive programming window can induce reproductive abnormalities such as those seen in human males including hypospadias, cryptorchidism and altered penile length (Welsh et al, 2008). EDC exposure during this sensitive window can also have effects that may not manifest until later in adulthood. The “Developmental Origins of Health and Disease” (DOHaD) hypothesis was underpinned by studies on maternal nutrition during pregnancy which altered the fetal environment and was associated with later risk for cardiovascular disease in adult offspring (Barker et al, 1989). More recently it has become recognised that a range of other health problems which manifest after birth, including male reproductive dysfunction, also have a developmental origin as the result of altered fetal programming by factors including EDCs (Rodprasert et al, 2021; Skakkebæk et al, 2001). Furthermore, there is evidence that effects of embryonic exposure to EDCs (vinclozolin and methoxychlor) during gonadal sex determination can have transgenerational consequences on male reproduction and sperm production (Anway et al, 2005). Human epidemiological studies which have examined the effects of prenatal exposure to certain EDCs on male reproductive function have again shown a range of effects. For example, maternal phthalate exposure has been associated with impaired Leydig cell function evidenced by lower total and free testosterone/luteinizing hormone (LH) ratios in a longitudinal Danish mother-child study of 100 young men aged between the years of 18–20 (Henriksen et al, 2023) and another study showed maternal serum concentrations of phthalate diesters were associated with reduced semen volume and motility in sons at 20–22 year of age and maternal serum BPA (during gestation) has been associated with increased sperm concentration and motility (Hart et al, 2018). The correlation between EDC exposure and declining male reproduction is still unclear, except for some rare cases of occupational or environmental accidents for example, where men with perinatal exposure to dioxin in Seveso, Italy (Mocarelli et al, 2011) or prenatal exposure to PCBs and polychlorinated dibenzofurans in Taiwan (YuCheng accident) (Guo et al, 2000) have shown a reduced semen quality. These exposure scenarios, however, are not representative of “everyday” human chemical exposure as most EDCs are only present in the environment at low individual concentrations. However, it is increasingly recognised that repeated exposure to low level of chemicals over time, or exposure to mixtures of chemicals even at low individual concentrations, can have additive or synergistic effects and/or within a chemical mixture ECs can be metabolized in vivo into derivatives with different modes of action to the parent chemical (Hass et al, 2012; Hass et al, 2017; Martin et al, 2021). So, while previous studies have been useful in establishing the link between prenatal EDC exposure and male reproductive health, interpretation of these studies in terms of the effects of “everyday” exposure to chemical mixtures is extremely difficult.

Rather than focusing on the effects of individual EDCs, there is a critical need to assess the effects of exposure to the real-life environmental chemical (EC) mixtures, which comprise multiple chemical classes, with multiple modes of action. Assessing risk from exposure to EC mixtures poses many unique problems however, including the near infinite number of possible chemical combinations, interactions between ECs within mixtures, and the attribution of responses to component chemicals and their metabolites (Bopp et al, 2019). Conventional chemical risk assessments are primarily based on testing individual chemicals to determine the “No Observable Adverse Effect Level (NOAEL)” usually in altricial species such as rodents, or cell-based assays, at doses which are orders of magnitude higher than that of human exposure. While NOAEL is invaluable in terms of individual chemical toxicological assessment, the toxicological profile of a mixture of chemicals can’t be determined by adding the NOAELs of each component. Some studies of the potential risk from exposure to mixtures of ECs have calculated risk using mathematical models of mixture toxicity based on the analysis of individual chemicals (Elcombe et al, 2022a), however, these calculations also do not account for synergistic or antagonistic interactions between chemicals where each chemical in a mixture is below the NOAEL. This is important because human exposure to ECs including EDCs, is not to single ECs but is chronic exposure to mixtures of ECs at low concentrations over multiple life stages. Given the lack of empirical data over the risk assessment of chemical mixtures, this has led to urgent calls for more appropriate assessment of the effects of exposure to low level EC mixtures, using more relevant experimental models to better understand their impact on human health including male reproductive health (Ribeiro et al, 2017). This review aims to highlight the results of several studies which have used a unique, “real-life” precocial animal model of EC exposure- the biosolids treated pasture (BTP) sheep model, to determine the effects of prenatal, low-level exposure to EC mixtures with a focus on the effects on male reproductive morphology and function.

Biosolids

All wastewater from domestic and industrial sources ends up at wastewater treatment plants where the aim of treatment is to recover water with a high enough quality to be released back into the environment, e.g. through rivers, or to be utilised again. Wastewater treatment is roughly organised into 4 process levels: preliminary treatment to remove coarse suspended materials and sand (Von Sperling, 2007), primary treatment to remove settleable solids and organic matter and secondary treatment to remove most of the organic matter and nutrients through biological processes (i.e. trickling filters, aeration tanks). Finally, tertiary treatment which usually involves advanced methods, such as ozonisation and ultraviolet radiation, the aim of which is removal of pathogens and pollutants (Von Sperling, 2007). In principle, the absence of chemical substances and organisms harmful to health are primary quality requirements for treated wastewater, irrespective of its subsequent use (Von Sperling, 2007). Sludge destined for application in agriculture undergoes further thermal drying to form biosolids. With an increasing number of wastewater treatment plants, over the last decade there has been a parallel increase in the production of biosolids (11.5 million tons d in 2010, 13.5 million tons in 2020, in the European Union alone) (Eurostat, 2017). Following the ban of dumping sewage sludge at sea in 1989, in the UK, the Sludge (Use in Agriculture) Regulations (SUiAR) were introduced to enact the European Commission Sludge Directive 1986 and to enable the spreading of sewage sludge to agricultural land. In the USA, biosolids use is regulated by the Environmental Protection Agency (EPA) Federal Water Pollution Control Act (Clean Water Act) and Code of federal regulations “Standards for the Use or Disposal of Sewage Sludge, 1993”. the application of processed biosolids to agricultural land, as a fertiliser, has thereafter been promoted globally as a cost-effective route for recycling the byproduct of wastewater treatment. Annually, biosolids are applied to around 150,000 ha in the UK, which equates to approximately 1.3% of UK farmland (BAS, 2019). Due to the origins of biosolids, they contain a diverse range of anthropogenic chemicals including heavy metals, personal care products, veterinary medicines and pharmaceuticals, BPA, phthalates, perfluoroalkyl and polyfluoroalkyl substances (PFAS), phthalates, flame retardants (FRs) like polybrominated biphenyl diethers (PBDEs) and organophosphorus flame retardants (OPFRs (Clarke & Smith, 2011)). The UK code of practice sets out statutory testing and monitoring of levels of potentially toxic elements (PTEs) in biosolids and soil, though these are mainly heavy metals and fluoride, while the EPA code has statutory monitoring of some additional pollutants e.g. dioxin and dioxin-like compounds, PCBs and aldrin/dieldrin in addition to several heavy metal pollutants. Importantly there is currently no statutory assessment of other anthropogenic chemicals including EDCs in biosolids or soil following application of biosolids to pasture in the UK or USA. While the UK and USA regulations have a 3 week, and 30 day, respectively, “no grazing” period for animals on pasture following biosolids application, the reason for these timeframes appears to be arbitrary rather than being based on scientific evidence of the fate of ECs in soil following the application of biosolids. The evidence that is available would suggest that many pollutants are still present in the soil at least 3 weeks after biosolids spreading albeit at very low individual concentrations. In 2023, a strategy for a new regulatory framework was devised by the UK government which will bring sludge/biosolids use under the Environmental Permitting (England and Wales) Regulations (EPR). One of the cited reasons for this change is that “they (SUiAR) do not take into account: modern risk-based regulation, the chemical complexity of current biosolids/sludge, the potential environmental impact such as from new hazards” however the new regulatory framework is still being developed.

ECs in biosolids

The application of biosolids to agricultural land can be a beneficial as it provides essential nutrients and organic matter and to improve soil properties (Clarke & Smith, 2011). However, biosolids are a sink for industrial and domestic chemicals including ECs that become sequestered in solids during wastewater treatment (Chari & Halden, 2012). Biosolids contain a complex mixture of ECs derived from a range of different sources including many of the ECs listed above and nanomaterials (Dalahmeh et al, 2018; Death et al, 2021; Maddela et al, 2022a; Maddela et al, 2022b). Most of these ECs have been widely reported in the soil (see Table 1)(Biel-Maeso et al, 2018; García Valverde et al, 2021; Rhind et al, 2013; Sun et al, 2016; Wu et al, 2012) and in diverse food crop plants following biosolids application to land (Christou et al, 2019; Hui et al, 2021; Li et al, 2015; Zhang et al, 2015). Importantly, Rhind et al 2013 demonstrated that a single applications of sewage sludge was sufficient to increase soil concentrations of some ECs, but repeated sludge applications, over 13 years, increased soil burdens of all of the EC groups measured, including all of the PBDE congeners that were assessed, to concentrations that may exert biological effects when different ECs act in combination. The study also concluded that changes in concentrations of DEHP and PBDEs 47 and 99 remained elevated for more than three weeks after application (when grazing animals are normally excluded from pasture). Studies by the same group also analysed pollutants in the tissues of animals grazed on pastures following sewage sludge application and showed measurable levels of some EDCs including PCBs, PBDEs and polycyclic aromatic hydrocarbons (PAHs) (Rhind et al, 2011), alkylphenols and phthalates (Rhind et al, 2005). Phthalates were also detectable in milk samples from animals grazed on BTP (Rhind et al, 2007) however these studies also found detectable levels of some EDCs in tissue samples from animals grazed on conventionally fertilised pastures. These results highlighted that many ECs are found ubiquitously in the environment and as such there is no clean ‘control’ that can be used and it should also be remembered that studies have often taken a targeted approach to assess EC content and have thus only measured a small number of the wide range of ECs that are present in biosolids. While the soil and grass burden of contaminants is difficult to quantify due to the need for chemical extraction and the complex interactions that can occur between the contaminants and soil/grass constituents, prevailing evidence shows that application of biosolids on agricultural land results in contamination of soils by a range of ECs, and that animals that graze on BTP are thus chronically exposed to low-level mixtures of anthropogenic chemicals including ECs.

Table 1.

Concentrations of different classes of selected ECs (range, mean±SD, or maximum) measured in agricultural soil and following application of biosolids/sewage sludge. The list in non-exhaustive. Data is taken from references in the “ECs in biosolids” section. PPCP = pharmaceuticals and personal care products.

Chemical	Concentration in Soil (dry matter)	Chemical	Concentration in Soil (dry matter)
PAHs (μg/kg)		PCBs (ng/Kg)
Naphthalene	26.3	28	1259
Acenaphthalene	3.63	52	95.5
Acenaphthene	4.79	101	224
Fluorene	6.03	118	275
Phenanthrene	89.1	138	309
Anthracene	12	153	447
Fluoranthene	132	180	219
Pyrene	87.1	PFAS substances (pg/g)
Benzo[a]anthracene	49	PFHxA	350±77
Chrysene	135	PFHpA	210±49
Benzo[b]fluoranthene	219	PFOA	410±90
Benzo[k]fluoranthene	55	PFOA	0.05–1.57*
Benzo[a]pyrene	95.5	PFNA	430±370
Indeno[1,2,3 cd]pyrene	75.9	PFUnDA	280±220
Dibenzo[a,h]anthracene	21.4	PFDoDA	120±42
Benzo[ghl]perylene	93.3	PFTriDA	95±92
PBDEs (ng/kg)		PFBS	95±54
28	49	PFOS	2600±360
47	5888	PFOS	2−483*
99	7762	MeFOSAA	235±95
100	1738	EtFOSAA	450±200
153	933	Combined PFOS and PFOA	6300†
154	741	PPCPs (ng/g)
183	224	Carbamazepine	94.7±13.7
BPA (ng/g)	12.89–167.9	Diphenhydramine	157±10.7
Phthalate esters, ng/g (max)		Triclocarban	820±88.0
DEHP	219*	Sulfamethoxazole	4.30*
DEHP	9190	Diclofenac	n.d.–5.60
DMP	71	Acetaminophen	n.d.– 5.95
DEP	90.6	Mefenamic acid	0.09–1.97
DiBP	474	Phenazone	n.d.–0.36
DnBP	1500	Caffeine	0.51–3.21
DMEP	40.1	Ornidazole	n.d.–0.47
BMPP	9.9	PBDEs (μg/Kg)
DEEP	151	∑BDE-47, BDE-99, BDE-209	15.20*
DPP	151	∑13PBDEs	<1.0–382*
DnHP	7.4	∑ OCPs, ng/g (max)	3520
BBP	12.2	p,p′-DDT, ng/g (max)	3240
DBEP	680
DCHP	265
DNOP	273

^*values are μg/Kg; †values are ng/g

Biosolids treated pasture sheep model of EC exposure

The mixture of ECs found in biosolids is representative of the human exposome (Rigby et al, 2021) and thus grazing genetically diverse (outbred) precocial animal species on BTP provides a real-life experimental model that is comparable to human exposure to EC mixtures. The BTP exposed sheep model is based on an experimental BTP which has been fertilised with biosolids at conventional spreading rates (4 tonnes/Ha twice per year in spring and autumn) over several years, and for comparison, a geographically separate pasture (to prevent run-off contamination) which is conventionally fertilised maintaining equivalent nitrogen levels but is exposed to the same environmental factors. The period and duration of grazing on BTP and control pastures can be easily manipulated and compared. BTP experimental sites were initially established by Stewart Rhind and colleagues at the James Hutton Institute (UK) in 1994 as part of the “Long Term Sludge Experiment (LTSE) at Hartwood”, a part of the UK Sewage Sludge Network to investigate the effects of adding treated waste water sludge to agricultural land, and the potential risk posed to grazing animals (Evans et al, 2014; Rhind, 2005). These studies were crucial in characterizing the presence and accumulation of pollutants, including EDCs, in the soil (Aitkenhead et al, 2014; Rhind et al, 2002; Zhang et al, 2015; Zhang et al, 2011) and tissues (Rhind et al, 2011; Rhind et al, 2007; Rhind et al, 2005) of animals following sludge application to pasture. In 2014, Bellingham and colleagues established the current experimental biosolids site at the University of Glasgow Cochno Farm and Research Centre, to continue the earlier work of Rhind with a focus on the impacts of maternal grazing of BTP on the offspring, with a particular focus on reproductive and metabolic health. Despite the potential variability in EC content in biosolids across batches, as well as potential variation in grazing patterns between animals, a wealth of studies over the last 20 years from Bellingham, Rhind and colleagues have documented measurable effects of mixed EC exposure in animals, and the F1 offspring of animals grazed on BTP during specific life stages (e.g. preconception only, during gestation and lactation only, or continuously before and during gestation/lactation) compared to animals grazed on control pastures. These studies have shown effects on several organs and physiological systems in all ages of offspring examined (fetal, prepubertal, adult). These include, in pregnant ewes, disruption of the metabolome (Thangaraj et al, 2023a) steroidal, inflammatory and oxidative milieu (Thangaraj et al, 2023b) which are influenced by fetal sex; in fetal offspring (110 days gestation), reduced bodyweight, altered thyroid gland structure, altered neuroendocrine regulatory centres and altered ovarian proteome and number of atretic follicles (Bellingham et al, 2013; Bellingham et al, 2016; Hombach-Klonisch et al, 2013; Lea et al, 2016; Paul et al, 2005); in juveniles, sex-specific effects on growth and body-weight (Evans et al, 2023) and emotional reactivity and exploratory behaviour (Erhard & Rhind, 2004); in adults, altered bone mineral density (Lind et al, 2009; Lind et al, 2010), altered liver lipid concentrations (Filis et al, 2019) and disrupted metabolic markers in the hypothalamus and adipose tissue (Ghasemzadeh-Hasankolaei et al, 2024). In addition to the effects listed above, one of the most consistent observations from BTP studies has been multiple effects on the reproductive system of males, in all ages examined, born to ewes exposed to BTP during gestation. The rest of this review will focus on studies using the BTP sheep model and the effects that have been observed on the male reproductive system.

Effects of gestational exposure to ECs via maternal grazing BTP on male reproduction

Effects in fetal offspring

It is well known that the fetal period is a crucial time when the male reproductive system is developing and sensitive to exogenous influences including the action of ECs which can disrupt normal development and affect function and fertility in adulthood. Studies of the hypothalamus, pituitary and testes of 110 day gestation (GD110) fetal offspring from ewes that were continuously exposed to BTP both prior to, and throughout gestation have documented alterations to the hypothalamic-pituitary axis with reduced kisspeptin gene expression and protein levels in the hypothalamus and pituitary and fewer kisspetin immunopositive cells colocalized with both LH beta and ER alpha in the pituitary gland (Bellingham et al, 2009). GD110 fetal offspring also had significantly reduced testes weights, fewer Sertoli cells, Leydig cells, and gonocytes, and reduced androgen receptor expression (Paul et al, 2005). In these animals it was also reported that circulating testosterone concentrations were reduced in BTP exposed fetuses. While the phenotypic effects in the fetal gonad reported by Paul et al (2005) could not be directly linked to the neuroendocrine alterations observed by Bellingham et al 2009, alterations in the expression of reproductive neuroendocrine regulatory genes during fetal life could impact on fetal testicular structure and function and such effects could affect post-natal reproductive function and fertility.

Studies using the BTP model have also examined how the timing of maternal exposure affects the male reproductive system in the late fetal animal (Bellingham et al, 2016; Lea et al, 2022). Specifically, GD110 fetal offspring from 4 separate groups of ewes with varying exposure to BTP or control pastures has been studied: 1) ewes exposed to control pastures both before conception and during gestation (CC) 2) ewes exposed to BTP prior to conception and throughout gestation (TT), these groups were comparable to the control and continuous exposure studies described above. 3) ewes exposed to BTP prior to conception, then maintained on control pastures during gestation (TC). One study showed that there was reduced hypothalamic GnRH and KISS1 mRNA expression in the prenatal-only exposure group (TC) and the continuously exposed (TT) group. In a separate experiment (Lea et al, 2022) Lea et al, examined the fetal testes at day 80 (2 cohorts, ewes exposed to C or BTP pastures) or day 140 (5 cohorts, 0-GD140 continuous BTP exposure; 0-GD80 early exposure; GD30 to GD110 mid-exposure; GD60 to GD140 late exposure and Control group 0 to 140 no BTP exposure). The study found that fetal testes from GD140 pregnant ewes that were exposed transiently for 80-day periods during early (0–80 days), mid (30–110 days), or late (60–140 days) pregnancy had fewer Sertoli cells and reduced testicular area stained for CYP17A1. Fetal testes from continuously exposed ewes were either unaffected at day 80 or exhibited a reduced area of testis immunostained for CYP17A1 protein at day 140.

The Bellingham et al, 2016 and Lea et al, 2022 studies both highlighted that the timing of fetal exposure may affect the later phenotype, so that even short exposures during sensitive developmental windows can induce effects, which may be greater than longer exposures.

Effects in juvenile offspring

The results by Lea et al 2022 suggested that longer term BTP exposure was associated with the fewest alterations in the male fetal testis and could suggest that there are compensatory mechanisms that exist that might mitigate effects later in life. In addition, it could be that the reproductive system may be able to recover or remodel where fetal EC exposure is not followed by exposure in post-natal life. Indeed, it is known that the neonatal and prepubertal life stages are crucial periods of developmental plasticity. Despite the observation of clear effects of EC exposure during fetal life, it is not possible to predict if or how alterations in the male fetal reproductive system observed as a result of gestational EC exposure, may manifest at later life stages. Therefore, studies using the BTP model have also investigated whether effects observed in the fetal male reproductive system continue to be evident after birth, in neonatal and prepubertal male offspring of ewes exposed to BTP one month prior to, and throughout gestation. Elcombe et al 2021, reported that in one day old male lambs, maternal BTP exposure was associated with morphological differences in testicular structure which are like those seen in human testicular dysgenesis syndrome (TDS), including a greater proportion of Sertoli cell-only seminiferous tubules, and fewer gonocytes (Elcombe et al, 2021). Plasma testosterone concentrations were also lower in the one day old BTP lambs compared to control animals, a result which agrees with the findings of Paul et al in the fetus (Paul et al, 2005). Transcriptome analysis in the neonatal testes highlighted reduced mammalian target of rapamycin complex (mTORC) signalling in BTP rams which has been shown to cause autophagy in response to xenobiotic induced mTORC attenuation and may explain the observed reduction in gonocyte number in these animals. As gonocytes are germ cells responsible for the production of spermatogonial stem cells (SSCs) later in life, reduced numbers in the neonatal lambs may impact spermatogenesis in adulthood. Elcombe et al 2021 also reported that there were different severities of the testicular phenotype in the BTP exposure group which were correlated with differential changes in testis transcriptome between BTP exposed males that were phenotypically “normal” or “abnormal”. The observation that some individuals thus appeared to be “susceptible” while others were “resistant” to the effects of EC exposure may be a consequence of the outbred nature of the study population and reflect individual (genetic) differences in EC action, metabolism and/or elimination.

Studies in prepubertal rams have indicated that a TDS phenotype is also maintained in males born to BTP grazed mothers despite chemical exposure only occurring during their prenatal development. Elcombe et al 2022 (Elcombe et al, 2022b) examined the morphology and transcriptome of gestationally BTP exposed prepubertal (8-week-old) male testes and observed adverse morphological and potential functional outcomes of BTP exposure. The results, which agreed with those seen in the neonatal male testis indicated that the testis of prepubertal males exposed to BTP during pregnancy contained fewer germ cells and had a greater proportion of Sertoli-cell-only seminiferous tubules. Moreover, alterations in the testicular transcriptome of BTP males, were shared with those seen when comparing the transcriptome of human testicular dysgenesis syndrome (TDS) patients and controls, including changes in genes which are involved in apoptotic and mTOR signalling. The study in the prepubertal BTP males also reported increased HIF1α activation and nuclear localisation in Leydig cells of BTP exposed animals. As HIF1α is reported to disrupt testosterone synthesis, these results provide a potential mechanism for the pathogenesis of this testicular phenotype, and TDS in humans. Comparison of the testicular transcriptomic profiles from fetal, neonatal, and pre-pubertal testes, suggest that multiple pathways may be perturbed across life following in utero EC exposure, and that alterations in mTOR and HIF1A may be key changes through which ECs can alter testicular function and later fertility.

Yet to be published results in prepubertal BTP males from the same study, also show greater testicular expression HSD3B1, HSD17B3, BMP4 and LH receptor and lower expression of androgen binding protein (ABP). They also show higher expression of GnRH and lower expression of NKB, and KISS1 in the. The results from this study also identified subgroups of the BTP animals which were more affected by EC exposure than others, as reported in (Elcombe et al, 2022b). GnRH and Kisspeptin are key neuroendocrine factors that regulate pubertal timing. A related study from the same experiment (Evans et al, 2023) temporally studied a separate cohort of male offspring from BTP ewes to assess the effect of exposure on the timing of puberty and found that despite having a smaller testis volume compared to controls in the initial growth stages (weeks 21–29) BTP males had higher concentrations of circulating testosterone, suggestive of earlier testicular activation and earlier puberty onset. Together these results suggest that prenatal EC exposure induces changes in hypothalamic-pituitary neuroendocrine regulators which are maintained or expressed in post-natal life such that alterations in testis morphology and pubertal timing may be explained by the effects of exposure to low level complex EC mixtures.

Effects in adult offspring

Bellingham et al 2012 (Bellingham et al, 2012) examined the testis in adult males (18 months old) following maternal EC exposure (gestational and lactational), and then direct exposure grazing on BTPs until 7 months old. The study found that testicular morphology was significantly altered with BTP rams showing a higher occurrence of Sertoli-cell-only seminiferous tubules, and lower numbers and volumes of germ cells (Bellingham et al, 2013). Interestingly these effects were not consistent in all animals, with only a subset of animals (5 of 12) showing a markedly altered phenotype, which is synonymous with the previous observed effects in the testes of the neonatal animals and may reflect the effects of EC exposure against the diverse genetic background of an outbred study population. In a later study Elcombe et al 2023 found that the testes of adult (11 month old) males born to ewes grazed on BTP one month prior and throughout pregnancy, had a higher proportion of seminiferous tubules at an early stage of spermatogenesis compared to rams born to control ewes but relative to the earlier study in adults (86), fewer significant phenotypic abnormalities were present. The different phenotypes observed between Bellingham et al 2012 and Elcombe et al 2023 could be explained if in the absence of post-natal EC exposure there is a “recovery”, but where there is longer term continuous BTP exposure (gestationally, and for 7 months during post-natal life, i.e., lactational / direct oral exposure) that results in the persistence and exacerbation of effects seen in utero and the development of a TDS-like phenotype in adulthood.

Limitations of BTP sheep model

The BTP sheep model is a relevant animal model to understand the effects of exposure to low-level, complex EC mixtures, akin to human EC exposure, but as with any model system it is not without its limitations. First, is the potential variation in the EC profile between batches of biosolids applied to pasture. As biosolids are made up from waste from a variety of anthropogenic sources there will be changes and differences in the EC profile both spatially and temporally. As a translational model, however, this could also be viewed as a strength, as the pattern of human EC exposure is also not constant and will vary with location and across time. Further it is worthy of note that we have been working with the BTP model for over 20 years and despite potential variation in the EC content of biosolids, as noted above some effects of grazing on BTP pasture appear to be consistent. Second, is that it is not possible to determine what each animal has been exposed to in terms of chemical mixtures. This is not only related to variation in biosolids EC profile, but also because the grazing pattern of individuals may vary across a site and the concentration and mixture of ECs across grazing areas may vary as a result of environmental conditions. As noted above this variation may again better replicate the natural variation that is seen in human exposure as we go about our normal business. Even if it were possible to capture individual grazing patterns, it is impossible to quantify the entire plethora of chemicals that the animals are exposed to and to consider how those ECs interact with one another and how they are metabolised within an individual. Thirdly, in the above studies the effects of grazing on BTP are compared to control pasture that has been treated with a conventional inorganic fertiliser, but due to the ubiquitous nature of ECs, that pasture is not a true “negative control” or “clean” site that is devoid of ECs. Indeed, the Rhind et al (2002 and 2013) studies reported that some ECs are also detectable in soil from “control” pastures albeit at lower concentrations. Thus, the comparison between both sites should always be considered as biosolids-treated versus conventionally fertilised which vary in the level of EC exposure. Despite the limitations mentioned, the BTP sheep model remains a more relevant animal model to examine the effects of real-life exposure in an outbred population.

Conclusion

It is clear from the studies using the BTP model discussed in this review, that low level exposure to EC mixtures during gestation has impacts on the male reproductive system across multiple life stages, relative to animals grazed on untreated pasture (see summary Figure 1). These studies have shown effects in fetal, neonatal, prepubertal and adult males at all levels within the hypothalamo-pituitary-gonadal axis. Interestingly, effects of prenatal EC exposure through maternal grazing on BTP at the level of the testes result in similarities in phenotypes and common transcript changes to those seen in human male testicular dysgenesis patients. These studies provide additional evidence that exposure to low level EC mixtures may explain, or contribute to, the increasing incidence of reproductive disorders and declining fertility in human males. The BTP studies also highlight the importance of the timing and duration of EC exposure relative to sensitive periods of development as this can influence the severity of effects, and that there may be compensatory mechanisms which can mitigate some of the effects of EC exposure.

Figure 1.

Summary of the effects of maternal grazing biosolids treated pasture one month prior to, and throughout pregnancy (gestational exposure, GE) on the reproductive system of male offspring at fetal, neonatal, juvenile (8 week old) and adult ages. Adult offspring (19 months) were also studied in a separate extended exposure (EE) experiment where offspring were exposed during lactation and up to 7 months after birth in addition to GE. Image created using BioRender.

Finally, the use of an outbred animal as the subject species with the BTP model has repeatedly resulted in the generation of different phenotypic cohorts within the population of exposed animals. This is of great interest as it demonstrates that some individual may be more or less sensitive to the effects of ECs. Current studies using the BTP model are assessing the transgenerational impacts of EC exposure on male reproductive health.

It is clear that declining male reproductive health is a global health issue. The role of everyday exposure to environmental pollutants remains a challenge. However, given the increasing human chemical burden, better risk assessment for emerging ECs of concern (eg PFAS) and concern regarding interactions between chemicals within mixtures, their biomonitoring is urgently required.