Biosolids, a by-product of wastewater treatment, are widely used as agricultural fertilizers and contain anthropogenically-derived chemical mixtures, often including obesogens such as perfluoroalkyl substances (PFAS), bisphenols, and phthalates. Given the potential for endocrine and metabolic disruption of biosolids’ complex chemical mixtures, we hypothesized that maternal exposure to biosolids-treated pastures would alter circulating chemical and steroid profiles and that these changes could influence adipogenic differentiation in preadipocytes. To test this, we characterized 125 environmental chemicals across 16 classes using ID-LC-MS/MS and adrenal- and ovarian-derived steroids using LC-MS/MS in plasma from pregnant sheep (n = 6/group) collected at gestational day 30. Biosolids exposure increased total concentrations of several chemical classes, notably phthalates, parabens, bisphenols, and PFAS. Steroid profiling revealed lower plasma pregnenolone and higher 17-OH-pregnenolone, DHEA, 17-OH-progesterone, and 18-OH-corticosterone. Adipogenic effects were assessed by differentiating 3T3-L1 preadipocytes in the presence of plasma. Plasma from biosolids-exposed females significantly increased 3T3-L1 lipid droplet count and BODIPY staining intensity compared to control plasma. No differences were observed between plasma from pregnancies with male vs. female fetuses. Lastly, LASSO regression revealed positive associations between acetamiprid and perfluorobutane sulfonic acid concentrations and lipid accumulation. In conclusion, exposure to biosolids-treated pastures altered chemical and steroid profiles in plasma from pregnant females and increased adipogenic differentiation in vitro, suggesting that circulating factors associated with environmental chemical mixtures may influence adipose development. These findings highlight the need to evaluate the safety of biosolids use and their potential contribution to metabolic risk.

Humans are chronically exposed to a mixture of environmental chemicals (ECs), many with metabolic and endocrine disrupting potential, contributing to non-communicable disease burden. Understanding the effects of chronic exposure to low-level mixtures of ECs requires an animal model that reflects real-world conditions, lags behind studies on single ECs. Biosolids, from wastewater treatment, offers a real-life model to investigate the developmental health risks from EC mixtures. Prenatal biosolids exposure studies have documented metabolic perturbations including heavier thyroid glands in male fetuses and reduced bodyweight in prepubertal male lambs followed by catchup growth. We hypothesized that maternal preconceptional and gestational exposure of sheep to biosolids programs sex-specific transcriptional and functional changes in the offspring liver. Ewes (F0) were grazed on either inorganic fertilizer (C) or biosolids-treated pastures (BTP) preconception till parturition. All lambs (n = 15/group with male n= 7/group and females n=8/group) were raised on Control pastures until euthanasia at 9.5 weeks. Next generation sequencing of liver RNA and DESeq2 was used to identify exposure-specific differentially expressed genes (DEG) and sex-differentially expressed genes (SDG). Liver function was assessed with markers of oxidative stress, triglyceride and fibrosis markers. Control lambs exhibited 647 SDGs confirming the inherent sexual dimorphism in hepatic gene expression. A sex-stratified analysis identified 10 DEG, mostly affecting metabolism, in male and none in female lambs. Biosolids exposure diminished the sexual dimorphism in hepatic gene expression barring 41 genes, potentially due to the increase in androgenic steroids found in F0 maternal circulation. Additionally, BTP male lambs showed elevated plasma triglyceride and a trend towards increased liver triglyceride concentrations. The identified effects of prenatal exposure to low-dose mixture of ECs via biosolids, in a precocial species paralleling human developmental patterns holds translational importance for understanding the sexually dimorphic origin of non-communicable diseases.

Female reproductive capacity is shaped by ovarian reserve and patterns of follicle development. Ovarian reserve depletion occurs by follicle activation and atresia, which are affected by environmental chemicals (ECs). Because humans are simultaneously exposed to hundreds of ECs, real-life exposure models are essential to assess patterns of atresia after EC exposure. Previous findings demonstrate maternal preconceptional and gestational EC exposure via biosolids increases activation rate and reduces primordial follicle pool in juvenile, but not adult sheep. We hypothesized that this shift involves changes in death and proliferative pathways that impact follicle atresia from juvenile to adult life. Ovaries were collected from juvenile (9.5 weeks) and adult (2.5 years) offspring from ewes grazed on biosolids-treated pasture (BTP) or inorganic fertilizer-treated pasture (Control). Follicular atresia was assessed through morphological characteristics and molecular death pathways, including expression of markers for apoptosis (CASP3), autophagy (LC3), ferroptosis (GPX4), and proliferation (Ki67). There were higher levels of apoptosis and autophagy, and lower proliferation, in juvenile BTP offspring compared to controls. In adult BTP offspring, apoptosis and proliferation were similar, autophagy was lower, and ferroptosis was higher compared to controls. Apoptosis was lower and ferroptosis was higher in adults than juveniles, regardless of treatment. Adult BTP offspring had lower autophagy and similar proliferation levels than juvenile BTP offspring. These findings suggest that lower autophagy and lack of decrease in proliferation contribute to normalization of activation rate and ovarian pool in BTP adult offspring and supportive of lasting impacts of gestational EC exposure on offspring follicular health.

The decline in human reproductive and metabolic health over the past 50 years is associated with exposure to complex mixtures of anthropogenic time and differs across geographical locations. Health-related issues include declining sperm quality, advanced puberty onset, premature ovarian insufficiency, cancer, obesity, and metabolic syndrome. Prospective animal studies with individual and limited EC mixtures support these observations and provide a means to investigate underlying physiological and molecular mechanisms. The greatest impacts of EC exposure are through programming of the developing embryo and/or fetus, with additional placental effects reported in eutherian mammals. Single-chemical effects and mechanistic studies, including transgenerational epigenetic inheritance, have been undertaken in rodents. Important translational models of human exposure are provided by companion animals, due to a shared environment, and sheep

Developmental exposure to environmental chemicals perturbs establishment and maintenance of the ovarian reserve across the reproductive lifetime, leading to premature follicle depletion and ovarian aging. Considering humans are exposed to a complex mixture of environmental chemicals, real-life models assessing their cumulative impact on the ovarian reserve are needed. Biosolids are a source of a real-life mixture of environmental chemicals. While earlier studies demonstrated that grazing pregnant sheep on biosolids-treated pastures did not influence establishment of the ovarian reserve in fetal life, its impact on subsequent depletion of ovarian reserve during reproductive life of offspring is unknown. We hypothesized that developmental exposure to biosolids accelerates depletion of ovarian reserve. Ovaries were collected from F1 juveniles (9.5 weeks) and adults (2.5 years) born to F0 ewes grazed on control inorganic fertilizer pastures or biosolids-treated pastures from before conception and throughout gestation. The impact on follicular density, activation rate, and anti-Müllerian hormone (mediator of activation) expression by immunohistochemistry was determined. Activation rate was increased in F1 biosolids-treated pastures juveniles with a corresponding reduction in primordial follicle density. In contrast, activation rate and ovarian reserve were similar between control and F1 biosolids-treated pastures adults. The density of anti-Müllerian hormone-positive antral follicles was lower in biosolids-treated pastures juveniles, whereas anti-Müllerian hormone expression tended to be higher in antral follicles of biosolids-treated pastures adults, consistent with the changes in the ovarian reserve. These findings of detrimental effects of developmental exposure to biosolids during juvenile life that normalizes in adults is supportive of a shift in activation rate likely related to peripubertal hormonal changes.

Environmental chemicals (ECs) have been associated with a broad range of disorders and diseases. Daily exposure to various ECs in the environment, or real-life exposure, has raised significant public health concerns. Utilizing the biosolids-treated pasture (BTP) sheep model, this study demonstrates that in-utero exposure to a real-life EC mixture disrupts hypothalamo-pituitary-gonadal (HPG) axis gene expression and reproductive traits in prepubertal (8-week-old, 8w) and adult (11-month-old) male sheep. Ewes were maintained on either BTP or pastures fertilized with inorganic fertilizer [control (C)] from approximately one month prior to insemination until around parturition. Thereafter, all animals were kept under control conditions. Effects on reproductive parameters including testosterone concentrations and the expression of key genes in the HPG axis were evaluated in eight-week-old and adult male offspring from both C and biosolids-exposed (B) groups. Results showed that, at 8w, relative to C (n = 11), B males (n = 11) had lower body weight, and altered testicular expression of HSD3B1, LHR and HSD17B3, BMP4, ABP, P27kip and CELF1. Principal component analysis (PCA) identified two 8w B subgroups, based on hypothalamic expression of GnRH, ESR1, and AR, and pituitary expression of KISSR. The two subgroups also exhibited different serum testosterone concentrations. The largest biosolids effects were observed in the hypothalamus of adult rams with NKB, ESR1, KISS1, AR, DLK1 and GNRH1 mRNA expression differing between B (n = 10) and C (n = 11) rams. Testicular steroidogenic enzymes CYP11A1 and HSD3B1 mRNA expression also differed between exposure groups. PCA identified two adult B subgroups, with BS1 (n = 6) displaying hypothalamic effects and BS2 (n = 4) both hypothalamic and testicular effects. The subgroups also differed in circulating testosterone concentrations. These findings demonstrate that exposure to a real-life EC mixture may predispose some males to infertility, by disrupting key functional HPG markers before puberty with consequent downstream effects on steroid hormones and spermatogenesis.

Abstract Disclosure: Y. Zhou: None. K.M. Halloran: None. M. Bellingham: None. N.P. Evans: None. R.G. Lea: None. K.D. Sinclair: None. V. Padmanabhan: None. The ovarian capacity to provide fertilizable oocytes, regulate folliculogenesis as well as activate and progress primordial follicles into preovulatory follicles are key contributing factors to female fertility and reproductive aging. In sheep, as in humans, while genetics play a role in establishing the ovarian reserve during fetal development, developmental insults such as inappropriate exposure to native steroids or environmental chemicals (ECs) can adversely impact the establishment of the ovarian reserve and, additionally can affect folliculogenesis. Considering humans are exposed to multiple ECs simultaneously which could have additive, synergistic, or antagonistic effects, real-life EC exposure models are needed to assess risks posed by EC exposure. Offspring from sheep grazed on biosolids-treated pasture offers one such real-life exposure model. Studies with day 140 fetuses found maternal exposure to a real-life EC mixture via biosolids increased the proportion of unhealthy transitory follicles (Lea et al., Sci Rep. 2016;6:22279). The present study tested if biosolids exposure during fetal life would impact folliculogenesis, follicle activation and follicle atresia during their adult life. Ovaries were derived from adult EasyCare ewes (age 31.4 ± 0.5 months) whose mothers were grazed on biosolids-treated (BTP, n=10) or inorganic fertilizer-treated (Control, n=10) pastures from preconception through to birth. Ovarian morphometry was undertaken to assess the distribution and health status of follicles. Follicular atresia was evaluated via the expression of Caspase-3, an apoptosis marker. Data analysis utilized Student's t-test for normally distributed variables and Mann-Whitney U tests for non-parametric comparisons. There were no differences in the absolute counts or the percentages of follicle classes at all stages of folliculogenesis, the density of primordial follicles in cortical tissue, or the follicular activation rate. An increase in the percentage of unhealthy activated follicles (post-primordial) was noted in the BTP group (p = 0.014), predominantly in the transitory stage (p = 0.021). Consistent with this, a higher density of Caspase-3 positive primary follicles was observed in the BTP group (p = 0.033). These findings indicate the adverse impacts of fetal exposure to an EC mixture via maternal biosolid exposure on the survival of early-stage activated follicles in adult sheep. The lack of difference in ovarian reserve between the control and BTP groups suggests compensatory mechanisms may be in place to prevent premature depletion of the ovarian reserve. Whether EC mixture exposure via biosolids also has an adverse effect on the quality of the surviving activated follicles remains to be determined. Funding source: NIH R01 ES030374. Presentation: 6/3/2024

Abstract Disclosure: K.M. Halloran: None. J.N. Ciarelli: None. Y. Zhou: None. M. Bellingham: None. R.G. Lea: None. N.P. Evans: None. K.D. Sinclair: None. P. Smith: None. V. Padmanabhan: None. Establishment of the fetal ovarian follicular reserve is a determinant of lifelong fertility. Environmental chemicals (ECs) are implicated in perturbation of early follicle development which may negatively impact adult reproductive function. Considering humans are exposed to a complex cocktail of ECs that may have additive, synergistic and/or antagonistic effects, real-life exposure models are needed to assess the impact of mixtures of ECs on ovarian reserve. Biosolids contain a complex mixture of ECs and earlier studies have shown that grazing pregnant sheep on biosolids-treated pastures (BTP) did not influence the ovarian follicular pool of fetal day 110 lambs. In contrast, an increased proportion of healthy primordial and reduced proportion of healthy transitory follicles, without an impact on their cortical density, was observed in late gestation fetuses (day 140). Collectively this suggests that in BTP sheep, while establishment of an ovarian reserve is not affected its depletion across the reproductive life span may be affected. In this study we tested the hypothesis that prenatal biosolid exposure would affect follicular depletion and negatively impact ovarian reserve in postnatal lambs. Ewes were grazed on pastures treated with either inorganic fertilizer (control; C) or biosolids (BTP) beginning a month before mating and throughout gestation until birth. After birth, ewes and lambs were moved to C pastures. Female lambs (10 C and 11 BTP) were euthanized at 8 weeks of age. Ovarian follicles were classified by stage (primordial, transitory, primary, preantral, and antral) and health status. The impact of exposure to mixture of ECs on follicular cortical density (n/mm2), distribution, health status, and activation rate were assessed. Anti-müllerian hormone (AMH) expression, which correlates with the number of growing follicles, was quantified by immunohistochemical staining. BTP lambs had reduced density (P=0.07) and proportion (P=0.001) of primordial follicles. Activation rate was increased in the BTP group (P=0.01). The density of unhealthy follicles was reduced in the primordial (P=0.04) and transitory (P=0.09) stages, but increased in preantral (P=0.02) and antral (P=0.09) stages. The percentage of unhealthy follicles was reduced in transitory (P=0.09), but increased in preantral (P=0.01) and antral (P=0.07) stages. Consistent with this, increased AMH staining intensity was evident in transitory plus primary follicles (P<0.05). Collectively these results provide evidence that lambs born to BTP ewes have a reduced ovarian follicular pool, increased follicular activation, and increased unhealthy activated follicles at 8 weeks of age. These findings demonstrate the detrimental effects of ECs in biosolids on ovarian health leading to premature depletion of follicular pool and raise concerns regarding the quality of activated follicles. Funding: NIH R01 ES030374 Presentation: 6/1/2024

Abstract Disclosure: S. Thangaraj: None. M. Bellingham: None. R. Lea: None. N. Evans: None. K. Sinclair: None. V. Padmanabhan: None. Humans are chronically exposed to a mixture of environmental chemicals (ECs), many with endocrine disrupting potential, contributing to the development of non-communicable diseases. The adverse health effects of exposure to single ECs have been extensively studied but less is known about the impact of chronic exposure to low levels of a mixture of ECs, which is the real-life exposure scenario. Biosolids, derived from human wastewater treatment, closely reflects the human exposome in both the array and concentration of ECs and is a novel, real-life model to investigate the health risks posed by developmental exposure to mixtures of ECs. Previous studies using the prenatal biosolids exposure model found an increased accumulation of the endocrine disrupting chemical diethylhexylphthalate in the fetal liver and metabolic perturbations including increased thyroid gland weight in the male fetus and reduced bodyweight in prepubertal male lambs. To understand the basis of this sexually dimorphic metabolic phenotype we investigated the transcriptome of liver, the primary metabolic organ, in prepubertal lambs of both sexes. Since many liver genes show sexual dimorphism, with sex-biased genes playing a pivotal role in lipid and drug metabolism, sex-differences in liver gene expression and the impact of prenatal biosolids exposure on it were examined. We hypothesized that maternal preconceptional and gestational exposure of sheep to biosolids programs sex-specific transcriptional changes in the offspring liver. Ewes (F0) were grazed on either inorganic fertilizer (C) or biosolids-treated pastures (BTP) from 1 month before mating till parturition. All lambs (F1) were raised on control pastures after lambing until euthanasia at 8 weeks of age. RNA from liver from Control male (n=7), Control female (n=8), BTP male (n=7) and BTP female (n=8) lambs were subjected to next generation sequencing and differentially expressed genes (DEG) identified with DESeq2 R package. A stratified sex-differential analysis identified 702 DEG between control males and females validating the sexual dimorphism in hepatic gene expression. A sex-stratified analysis between C and BTP identified 5 downregulated DEG - WNT2B, LCN2, TEAD4, QSOX1 and ITIH4 in male and none among female lambs. Biosolids exposure dissipated the sexual dimorphism seen in hepatic gene expression in the controls barring 3 genes involved in cell growth/differentiation (WNT2B) and chromatin regulation (KDM6A, SS18). This may relate to the increase in androgenic steroids found in F0 maternal circulation (Sci Total Environ 2023, 900:165674). The findings of dissipation of the sex-differences in hepatic gene expression by real-life exposure to ECs in a precocial model with a developmental trajectory similar to humans is of translational significance in the context of sexually dimorphic origin of non-communicable diseases.Funding NIH R01ES030374 Presentation: 6/1/2024

BACKGROUND:Outcome measures are extensively used within human physiotherapy, but a widely accepted issue in veterinary physiotherapy is that outcome measures lack sufficient evaluation and standardisation in terms of how they are implemented. This cross-sectional study aimed to provide clarity on (1) the current selection of outcome measures in canine and equine physiotherapy and (2) investigate external influences on outcome measure selection, including comparative literature availability, professional memberships and background.METHODS:A structured scoping literature review consolidated current understanding and limitations. This informed a survey of qualified veterinary physiotherapists (n = 40). The statistical analysis comprised descriptive statistics.RESULTS:Key observations included (1) a lack of difference in outcome measure application between veterinary physiotherapists with and without a human physiotherapy background, (2) enhanced outcome measure utilisation by registry body members and (3) an overall skew towards subjective, rather than objective, outcome measure use.LIMITATIONS:The study was limited by the absence of a defined veterinary physiotherapist population and subsequent convenience sample size.CONCLUSION:The apparent skew towards subjective outcome measures highlights objective outcome measure underutilisation and the need for a more extensive evidence base. In conclusion, there is a need to develop comprehensive professional development resources promoting the use of repeatable outcome measures such as goniometers and the Liverpool osteoarthritis scoring.

Over recent decades, an extensive array of anthropogenic chemicals have entered the environment and have been implicated in the increased incidence of an array of diseases, including metabolic syndrome. The ubiquitous presence of these environmental chemicals (ECs) necessitates the use of real-life exposure models to the assess cumulative risk burden to metabolic health. Sheep that graze on biosolids-treated pastures are exposed to a real-life mixture of ECs such as phthalates, per- and polyfluoroalkyl substances, heavy metals, pharmaceuticals, pesticides, and metabolites thereof, and this EC exposure can result in metabolic disorders in their offspring. Using this model, we evaluated the effects of gestational exposure to a complex EC mixture on plasma triglyceride (TG) concentrations and metabolic and epigenetic regulatory genes in tissues key to energy regulation and storage, including the hypothalamus, liver, and adipose depots of 11-month-old male offspring. Our results demonstrated a binary effect of EC exposure on gene expression particularly in the hypothalamus. Principal component analysis revealed two subsets (B-S1 [n = 6] and B-S2 [n = 4]) within the biosolids group (B, n = 10), relative to the controls (C, n = 11). Changes in body weight, TG levels, and in gene expression in the hypothalamus, and visceral and subcutaneous fat were apparent between biosolid and control and the two subgroups of biosolids animals. These findings demonstrate that gestational exposure to an EC mixture results in differential regulation of metabolic processes in adult male offspring. Binary effects on hypothalamic gene expression and altered expression of lipid metabolism genes in visceral and subcutaneous fat, coupled with phenotypic outcomes, point to differences in individual susceptibility to EC exposure that could predispose vulnerable individuals to later metabolic dysfunction. Developmental exposure to environmental chemical mixture within biosolids affects energy and lipid metabolism in adult male offspring.image

Exposure to environmental chemicals during fetal and pre-pubertal development has been associated with perturbed mammalian reproductive development and function. Indeed, there is increasing evidence that chemical contaminants adversely impact the developing fetus in utero, the pre-pubertal infant and the adult. In the male, chemical exposures have been associated with sub-fertility, malformations at birth and testicular cancer and in the female, with precocious puberty, premature menopause and polycystic ovarian disease. Although temporal changes and geographical differences in human reproductive function are indicative of an environmental aetiology, determining underlying mechanisms and true cause and effect is problematic. Developmental and functional impacts of individual or chemical mixtures are explored using three animal models. (1) Pregnant ewes exposed to chemical mixtures in widely used sewage sludge derived fertiliser (biosolids) provide a model of real-life human exposure. (2) Companion dogs that share our homes serve as a sentinel species for human exposure to household pollutants. (3) Neonatal mouse gonads exposed in vitro to selected chemicals provide information on subtle perturbations of early gonadal development. (1: sheep) Exposure induced changes in the transcriptome (testis and ovary) are reported combined with altered fetal gonadal morphology and endocrine function. (2: dog) a decline in dog semen quality over 26 years parallels that reported in the human. Regional differences in chemical profiles and morphology also approximate those reported in the human. Specific chemical contaminants alter semen function at environmental/tissue concentrations. (3: mouse) gonadal concentrations of chemicals alter follicle development in the early post-natal ovary and perturb cellular development in the testis. Details on each of these paradigms will be presented. This combination of animal models has provided key insights into possible chemical perturbations of human fertility and reproductive health.

Veterinary RecordVolume 192, Issue 4 p. 174-175 Veterinary Ethics Promoting good ethical practice David Morton, Corresponding Author David Morton group chair [email protected] Search for more papers by this authorKate Allen, Kate Allen group secretary [email protected] Search for more papers by this authorBeth Jinks, Beth Jinks RCVS representativesSearch for more papers by this authorMike Herrtage, Mike Herrtage RCVS representativesSearch for more papers by this authorMichael Clark, Michael Clark University of Nottingham representativesSearch for more papers by this authorRichard Lea, Richard Lea University of Nottingham representativesSearch for more papers by this authorEddie Clutton, Eddie Clutton University of Edinburgh representativesSearch for more papers by this authorKiterie Faller, Kiterie Faller University of Edinburgh representativesSearch for more papers by this authorStephen Greenhalgh, Stephen Greenhalgh University of Edinburgh representativesSearch for more papers by this authorPeter Fordyce, Peter Fordyce University of Cambridge representativeSearch for more papers by this authorSarah Wolfensohn, Sarah Wolfensohn University of Surrey representativesSearch for more papers by this authorChris Stevens, Chris Stevens University of Surrey representativesSearch for more papers by this authorPamela Gillick, Pamela Gillick University College Dublin representativeSearch for more papers by this authorNicola Menzies-Gow, Nicola Menzies-Gow Royal Veterinary College representativeSearch for more papers by this authorDavid Killick, David Killick University of Liverpool representativeSearch for more papers by this authorJoanna Morris, Joanna Morris University of Glasgow representativeSearch for more papers by this authorDavid Martin, David Martin IVC Evidensia representativeSearch for more papers by this authorLuisa De Risio, Luisa De Risio Linnaeus representativeSearch for more papers by this authorJo Murrell, Jo Murrell CVS Group representativeSearch for more papers by this author David Morton, Corresponding Author David Morton group chair [email protected] Search for more papers by this authorKate Allen, Kate Allen group secretary [email protected] Search for more papers by this authorBeth Jinks, Beth Jinks RCVS representativesSearch for more papers by this authorMike Herrtage, Mike Herrtage RCVS representativesSearch for more papers by this authorMichael Clark, Michael Clark University of Nottingham representativesSearch for more papers by this authorRichard Lea, Richard Lea University of Nottingham representativesSearch for more papers by this authorEddie Clutton, Eddie Clutton University of Edinburgh representativesSearch for more papers by this authorKiterie Faller, Kiterie Faller University of Edinburgh representativesSearch for more papers by this authorStephen Greenhalgh, Stephen Greenhalgh University of Edinburgh representativesSearch for more papers by this authorPeter Fordyce, Peter Fordyce University of Cambridge representativeSearch for more papers by this authorSarah Wolfensohn, Sarah Wolfensohn University of Surrey representativesSearch for more papers by this authorChris Stevens, Chris Stevens University of Surrey representativesSearch for more papers by this authorPamela Gillick, Pamela Gillick University College Dublin representativeSearch for more papers by this authorNicola Menzies-Gow, Nicola Menzies-Gow Royal Veterinary College representativeSearch for more papers by this authorDavid Killick, David Killick University of Liverpool representativeSearch for more papers by this authorJoanna Morris, Joanna Morris University of Glasgow representativeSearch for more papers by this authorDavid Martin, David Martin IVC Evidensia representativeSearch for more papers by this authorLuisa De Risio, Luisa De Risio Linnaeus representativeSearch for more papers by this authorJo Murrell, Jo Murrell CVS Group representativeSearch for more papers by this author First published: 17 February 2023 https://doi.org/10.1002/vetr.2761Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume192, Issue418/25 February 2023Pages 174-175 RelatedInformation

Background: Gestational exposure to environmental chemicals (ECs) is associated with adverse, sex-specific offspring health effects of global concern. As the maternal steroid, cytokine and oxidative stress milieus can have critical effects on pregnancy outcomes and the programming of diseases in offspring, it is important to study the impact of real-life EC exposure, i.e., chronic low levels of mixtures of ECs on these milieus. Sheep exposed to biosolids, derived from human waste, is an impactful model representing the ECs humans are exposed to in real life. Offspring of sheep grazed on biosolids-treated pasture are characterized by reproductive and metabolic disruptions. Objective: To determine if biosolids exposure disrupts the maternal steroid, cytokine and oxidative stress milieus, in a fetal sex-specific manner.Methods: Ewes were maintained before mating and through gestation on pastures fertilized with biosolids (BTP), or inorganic fertilizer (Control). From maternal plasma collected mid-gestation, 19 steroids, 14 cytokines, 6 oxidative stress markers were quantified. Unpaired t-test and ANOVA were used to test for differences between control and BTP groups (n = 15/group) and between groups based on fetal sex, respectively. Correlation between the different markers was assessed by Spearman correlation.Results: Concentrations of the mineralocorticoids deoxycorticosterone, corticosterone, the glucocorticoids deoxycortisol, cortisol, cortisone, the sex steroids androstenedione, dehydroepiandrosterone, 16-OH -progesterone and reactive oxygen metabolites were higher in the BTP ewes compared to Controls, while the proinflammatory cytokines IL-1 & beta; and IL-17A and anti-inflammatory IL-36RA were decreased in the BTP group. BTP ewes with a female fetus had lower levels of IP-10.Discussion: These findings suggest that pre-conceptional and gestational exposure to ECs in biosolids increases steroids, reactive oxygen metabolites and disrupts cytokines in maternal circulation, likely contributors to the aberrant phenotypic outcomes seen in offspring of BTP sheep a translationally relevant precocial model.

Background: Everyday, humans are exposed to a mixture of environmental chemicals some of which have endocrine and/or metabolism disrupting actions which may contribute to non-communicable diseases. The adverse health impacts of real-world chemical exposure, characterized by chronic low doses of a mixture of chemicals, are only recently emerging. Biosolids derived from human waste represent the environmental chemical mixtures humans are exposed to in real life. Prior studies in sheep have shown aberrant reproductive and metabolic phenotypes in offspring after maternal biosolids exposure.Objective: To determine if exposure to biosolids perturbs the maternal metabolic milieu of pregnant ewes, in a fetal sex specific manner.Methods: Ewes were grazed on inorganic fertilizer (Control) or biosolids-treated pastures (BTP) from before mating and throughout gestation. Plasma from pregnant ewes (Control n = 15, BTP n = 15) obtained mid-gestation were analyzed by untargeted metabolomics. Metabolites were identified using Agilent MassHunter. Multivariate analyses were done using MetaboAnalyst 5.0 and confirmed using SIMCA.Results: Univariate and multivariate analysis of 2301 annotated metabolites identified 193 differentially abundant metabolites (DM) between control and BTP sheep. The DM primarily belonged to the super-class of lipids and organic acids. 15-HeTrE, oleamide, methionine, CAR(3:0(OH)) and pyroglutamic acid were the top DM and have been implicated in the regulation of fetal growth and development. Fetal sex further exacerbated differences in metabolite profiles in the BTP group. The organic acids class of metabolites was abundant in animals with male fetuses. Prenol lipid, sphingolipid, glycerolipid, alkaloid, polyketide and benzenoid classes showed fetal sex-specific responses to biosolids.Discussion: Our study illustrates that exposure to biosolids significantly alters the maternal metabolome in a fetal sex specific manner. The altered metabolite profile indicates perturbations to fatty acid, arginine, branched chain amino acid and one-carbon metabolism. These factors are consistent with, and likely contribute to, the adverse phenotypic outcomes reported in the offspring.

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Environmental chemical (EC) exposure may be impacting male reproductive health. The translationally relevant biosolids treated pasture (BTP) sheep model was used to investigate gestational low-level EC mixture exposure on the testes of F1 male offspring. Adult rams from ewes exposed to BTP 1 month before and throughout pregnancy had more seminiferous tubules with degeneration and depletion of elongating spermatids, indicating possible "recovery" from previously reported testicular dysgenesis syndrome-like phenotype in neonatal and pre-pubertal BTP lambs. Expression of transcription factors CREB1 (neonatal) and BCL11A and FOXP2 (pre-pubertal) were significantly higher in the BTP exposed testes, with no changes seen in adults. Increased CREB1, which is crucial for testes development and regulation of steroidogenic enzymes, could be an adaptive response to gestational EC exposure to facilitate the phenotypic recovery. Overall, this demonstrates that testicular effects from gestational exposure to low-level mixtures of ECs can last into adulthood, potentially impacting fertility and fecundity.