Periodic Reporting for period 4 - FREIA (Female Reproductive toxicity of EDCs: a human evidence-based screening and Identification Approach)
Reporting period: 2023-07-01 to 2024-06-30
Endocrine disrupting chemicals (EDCs) are human-made chemicals that disturb our own hormones in our bodies. Unfortunately, knowledge and methods are lacking to properly test for the effects of EDCs on women’s reproductive health. As a consequence, EDC effects on females are often overlooked in regulatory chemical safety assessments, which puts women’s reproductive health is at risk globally. Our EU-funded project FREIA addressed this by providing (i) knowledge on the effects of EDCs on the human ovary, and the eggs (oocytes) it contains, at different ages, (ii) test methods and endpoints that specifically focus on the effects of EDCs on female reproduction, and (iii) clear recommendations to reduce EDC exposure and protect women’s reproductive health.
New knowledge
Human fetal, child and adult ovaries were used to study the biology of ovaries at different ages. We showed that different types of cells and their function change dramatically with age. This means that sensitivity to EDCs changes with age. We found that up to one-third of follicles, structures that contain the eggs, in the human ovary help with communication between cells in the ovary rather than making eggs. We also found that the store of eggs in the ovary, the “ovarian reserve”, changes during puberty, while it was thought to be dormant. These findings highlight the importance of considering age-dependent differences when studying the effects of EDCs. The pathways leading to harmful effects on reproduction need to be properly mapped for each age. We provided several descriptions of such pathways, called adverse outcome pathways (AOPs) and knowledge networks.
Test methods
We studied the well-characterised EDCs diethylstilbestrol (DES, which activates the estrogen receptor) and ketoconazole (KTZ, which blocks steroid hormone production), and the suspected EDC propylparaben (PrPB, mixed actions). This enabled us to identify several biological characteristics, or biomarkers, of female reproductive toxicity. We showed the need for female reproduction endpoints that are more sensitive to detect an endocrine disrupting effect than those currently measured in rat studies for regulatory assessment. We found that measuring blood levels of steroid hormones does not help to identify EDCs in rat studies. Other endpoints, such as mammary gland development and release of signalling molecules (neuropeptides) in the brain are sensitive and predictive of an EDC effect. Although no obvious structural effects were detectable, many molecular changes were measured in the rat ovaries exposed to our test EDCs. We also performed experiments with cells of human fetal and adult ovaries and immortalized ovarian cells. These showed that the production of cholesterol, the building block for sex steroid hormones like oestradiol, and sex steroid hormones themselves are affected by EDCs. Our results indicate a consistent and direct effect of EDCs on reproductive cells, which will not be captured by testing gross morphological changes in rats. Nevertheless, our studies show ‘subtle’ effects that indicate negative consequences for reproductive function and fertility. These molecular changes are age-dependent and species-specific and need further studies to show their value in toxicological studies as well as in clinical settings.
We have developed and employed several cell- and computer-based methods covering critical events in female reproductive development and function. These include an oestrogen receptor-beta (ER-beta, a specialised oestrogen receptor) assay, a high-content assay for ovarian cell toxicity, a cow oocyte maturation and competency assay, and computer (QSAR) models that predict EDC interactions with aromatase (enzyme that makes oestrogens), PPAR-gamma (involved in lipid metabolism, inflammation, and cell proliferation), and ER-beta. Based on our studies, an ovarian-specific steroidogenesis assay, to determine if a chemical disrupts sex steroid hormone formation in the ovary, is unlikely to be useful for regulatory purposes. Our studies also show that the commercially available KGN cell line may be suitable to test for EDC effects on human adult ovary cells.
Overall, FREIA has provided new and improved avenues to identify EDCs that cause female reproductive toxicity, although our outcomes are at different stages of development. Some methods we developed are scientifically sound (e.g. biomarkers, ER-beta assay, neuropeptide release, ovarian surface photo counting for fast assessment of ovarian effects) and now need to be tested in multiple laboratories to show their suitability for regulatory testing. Some are already discussed within the international standard-setting Organisation for Economic Co-operation and Development (OECD), such as the inclusion of whole-mount mammary gland analysis and expansion of the existing H295R steroidogenesis assay. Some outcomes, like the QSARs and AOPs, are ready to use. Even so, our outcomes can already support EDC identification processes; we have described our assays and endpoints in the context of existing test strategies to facilitate easy implementation in European regulatory frameworks for EDC identification.
Protect women’s reproductive health
By studying the levels of several EDCs in the ovaries of women undergoing fertility treatment, we provided evidence that a plastics additive, DEHP, alters women’s ovaries and reduces female fertility. In addition, several other chemicals, i.e. methylparaben and some PFAS, were identified that may harm ovarian function and contribute to female infertility. We also identified several lifestyle factors, such as frequent use of perfumes, that led to higher exposure of these women to certain EDCs. Additionally, our measurements show EDC levels in fetal liver and placenta that are close to the EDC levels that show harmful effects in our studies. We used these findings, combined with an evaluation of the scientific literature and results from our survey among the Dutch population, to formulate recommendations on how to successfully reduce exposure and protect our reproductive health against EDCs.
On the FREIA website (www.freiaproject.eu) general background information on EDCs and female reproductive health can be found, as well as project-specific information, including webinar recordings, peer-reviewed scientific publications, databases, FREIA factsheets, infographics and reports. FREIA is one of the eight projects on test method development for EDC identification within the EURION cluster (www.eurion-cluster.eu).
Human fetal, child and adult ovaries were used to study the biology of ovaries at different ages. We showed that different types of cells and their function change dramatically with age. This means that sensitivity to EDCs changes with age. We found that up to one-third of follicles, structures that contain the eggs, in the human ovary help with communication between cells in the ovary rather than making eggs. We also found that the store of eggs in the ovary, the “ovarian reserve”, changes during puberty, while it was thought to be dormant. These findings highlight the importance of considering age-dependent differences when studying the effects of EDCs. The pathways leading to harmful effects on reproduction need to be properly mapped for each age. We provided several descriptions of such pathways, called adverse outcome pathways (AOPs) and knowledge networks.
Test methods
We studied the well-characterised EDCs diethylstilbestrol (DES, which activates the estrogen receptor) and ketoconazole (KTZ, which blocks steroid hormone production), and the suspected EDC propylparaben (PrPB, mixed actions). This enabled us to identify several biological characteristics, or biomarkers, of female reproductive toxicity. We showed the need for female reproduction endpoints that are more sensitive to detect an endocrine disrupting effect than those currently measured in rat studies for regulatory assessment. We found that measuring blood levels of steroid hormones does not help to identify EDCs in rat studies. Other endpoints, such as mammary gland development and release of signalling molecules (neuropeptides) in the brain are sensitive and predictive of an EDC effect. Although no obvious structural effects were detectable, many molecular changes were measured in the rat ovaries exposed to our test EDCs. We also performed experiments with cells of human fetal and adult ovaries and immortalized ovarian cells. These showed that the production of cholesterol, the building block for sex steroid hormones like oestradiol, and sex steroid hormones themselves are affected by EDCs. Our results indicate a consistent and direct effect of EDCs on reproductive cells, which will not be captured by testing gross morphological changes in rats. Nevertheless, our studies show ‘subtle’ effects that indicate negative consequences for reproductive function and fertility. These molecular changes are age-dependent and species-specific and need further studies to show their value in toxicological studies as well as in clinical settings.
We have developed and employed several cell- and computer-based methods covering critical events in female reproductive development and function. These include an oestrogen receptor-beta (ER-beta, a specialised oestrogen receptor) assay, a high-content assay for ovarian cell toxicity, a cow oocyte maturation and competency assay, and computer (QSAR) models that predict EDC interactions with aromatase (enzyme that makes oestrogens), PPAR-gamma (involved in lipid metabolism, inflammation, and cell proliferation), and ER-beta. Based on our studies, an ovarian-specific steroidogenesis assay, to determine if a chemical disrupts sex steroid hormone formation in the ovary, is unlikely to be useful for regulatory purposes. Our studies also show that the commercially available KGN cell line may be suitable to test for EDC effects on human adult ovary cells.
Overall, FREIA has provided new and improved avenues to identify EDCs that cause female reproductive toxicity, although our outcomes are at different stages of development. Some methods we developed are scientifically sound (e.g. biomarkers, ER-beta assay, neuropeptide release, ovarian surface photo counting for fast assessment of ovarian effects) and now need to be tested in multiple laboratories to show their suitability for regulatory testing. Some are already discussed within the international standard-setting Organisation for Economic Co-operation and Development (OECD), such as the inclusion of whole-mount mammary gland analysis and expansion of the existing H295R steroidogenesis assay. Some outcomes, like the QSARs and AOPs, are ready to use. Even so, our outcomes can already support EDC identification processes; we have described our assays and endpoints in the context of existing test strategies to facilitate easy implementation in European regulatory frameworks for EDC identification.
Protect women’s reproductive health
By studying the levels of several EDCs in the ovaries of women undergoing fertility treatment, we provided evidence that a plastics additive, DEHP, alters women’s ovaries and reduces female fertility. In addition, several other chemicals, i.e. methylparaben and some PFAS, were identified that may harm ovarian function and contribute to female infertility. We also identified several lifestyle factors, such as frequent use of perfumes, that led to higher exposure of these women to certain EDCs. Additionally, our measurements show EDC levels in fetal liver and placenta that are close to the EDC levels that show harmful effects in our studies. We used these findings, combined with an evaluation of the scientific literature and results from our survey among the Dutch population, to formulate recommendations on how to successfully reduce exposure and protect our reproductive health against EDCs.
On the FREIA website (www.freiaproject.eu) general background information on EDCs and female reproductive health can be found, as well as project-specific information, including webinar recordings, peer-reviewed scientific publications, databases, FREIA factsheets, infographics and reports. FREIA is one of the eight projects on test method development for EDC identification within the EURION cluster (www.eurion-cluster.eu).
FREIA uniquely provided the opportunity to investigate hormonal processes in human ovaries from fetal to adult age and improve scientific knowledge on the causes of female reproductive toxicity. Our committed collaborators for policy, advocacy and communicating actions to promote women’s health and a healthy society allowed FREIA to have a substantial societal impact. The FREIA approach supports the work of European regulatory agencies, or even globally, through the EURION cluster activities. We have developed tools that fit the needs of modern-day toxicity testing with a clear regulatory application in mind. Together, the FREIA outcomes support testing, identification and assessment of EDCs that are toxic for female reproduction. Importantly, our outcomes contribute to protecting women’s health against EDCs globally.