Environmental chemicals and their effects on female reproductive health: Searching for molecular mechanisms and effect biomarkers

Research output: Book/ReportPh.D. thesis – Annual report year: 2017Research

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Incorrect developmental programming of the female reproductive tract can lead to compromised reproductive fitness later in life. It has been suggested that exposure to endocrine disrupting chemicals (EDCs) in utero can disrupt ovarian programming in humans, which is supported by several animal studies. However, it remains unclear which specific processes during development are affected, and if there are particular sensitive developmental windows. Most of the etiological evidence derives from rodent studies, whereas cause-effect relationships in humans are extremely difficult to obtain, not least due to the fact that there is a significant lag time between exposure during fetal life and disease symptoms in adulthood. Furthermore, humans are typically exposed to chemicals at a much lower dose than those of experimental studies, but exposed to a large number of different chemicals. This may lead to combination or mixture effects, where chemicals present at doses that would not cause effects on their own, can add up and cause an effect. The aim of the PhD project was to identify early biomarkers and sensitive windows for late life effects on the ovary after chemical exposure to mixtures of EDCs during early development.
A comprehensive literature review was synthesized to obtain an overview over current knowledge on the effects environmental chemicals can have on the developing ovary. This work identified four potentially sensitive windows of reproductive programming in females; i) primordial germ cell migration and gonadal sex determination, ii) meiosis, iii) follicle assembly, and iv) early folliculogenesis. For the experimental work, which aimed at identifying potential early biomarkers for late life diseases, two general approaches were adopted; a targeted approach looking at specific endpoints and a selection of effect biomarkers, and a more open-ended screening approach looking for potentially novel biomarkers. In the targeted approach, endpoints known to be important for reproductive function and ovary health were investigated at the molecular and morphological levels in neonatal, pre-pubertal and adult rat ovaries exposed to mixtures of EDCs during development. In the screening approach, a proteomics screen was performed to investigate differentially expressed proteins in the rat ovary after developmental exposure to mixtures of EDCs.
In the initial targeted approach, rat dams were exposed to a mixture of phthalates, pesticides, UV-filters, bisphenol A, butyl-paraben, as well as the mild analgesic paracetamol (PM). The compounds were tested all together (Totalmix) or in subgroups with anti-androgenic (AAmix) or estrogenic (Emix) properties. PM was tested separately. Reproductive endpoints were investigated in offspring at pre-puberty (PD22) and adulthood (approx. 1 year of age). In pre-pubertal animals a significant reduction in primordial follicle numbers was seen after AAmix and PM exposure, whereas in the 1 year old animals reduced ovary weights were seen in Totalmix-, AAmix-, and PM-groups. Finally, animals in the Totalmix group showed a higher incidence rate of irregular estrous cycles than control animals.
The reduction in primordial follicles after AAmix exposure was suspected to be caused by interruption to follicle assembly. Thus, a small pilot study, exposing explanted neonatal ovaries to AAmix, submixtures (pesticide mix (PEmix), phthtalate mix (PHmix)), and mono(2-ethylhexyl)phthalate (MEHP), was conducted. No significant effects were seen on gene expression, but histological evaluation showed that primordial follicles were reduced in the PEmix exposed ovaries.
For the proteomics screening study, a shotgun proteomics approach was performed on PD17 ovaries from offspring corresponding to those of the initial targeted study. Protein extracts were analyzed by LC-MS/MS, and evaluation of the data for potential effect biomarkers showed that three proteins, Trimethyllysine dioxygenase (TMLH), Keratin, type II cytoskeletal 8 (KRT8), and anti-Müllerian hormone (AMH) were dysregulated in all exposure groups. Also, ingenuity pathway analysis revealed canonical pathways known to be involved in ovary function, such as mTOR and HIPPO signaling, to be affected in all exposure groups.
In conclusion, the studies conducted for this PhD revealed that follicle count in pre-pubertal rats can potentially be used as a marker for early life affected ovary development caused by EDC mixture exposure, leading to reproductive senescence later in life. Furthermore, three proteins were identified as possible biomarkers for effects on the developing ovary, and potentially for late life adverse effects.

Original languageEnglish
Place of PublicationSøborg
PublisherNational Food Institute, Technical University of Denmark
Number of pages107
ISBN (Electronic)978-87-93109-96-4
Publication statusPublished - 2016

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