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Relationship: 3142

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Reduced granulosa cell proliferation leads to disrupted, ovarian cycle

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Reduced granulosa cell proliferation

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

disrupted, ovarian cycle

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Androgen receptor (AR) antagonism leading to decreased fertility in females	adjacent	High	Low	Terje Svingen (send email)	Under development: Not open for comment. Do not cite	Under Development

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
mammals	mammals	High	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Female	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Adult, reproductively mature	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

This KER connects reduced granulosa cell proliferation of early-stage follicles (gonadotropin-independent, up to antral stage) to ovarian cycle irregularities, which include disturbances in the ovarian cycle (e.g. longer cycle) and/or ovulation problems (deferred ovulation or anovulation). Reduced granulosa cell proliferation manifests as reduced follicle growth, which can be observed by follicle counting or staging.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence was first collected with a scoping search that provided information for biological plausibility and studies used as empirical evidence. To gather additional empirical evidence, targeted searches were also performed. In short, the first approach combined results from two separate search strings including search terms related to granulosa, follicles, estrus/menstrual cycle, and ovulation. The search resulted in a high number of hits, which were then limited to ones tagged as ‘exposure’ using the RAYYAN tool. The second approach involved using a search string that combined the above strategy with defined chemical compounds resulting in fewer hits. A semi-systematic approach was deemed appropriate since it is considered common knowledge that disturbances in granulosa growth and subsequently follicular growth will also affect the availability of later stages and ultimately ovulation.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Ovarian follicles are composed of a centrally located immature oocyte surrounded by supporting somatic cells where granulosa cells make up the inner layer most closely associated with the oocyte. In humans, it is important to emphasize that all follicles are formed during fetal life and this pool of primordial follicles makes up the ovarian reserve. Once primordial follicles are activated, they can grow into primary follicles as granulosa cells proliferate and change in morphology. This transition is driven by factors produced by either the granulosa cells or the oocytes, notably Kit ligand (KITL). As granulosa cells continue to proliferate, the follicles become secondary, a process regulated by factors including Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15). As more layers of granulosa cells are established, the follicle forms an antral cavity. Up to this stage, the growth and survival of the follicles is not dependent on gonadotropins, and thus do not involve the hypothalamus-pituitary-gonadal signaling axis, which is activated during puberty.

For later stages of follicular maturation and ovulation, the gonadotrophins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are essential. They are secreted from the anterior pituitary gland upon stimulation from the gonadotropin-releasing hormone (GnRH) from the hypothalamus. LH stimulates androgen production in theca cells of late-stage ovarian follicles, and is used by the neighbor granulosa cells to produce estrogens upon FSH stimulation. The hormones produced by these late-stage follicles and the corpora lutea formed by ovulated follicles, control the release of hormones from hypothalamus and anterior pituitary. The ovarian cycle results from the cyclic changes that occur in the female reproductive tract and are initiated and regulated by the hypothalamic-pituitary-ovarian (HPO) axis.

All stages of follicles can be found in the ovaries of reproductively active adult mammals, with the majority of the follicles being primordial. Only a minority of the primordial follicles ever complete folliculogenesis, with the majority dying by atresia. The granulosa cells are key determinants of follicle fate. Therefore, disruption of granulosa cell proliferation can halt follicular growth. By reducing the number of available earlier-stage gonadotropin-independent follicles, this will also affect the pool of more mature follicles. Consequently, fewer steroid hormones that regulate the ovarian cycle will be produced, leading to disturbances such as longer cycles. Additionally, less mature follicles will be available for ovulation, leading to e.g. deferred ovulation or anovulation. Biological evidence in humans can be provided by the irregular menstrual cycles observed in perimenopausal individuals (O’Connor et al., 2001).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Selected studies demonstrating an effect on early follicles by reducing their population and leading to effects on estrus cyclicity were selected as empirical evidence for the KER. An effect was observed by histologically identifying, staging, and counting the follicles in all of the included studies. Studies that observed a decrease in primordial follicle counts were excluded, as this indicates an effect on the follicular reserve, rather than on follicle growth.

Table 1. In vivo rodent exposure studies demonstrating reduced granulosa cell proliferation leading to ovarian cycle irregularities.

Species	Exposure	Dose	Duration and Age	Reduced granulosa proliferation	Ovarian cycle irregularities	Reference
Wistar rats	2,4,6-trinitrotoluene (TNT)	40, 80 g	Single explosion (6 w.o.)	Preantral proportions	Longer diestrus	(Lin et al., 2023)
ICR mice	Perfluorohexane sulfonate (PFHxS)	5 mg/kg/day intragastrical	42 days (8 w.o.)	Secondary and antral follicles	Longer diestrus	(Yin et al., 2021)
ICR mice	Pueraria murifica	100 mg/kg/day in water	8 weeks (∼10 w.o.)	Primary, secondary, Graffian	Longer estrus	(Jaroenporn et al., 2007)
Sprague Dawley albino rats	Triclocarban	0.5 mg/l/day in water	GD 5 to PND 21	Primary, secondary, antral Reduced Ki67 on granulosa	Longer estrus	(Mandour et al., 2021)
Sprague Dawley rats	Di-(2-ethylhexyl)-phthalate (DEHP)	600 mg/kg/alternate day oral gavage	60 days (6 w.o.)	Primary, secondary	Longer estrus cycle	(Xu et al., 2010)
CD-1 mice	Phthalate mixture	20, 200 µg/kg/day oral dosing	GD 10 to birth	Preantral percentage in F3	Longer metestrus, diestrus in F3	(Brehm et al., 2020)
CD-1 mice	Di-(2-ethylhexyl)-phthalate (DEHP)	200 µg/kg/day 500mg/kg/day oral dosing	GD 11 to birth	Preantral percentage in F3	Shorter prestrus, longer metestrus/diestrus	(Brehm et al., 2018)
ICR mice	Perfluorooctane sulfonate (PFOS)	0.1 mg/kg/day in water	4 months (12 w.o.)	Antral	Longer diestrus	(Feng et al., 2015)
Sprague Dawley rats	Perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS)	0.1 mg/kg/day subcutaneous injection	5 days (PND 1–5 or PND 26–30)	Primary and secondary	Longer diestrus, acyclicity	(Du et al., 2019)
Sprague Dawley rats	3,3′,4,4′,5-Pentachlorobiphenyl(PCB126)	250 ng/kg/day 7.5 μg/kg/day oral dosing	Day 13-19 post-conception	Antral	Longer diestrus	(Muto et al., 2003)
Wistar rats	1-bromopropane	400 ppm inhalation 8h/day	12 weeks (10 w.o.)	Primary, secondary, antral	Longer diestrus	(Yamada et al., 2003)

Table 2. In vivo rodent model demonstrating reduced granulosa cell proliferation leading to ovarian cycle irregularities.

Model	Reduced granulosa proliferation	Ovarian cycle irregularities	Reference
DHT-treated NOD/ShiLtJ mice (polycystic ovarian syndrome model)	Preantral, small antral	Longer metestrus/diestrus, shorter proestrus	(Binder et al., 2023)
Maternal high-fat diet Sprague-Dawley rats	Secondary	Irregular cycle	(Zhou et al., 2019)
Akt1^-/- mice	Early antral, antral, Graffian Reduced BrdU incorporation in granulosa of secondary	Longer diestrus	(Brown et al., 2010)
GCARKO^Ex2	Antral	Longer estrus cycle	(Sen & Hammes, 2010)
GCARKO^Ex3	Preantral, antral	Longer estrus cycle	(Walters et al., 2012)

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

The studies included as empirical evidence report decreased numbers of growing follicles of early stages. However, some of the studies also report increased atresia, which could imply that the observations of altered follicle numbers are a result of stage-specific atresia rather than decreased follicular growth.

During our evidence collection, we identified studies that observed changes in follicle numbers with no effects on the ovarian cycle (Guerra et al., 2011; Ulker et al., 2020). An additional uncertainty is that estrus cyclicity is an endpoint potentially affected by different experimental set-ups, for example, group size, study length and statistical analyses (Goldman et al., 2007). Lastly, ovarian cycle irregularities indicate disturbances in any parts of the Hypothalamic-Pituitary-Ovarian (HPO) axis, which regulates reproductive processes. Therefore, direct effects on hypothalamus and pituitary can lead to uncertainties.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

The nature of the response-response relationship between reduced granulosa cell proliferation and ovarian cycle irregularities is not clear, therefore, at present, the quantitative understanding of this KER is rated ‘low’.

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Studies included in establishing this KER exhibit observed changes upon a singular acute exposure, in the case of the TNT study presented in the empirical evidence (Lin et al., 2023).

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

During the ovarian cycle, there is a feedback loop between the hypothalamus, anterior pituitary gland and the ovary. In brief, the hypothalamus produces gonadotropin-releasing hormone (GnRH), stimulating the anterior pituitary to produce gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that in turn stimulate growth of ovarian follicles and the production of androgens in theca cells and estrogens in granulosa (Jamnongjit & Hammes, 2006). The estrogens produced by the granulosa along with the progesterone from the corpus luteum feedback to hypothalamus and anterior pituitary gland, to control their release.

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

This KER refers to females, as it refers to the primary component of the female reproductive system, the ovary and it includes all mammals. Although follicle activation and growth can occur at all ages, the ovarian cycle can only be completed upon sexual maturation, therefore the life stage applicability is defined as ‘Adult, reproductively mature’. The supporting empirical evidence includes rodent studies and biological plausibility additionally includes humans. However, the taxonomic applicability can be expanded to other mammals as follicle growth is the basic principle driving the ovarian cycle in all mammalian species.

References

List of the literature that was cited for this KER description. More help

Binder, A. K., Peecher, D. L., Qvigstad, A. J., Gutierrez, S. D., Magaña, J., Banks, D. B., & Korach, K. S. (2023). Differential Strain-dependent Ovarian and Metabolic Responses in a Mouse Model of PCOS. Endocrinology (United States), 164(4). https://doi.org/10.1210/endocr/bqad024

Brehm, E., Rattan, S., Gao, L., & Flaws, J. A. (2018). Prenatal exposure to Di(2-ethylhexyl) phthalate causes long-term transgenerational effects on female reproduction in mice. Endocrinology, 159(2), 795–809. https://doi.org/10.1210/en.2017-03004

Brehm, E., Zhou, C., Gao, L., & Flaws, J. A. (2020). Prenatal exposure to an environmentally relevant phthalate mixture accelerates biomarkers of reproductive aging in a multiple and transgenerational manner in female mice. Reproductive Toxicology, 98, 260–268. https://doi.org/10.1016/j.reprotox.2020.10.009

Brown, C., Larocca, J., Pietruska, J., Ota, M., Anderson, L., Smith, S. D., Weston, P., Rasoulpour, T., & Hixon, M. L. (2010). Subfertility Caused by Altered Follicular Development and Oocyte Growth in Female Mice Lacking PKBalpha/Akt1 1. BIOLOGY OF REPRODUCTION, 82, 246–256. https://doi.org/10.1095/biolreprod.109.077925

Du, G., Hu, J., Huang, Z., Yu, M., Lu, C., Wang, X., & Wu, D. (2019). Neonatal and juvenile exposure to perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS): Advance puberty onset and kisspeptin system disturbance in female rats. Ecotoxicology and Environmental Safety, 167, 412–421. https://doi.org/10.1016/j.ecoenv.2018.10.025

Feng, X., Wang, X., Cao, X., Xia, Y., Zhou, R., & Chen, L. (2015). Chronic exposure of female mice to an environmental level of perfluorooctane sulfonate suppresses estrogen synthesis through reduced histone h3k14 acetylation of the StAR promoter leading to deficits in follicular development and ovulation. Toxicological Sciences, 148(2), 368–379. https://doi.org/10.1093/toxsci/kfv197

Goldman, J. M., Murr, A. S., & Cooper, R. L. (2007). The rodent estrous cycle: Characterization of vaginal cytology and its utility in toxicological studies. In Birth Defects Research Part B - Developmental and Reproductive Toxicology (Vol. 80, Issue 2, pp. 84–97). https://doi.org/10.1002/bdrb.20106

Guerra, M. T., de Toledo, F. C., & Kempinas, W. D. G. (2011). In utero and lactational exposure to fenvalerate disrupts reproductive function in female rats. Reproductive Toxicology, 32(3), 298–303. https://doi.org/10.1016/j.reprotox.2011.08.002

Jamnongjit, M., & Hammes, S. R. (2006). Ovarian Steroids: The Good, the Bad, and the Signals that Raise Them. Cell Cycle. https://doi.org/10.4161/cc.5.11.2803

Jaroenporn, S., Malaivijitnond, S., Wattanasirmkit, K., Watanabe, G., Taya, K., & Cherdshewasart, W. (2007). Assessment of Fertility and Reproductive Toxicity in Adult Female Mice after Long-Term Exposure to Pueraria mirifica Herb. In Journal of Reproduction and Development (Vol. 53, Issue 5).

Lin, D., Chen, Y., Liang, L., Huang, Z., Guo, Y., Cai, P., & Wang, W. (2023). Effects of exposure to the explosive and environmental pollutant 2,4,6-trinitrotoluene on ovarian follicle development in rats. Environmental Science and Pollution Research, 30(42), 96412–96423. https://doi.org/10.1007/s11356-023-29161-w

Mandour, D. A., Aidaros, A. A. M., & Mohamed, S. (2021). Potential long-term developmental toxicity of in utero and lactational exposure to Triclocarban (TCC) in hampering ovarian folliculogenesis in rat offspring. Acta Histochemica, 123(6). https://doi.org/10.1016/j.acthis.2021.151772

Muto, T., Imano, N., Nakaaki, K., Takahashi, H., Hano, H., Wakui, S., & Furusato, M. (2003). Estrous cyclicity and ovarian follicles in female rats after prenatal exposure to 3,3′,4,4′,5-pentachlorobiphenyl. Toxicology Letters, 143(3), 271–277. https://doi.org/10.1016/S0378-4274(03)00175-9

O’Connor, K. A., Holman, D. J., & Wood, J. W. (2001). Menstrual cycle variability and the perimenopause. American Journal of Human Biology, 13(4), 465–478. https://doi.org/10.1002/ajhb.1078

Sen, A., & Hammes, S. R. (2010). Granulosa cell-specific androgen receptors are critical regulators of ovarian development and function. Molecular Endocrinology, 24(7), 1393–1403. https://doi.org/10.1210/me.2010-0006

Ulker, N., Yardimci, A., Kaya Tektemur, N., Bulmus, O., Kaya, S. O., Bulmus, F. G., Turk, G., & Canpolat, S. (2020). Irisin may have a role in pubertal development and regulation of reproductive function in rats. https://doi.org/10.1530/REP

Walters, K. A., Middleton, L. J., Joseph, S. R., Hazra, R., Jimenez, M., Simanainen, U., Allan, C. M., & Handelsman, D. J. (2012). Targeted loss of androgen receptor signaling in murine granulosa cells of preantral and antral follicles causes female subfertility. Biology of Reproduction, 87(6). https://doi.org/10.1095/biolreprod.112.102012

Xu, C., Chen, J. A., Qiu, Z., Zhao, Q., Luo, J., Yang, L., Zeng, H., Huang, Y., Zhang, L., Cao, J., & Shu, W. (2010). Ovotoxicity and PPAR-mediated aromatase downregulation in female Sprague-Dawley rats following combined oral exposure to benzo[a]pyrene and di-(2-ethylhexyl) phthalate. Toxicology Letters, 199(3), 323–332. https://doi.org/10.1016/j.toxlet.2010.09.015

Yamada, T., Ichihara, G., Wang, H., Yu, X., Maeda, K.-I., Tsukamura, H., Kamijima, M., Nakajima, T., & Takeuchi, Y. (2003). Exposure to 1-Bromopropane Causes Ovarian Dysfunction in Rats. https://academic.oup.com/toxsci/article/71/1/96/1639572

Yin, X., Di, T., Cao, X., Liu, Z., Xie, J., & Zhang, S. (2021). Chronic exposure to perfluorohexane sulfonate leads to a reproduction deficit by suppressing hypothalamic kisspeptin expression in mice. Journal of Ovarian Research, 14(1). https://doi.org/10.1186/s13048-021-00903-z

Zhou, Z., Lin, Q., Xu, X., Illahi, G. S., Dong, C., & Wu, X. (2019). Maternal high-fat diet impairs follicular development of offspring through intraovarian kisspeptin/GPR54 system. Reproductive Biology and Endocrinology, 17(1). https://doi.org/10.1186/s12958-019-0457-z