Decreased, GnRH pulsatility/release

D007987

MESH Gonadotropin Releasing Hormone

CHEBI:81568

CHEBI Luteinizing hormone

FMA:67343

FMA Ovum

CHEBI:23965

CHEBI estradiol

FMA:86488

FMA Glandular part of endometrium

UBERON:0001344

UBERON epithelium of vagina

GO:0046879

GO hormone secretion

GO:0033684

GO regulation of luteinizing hormone secretion

GO:0030728

GO ovulation

GO:0035938

GO estradiol secretion

GO:0070268

GO cornification

WIKI decreased

WIKI increased

WIKI occurrence

WikiUser_17

mammals

WikiUser_29

Invertebrates

WikiUser_6

ApacheUser fish

8782

NCBI Aves

WikiUser_28

Vertebrates

Decreased, GnRH pulsatility/release

Cellular

Biological state At puberty, release of GnRH (gonadotropin releasing hormone) by specific brain areas stimulates the pituitary release of luteinising hormone (LH) and follicle stimulating hormone (FSH) that in turn stimulates gonads (ovary and testes) to release sex hormones (androgens, estrogens, progesterone). This is called hypothalamus pituitary gonads (HPG) axis (Fig. 1). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/02/58k7b8zy8_Figure_1.png" /> Figure 1. Regulation of reproductive function by the hypothalamus- pituitary-gonadal (HPG) axis. Arrows with solid and broken lines indicate stimulatory and suppressive effects, respectively. ARC, arcuate nucleus; AVPV, anteroventral periventricular nucleus; FSH, follicle-stimulating hormone; GnRH, gonadotropin- releasing hormone; LH, luteinizing hormone; POA, preoptic area (from Matsuda F. et al., 2019) The master position of GnRH neurons in the hierarchy of signals controlling the gonadotropic axis makes it the final target of a large number of regulators of central (e.g., glutamate, g-amino-butyric acid, neuropeptide Y, noradrenaline) and peripheral (e.g., gonadal steroids, metabolic hormones) origin (Tovar et al., 2006) (Fig. 2). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/02/8s1qqsw68p_Figure_2.png" style="height:394px; width:668px" /> Figure 2. Mechanisms regulating POA/AVPV kisspeptin neurons, ARC kisspeptin neurons and GnRH neurons. Arrows with solid and broken lines indicate stimulatory and suppressive effects, respectively. ARC, arcuate nucleus; AVP, argi- nine vasopressin; AVPV, anteroventral periventricular nucleus; ERα, estrogen receptor alpha; GABA, γ-aminobutyric acid; GnRH, gonadotropin- releasing hormone; POA, preoptic area; VIP, vasoactive intestinal polypeptide from Matsuda F. et al., 2019. J. Obstet. Gynaecol. Res. Vol. 45, No. 12: 2318–2329, doi:10.1111/JOG.14124. A key role is played, for example, by estrogens which, however, do not act directly on the GnRH neurons (which do not express E2 receptors), but by modulating the kisspeptin neurons (see also KE:968 in AOP-Wiki). Kisspeptin ESR1- expressing neurons innervating GnRH neurons are located primarily in the median preoptic nucleus, anteroventral preoptic area (AVPV) and preoptic periventricular nucleus (PeN), 3 contiguous brain regions termed the rostral periventricular area of the third ventricle (RP3V) (see also Fig. 3). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/02/1ksqrqd304_Figure_3.png" style="height:381px; width:918px" /> Schematic representation of a parasagittal section of the macaque and human hypothalamus. The arcuate nucleus may be referred to as the infundibular nucleus in the human. The anteroventral nucleus in the preoptic area and the arcuate nucleus in the more caudal medio basal hypothalamus are highlighted in magenta. Redrawn from Yen, 2004 with permission. From Plant, 2012. Right: Classical model of the hypothalamic control of LH secretion during the ovarian cycle of the rat overlayed on a schematic representation of a parasagittal section of the rat hypothalamus (MBH). The preovulatory LH surge, on the other hand is triggered by a daily circadian signal that originates within the preoptic area, and which is relayed to the GnRH neuronal network only when gated by preovulatory levels of estradiol by the so-called positive feedback action of this steroid. This ensemble of ESR1 neurons innervating the GnRH neuron and their associated glia and afferent inputs is considered here to represent the GnRH surge generator. From Plant, 2012. The activation of ERα in kisspeptin neurons is an obligatory step in the neural mechanisms mediating release of E2-induced GnRH and LH surges (DuBois et al., 2015). It has been demonstrated that ERα in kisspeptin neurons is required for the positive, but not negative feedback actions of E2 on GnRH/LH secretion in adult female mice (Dubois et al., 2015). Actually, GnRH neurons do not reside within a discrete brain region but form a dispersed longitudinal array of cells within the medial septum, preoptic area, and hypothalamus. Species differences exist in the caudal limits of this continuum, as most GnRH cells are found in the preoptic area of sheep and rats, but significant numbers exist within the basal hypothalamus of primates. However, retrograde labelling studies have now shown that GnRH neurons throughout this continuum project to the median eminence in rats, sheep, and monkeys (Herbison, 1998). Biological compartment GnRH is produced in hypothalamus and released in hypophyseal portal blood system. General role in biology The secretion of GnRH triggers sexual maturation, or puberty. GnRH is released in a pulsatile manner into the hypophyseal portal blood system. In the anterior pituitary, GnRH binds to its receptor expressed by gonadotropic cells and induces the release of the two gonadotropins, LH and FSH (Franssen et al., 2021). There is remarkable consistency across mammals in the pattern of pulsatile secretion with one pulse generated per hour during the follicular/diestrous phase of the cycle and a slower rate of one pulse every 3–4 h following ovulation in the estrous/luteal phase (Herbison, 2018). The only other major change that occurs during the cycle is the occurrence of an abrupt and massive outpouring of GnRH that generates the preovulatory GnRH/LH surge in all spontaneously ovulating mammals (Karsch, et al., 1997, Plant 2012). Thus, the brain and pituitary produce an on-going pulsatile pattern of gonadotropin secretion that slows on estrous to allow appropriate follicular development and a surge pattern of secretion at mid-cycle to initiate ovulation. Studies undertaken in rodents suggest that <100 GnRH neurons are sufficient for pulsatile LH secretion and that the most caudally positioned GnRH neuron cell bodies might be preferentially involved in pulse generation (in Herbison, 2016). Fluctuations in this pattern of GnRH release, combined with alterations in the secretory capacity of the pituitary gonadotrophs, generate the marked changes in LH secretion profile observed over the course of the ovarian cycle (Herbison, 1998; Goodman, 1994). Low levels of estradiol (E2) inhibit GnRH expression and secretion. In ovariectomized animals, GnRH expression and secretion increase. In primate (non-human) and mouse both the duration and amplitude of the circulating estradiol signal are critical for the generation of the normal LH surge.

<ul> <li>selective visualization of GnRH neurons with β-galactosidase (Skinner et al., 1999)</li> <li>detect living cells tagged with green fluorescent protein (GFP) (Spergel et al., 1999; Kato et al., 2003) or calcium sensors in acute brain-slice preparations (Jasoni et al., 2007).</li> <li>sampling of portal blood on a minute-by-minute basis (Evans, 1995).</li> <li>microdialysis in rat (Sisk, et al., 2001).</li> <li>Immunohistochemistry</li> <li>ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS)</li> <li>in situ hybridization was performed to examine total GnRH mRNA and the primary GnRH heterogeneous nuclear RNA transcript.</li> <li>GnRH immunoreactivity and total peptide levels were measured in hypothalamic tissue </li> <li>In vitro: commercial RIA kit, sensitivity of the assay was 1 pg/tube.</li> <li>Hypothalamic explants</li> </ul> Other The development of GnRH-secreting neurons from human pluripotent stem cell (hPSC) could be relevant in the future, but these new cells require characterization of their pulsatile secretory properties (Lund et al., 2016 and Lund et al., 2020).

Biological domains of applicability  Endocrine systems with respect to HPG axis, hormone structure, receptors, synthesis pathways, hormonal axes and degradation pathways are well conserved across vertebrate taxa. Life Stage: Adult, reproductively mature and juveniles. Sex: Applies to both males and females. Taxonomic: Primarily studied in laboratory rodents and humans.  Plausible for most mammals due to conserved hormone pathways regulating hypothalamus-pituitary-gonadal axis processes.  GnRH widespread among vertebrates, including amphibians, reptiles, birds, and mammals (Duan and Allard 2020).

UBERON:0001898

UBERON hypothalamus

CL:0000437

CL gonadtroph

Not Specified Unspecific

Not Specified

All life stages

Not Specified Not Specified Not Specified Not Specified

Duan C, Allard J. 2020.  Gonadotropin-releasing hormone neuron development in vertebrates. General and Comparative Endocrinology. 292: 113465 Dubois SL, Acosta-Martínez M, DeJoseph MR, Wolfe A, Radovick S, Boehm U, Urban JH and Levine JE, 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology, 156:1111-1120. doi: 10.1210/en.2014-1851 Evans NP, McNeilly JR and Webb R, 1995. Effects of indirect selection for pituitary responsiveness to gonadotropin-releasing hormone on the storage and release of luteinizing hormone and follicle-stimulating hormone in prepubertal male lambs. Biol Reprod, 53:237-243. doi: 10.1095/biolreprod53.2.237 Franssen D, Svingen T, Lopez Rodriguez D, Van Duursen M, Boberg J and Parent AS, 2022. A Putative Adverse Outcome Pathway Network for Disrupted Female Pubertal Onset to Improve Testing and Regulation of Endocrine Disrupting Chemicals. Neuroendocrinology, 112:101-114. doi: 10.1159/000515478 Goodman RL (Knobil E NJe), 1994. The neuroendocrine control of the ovine estrous cycle. New York, Raven Press. 659–709 pp. Herbison AE, 1998. Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr Rev, 19:302-330. doi: 10.1210/edrv.19.3.0332 Herbison AE, 2016. Control of puberty onset and fertility by gonadotropin-releasing hormone neurons. Nat Rev Endocrinol, 12:452-466. doi: 10.1038/nrendo.2016.70 Herbison AE, 2018. The Gonadotropin-Releasing Hormone Pulse Generator. Endocrinology, 159:3723-3736. doi: 10.1210/en.2018-00653 Jasoni CL, Todman MG, Strumia MM and Herbison AE, 2007. Cell type-specific expression of a genetically encoded calcium indicator reveals intrinsic calcium oscillations in adult gonadotropin-releasing hormone neurons. The Journal of neuroscience: the official journal of the Society for Neuroscience, 27:860-867. doi: 10.1523/jneurosci.3579-06.2007 Karsch FJ, Bowen JM, Caraty A, Evans NP and Moenter SM, 1997. Gonadotropin-releasing hormone requirements for ovulation. Biol Reprod, 56:303-309. doi: 10.1095/biolreprod56.2.303 Kato M, Ui-Tei K, Watanabe M and Sakuma Y, 2003. Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats. Endocrinology, 144:5118-5125. doi: 10.1210/en.2003-0213 Lund C, Pulli K, Yellapragada V, Giacobini P, Lundin K, Vuoristo S, Tuuri T, Noisa P and Raivio T, 2016. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells. Stem Cell Reports, 7:149-157. doi: 10.1016/j.stemcr.2016.06.007 Lund C, Yellapragada V, Vuoristo S, Balboa D, Trova S, Allet C, Eskici N, Pulli K, Giacobini P, Tuuri T and Raivio T, 2020. Characterization of the human GnRH neuron developmental transcriptome using a GNRH1-TdTomato reporter line in human pluripotent stem cells. Disease Models & Mechanisms, 13 Matsuda F, Ohkura S, Magata F, Munetomo A, Chen J, Sato M, Inoue N, Uenoyama Y and Tsukamura H, 2019. Role of kisspeptin neurons as a GnRH surge generator: Comparative aspects in rodents and non-rodent mammals. Journal of Obstetrics and Gynaecology Research, 45:2318-2329. doi: <a href="https://doi.org/10.1111/jog.14124">https://doi.org/10.1111/jog.14124</a> Plant TM, 2012. A comparison of the neuroendocrine mechanisms underlying the initiation of the preovulatory LH surge in the human, Old World monkey and rodent. Front Neuroendocrinol, 33:160-168. doi: 10.1016/j.yfrne.2012.02.002 Sisk CL, Richardson HN, Chappell PE and Levine JE, 2001. In vivo gonadotropin-releasing hormone secretion in female rats during peripubertal development and on proestrus. Endocrinology, 142:2929-2936. doi: 10.1210/endo.142.7.8239 NOTE: Italics indicate edits from John Frisch October 2025.  A full list of updates can be found in the Change Log on the View History page. AOPWiki 2016-11-29T18:41:25

2026-01-28T14:39:37

Decreased, LH Surge

Cellular

Some sections of the KE description were adapted from AOPs 309. Luteinizing hormone (LH), together with follicle-stimulating hormone (FSH,) is one of the glycoprotein hormones, called gonadotropins, that control gonadal functions interacting trough specific receptors.  LH and FSH are secreted from the anterior pituitary gland in response to GnRH and play a complementary role in follicle development and ovulation. In many species, LH receptor (LHR) is primarily expressed in reproductive organs and functions coordinately to control steroidogenesis and ovulation. LHR is expressed primarily in the theca and granulosa cells of preovulatory ovarian follicles. In ovarian theca cells, LH through interaction with LHR, stimulates the secretion of androgens that are transferred to granulosa cells to be converted to oestradiol (E2) by aromatase. In granulosa cells, FSH stimulates the development of ovarian follicles, while LH action is involved in follicle development and maturation. LH regulates the expression of a variety of genes essential for ovulation which convert from cholesterol to pregnenolone resulting the synthesis of progesterone.  In fish, the nuclear progesterone receptor, which is a member of the nuclear receptor transcription factor superfamily, has been suggested as an essential factor for LH-dependent ovulation. In conclusion, A deficiency in LH and FSH production or action (e.g., receptor activation) compromises gametogenesis and gonadal steroid production thereby reducing female fertility.

(see also KE129) <ul> <li>Circulating concentrations of gonadotropins in humans and common mammalian models (e.g., rodents, many livestock species) can be directly measured using either commercial or custom immunoassays (e.g., enzyme-linked immunosorbent assays, radioimmunoassay, etc.).</li> <li>Similar immunoassay-based methods have been developed for quantifying gonadotropins in fish (e.g., (Govoroun et al., 1998; Amano et al., 2000; Kah et al., 1989; Prat et al., 1996)). However, at present, antibodies specific for distinguishing LH and FSH are only available for a limited number of species, primarily salmonids (Levavi-Sivan et al., 2010).</li> <li>Expression of mRNAs coding for luteinizing hormone beta subunit (LHb) and follicle-stimulating hormone beta subunit (FSHb) tend to fluctuate in parallel in repeat-spawning fish and plasma concentrations LH and FSH in tilapia were also shown to fluctuate in parallel (reviewed in (Levavi-Sivan et al., 2010)). Consequently, the two gonadotropins are treated non-specifically for the purposes of the current key event.</li> <li>For small fish species limited plasma volumes relative to the sensitivity of the available immunoassay methods may impose limits on the ability to measure this key event directly and reliably.</li> </ul> Pulsatile LH secretion <ul> <li>urine LH ➔ immunoassay</li> <li>serum LH ➔a time-resolved immunofluorometric assay (TRIFMA) (Bang 2017)</li> <li>intermittent jugular vein or tail tip bleeding</li> <li>in vivo GCaMP imaging (McQuillan, et al., 2019)  </li> <li>LbT2 cells is the major homologous cell line available for the study of FSH and LH synthesis and secretion ➔ELISA is used.</li> </ul>

In the context of AOP566, the taxonomic applicability is restricted to mammals. However, LH is also present in other species invertebrate species and fish and avian.

UBERON:0000007

UBERON pituitary gland

CL:0000437

CL gonadtroph

Not Specified Unspecific

Not Specified

All life stages

Not Specified Not Specified Not Specified Not Specified

Bang AK, Nordkap L, Almstrup K, Priskorn L, Petersen JH, Rajpert-De Meyts E, Andersson AM, Juul A and Jørgensen N, 2017. Dynamic GnRH and hCG testing: establishment of new diagnostic reference levels. Eur J Endocrinol, 176:379-391. doi: 10.1530/eje-16-0912 McQuillan HJ, Han SY, Cheong I and Herbison AE, 2019. GnRH Pulse Generator Activity Across the Estrous Cycle of Female Mice. Endocrinology, 160:1480-1491. doi: 10.1210/en.2019-00193 AOPWiki 2016-11-29T18:41:25

2025-01-13T16:37:30

Impaired ovulation

Organ

For rodent studies, a few methods of measurement of ovulation are available. Vaginal cytology is a method to assess the stage in the estrous cycle. This is done by flushing the vagina or collecting a vaginal swab followed by staining to observe cytology and determine the stage. Another determinant of ovulation is the presence of corpus lutea. After sacrifice of the female mouse or rat, sections of the reproductive organs are stained, and histological examination is performed to count and identify corpus luteum. Finally, the most direct method is the flushing of the oviduct. Once the estrous stage is determined, female mice can be placed in presence of males to induce ovulation. This is determined the next morning due to the presence of a vaginal plug. The mice are then euthanized, and the oviducts are flushed. The number of ova (mature oocytes) are counted to determine the quality of ovulation. The number of females that have ova in the oviduct is an additional measurement that may be taken into account (Ajayi and Akhigbe, 2020; Byers et al., 2012; Caligioni, 2009). Regarding in vitro studies, involving human or rat granulosa cells for example, ovulation is not measured the same way since it does not occur in these systems. However, mRNA expression and profiles of ovulation markers can be investigated. Certain genes are overexpressed during ovulation and are considered markers of ovulation, such as AREG and EREG, shown in many studies (Fa et al., 2013; Pogrmic-Majkic et al., 2018).

While in many species, ovulation is dependent on LH surge, some species differences exist in regulation of this process. Ovulation is applicable to females starting from puberty and during adult life.

UBERON:0000992

UBERON female gonad

Not Specified Female

Not Specified

Adults

Not Specified

2025-01-03T15:38:51

Increased plasma estradiol to progesterone ratio (estrogen dominance/unopposed estrogen)

Plasma estradiol/progesterone ratio, increase

Organ

The ovarian steroids, estrogens (principally estradiol 17β (E2)) and progestogens (principally progesterone (P4)), regulate female reproduction and have tissue-selective effects in most organs and cell types in the body, including the ovary. In brief, ovarian steroids are synthesized in the granulosa and thecal cell layers of the ovarian follicle and by the corpus luteum after ovulation (McKenna, 2015). Changes in enzyme expression and activity mediated by LH surge shift the balance of steroid hormone synthesis from primarily estrogens before the LH surge to primarily progesterone after the LH surge (Duffy et al., 2019) Fig. 15 in KER3. Synthesised hormones are secreted into ovarian vein and reach the systemic circulation (Levine, 2015). In cycling females, E2 and P4 levels are under the control of the hypothalamus-pituitary-ovary axis and their plasmatic levels varies according to different stages of the estrous cycle in rodents or the different phases of the menstrual phase in women. In cycling female rodents, an increase in E2 begins on the second day of diestrous, which peaks midday on the day of proestrous and then fall during the afternoon of proestrous.  The LH surge, which closely follows the estrogen peak, occurs during the afternoon of proestrous and triggers ovulation approximately 10-12 hours later. After ovulation, luteinisation of the follicular granulosa and thecal cells occurs resulting in the formation of the corpus luteum. The rat corpus luteum secretes progesterone autonomously for approximately 48 hours before becoming non-functional and degenerating over the course of several subsequent estrous cycles. Following lysis of the corpus luteum, a new wave of follicular development begins, and the cycle is repeated (OECD 2009). Consequently, in normally cycling rodents the estradiol to progesterone ratio (E2/P4) varies according to cycle stages being lower during diestrous (Lu, 1979, Nelson, 1981) (see also Fig. 14). Similarly, in women plasma levels of E2 and P4 vary during the menstrual cycle, E2 level increase during the second half of the follicular phase reaches its highest level immediately before ovulation. Plasma levels of progesterone are low during the follicular phase and begin to increase just before the onset of the LH surge and then increase progressively to peak levels 6 to 8 days after ovulation. Consequently, the E2/P4 ratio is lower during the luteal phase (see also Fig. 15). In case of delayed ovulation, the ovarian Graafian follicles persist and continue to produce estrogen while corpora lutea that produce progesterone do not develop resulting in a deficit of progesterone. The E2/P4 ratio is therefore elevated for prolonged periods before ovulation resulting in hormonal imbalance (i.e., relative estrogen dominance/ non-opposed estrogen) (Finch, 2014, Westwood 2008). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/170q4njqx3_Figure_14.png" style="height:516px; width:594px" /> Figure 14. Schematic pattern of typical endocrine changes during the rat estrous cycle (Anderson, 2013) <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/2vie7x0coj_Figure_15.png" style="height:599px; width:551px" /> Figure 15. Estrogen level during estrous cycle and menstrual cycle (Hong and Choi, 2018). (A) The estrous cycle is divided into four stages in mice: proestrous, estrous, metestrous, and diestrous. (B) The menstrual cycle is divided into two phases in humans: follicular phase and luteal phase In aging rodents showing persistent estrous characterized by persistent vaginal cornification (PVC), the hormonal profile is defined by sustained E2 and low P4 (Finch, 2014). The levels of E2 are comparable to the basal values of younger cycling females while P levels are lower. This results in a 2-fold or more increased E2/P4 ratio in aging PVC females compared to the average value of younger cycling females (Lu, 1979; Nelson, 1981). In women, perimenopause is characterized by major hormonal changes. Estradiol levels become erratic and often high, while progesterone levels decrease (in normally ovulatory, short luteal phase or anovulatory cycles) resulting in increased estradiol to progesterone ratio (Prior, 2011). Given the length of time women spend in the transition to menopause, women are exposed to unopposed estrogen (O’Connor, 2009).

Standard methods for serum estrogen and progesterone analysis include Standard methods for serum estrogen and progesterone analysis include <ul> <li>radioimmunoassay (RIA),</li> <li>enzyme-linked immunosorbent assay (ELISA), and</li> <li>multiplex immunoassay.</li> <li>Liquid chromatography/mass spectrometry (LC/MS)-based methods are also becoming more widely used (as cost and sample size requirements decrease), particularly for measurement of estrogens and estrogen metabolites. For P4 and E2, rodent-specific immunoassays are commercially available (Andersson, 2013). </li> </ul>   As estradiol and progesterone levels fluctuate across the ovarian cycle, the stage of estrous cycle at the time of blood collection should be determined to allow appropriate interpretation of the variations. Circadian rhythm should also be taken into consideration i.e., blood sampling should be accomplished in a 3-h time window in the morning and the method of blood sampling should guarantee the lowest possible stress level (ECHA and EFSA, 2018). In OECD TG dedicated to repeated dose toxicity and reproduction, sex hormones data are not routine endpoints. In OECD TG 408, measurement of sexual hormones is optional and should be considered on a case-by-case basis. However, it is not recommended to include female reproductive hormonal measurements in first-tier toxicity studies of standard design. Indeed, due to the limited standard number of animals per group the average number of each animal in each stage of the cycle is generally too few to permit conclusions (Stanislaus, 2012). Specifically designed and statistically powered investigative studies (with appropriate animal numbers, sacrifice at optimum stage of the cycle) are best suited to measure serum hormones in female rodents (Andersson, 2013).

The majority of the information on this KE comes from in vivo studies with rodents. In view of the evolutionary conservation of the steroid hormones and the importance of the balance between of estradiol and progesterone in the reproductive cycles (estrous cycle, menstrual cycle), this key event is applicable to most to other mammalian species. Regulatory Significance of the KE Estrogen and progesterone are steroid hormones that play a pivotal role in the regulation of female reproductive function. Any prolonged imbalance, and especially sustained estrogen dominance may lead to different adverse outcomes on the reproductive system. Changes in hormone levels are not considered as an adverse outcome per se even when they are measured in OECD CF level 4 and 5 assays as defined in The OECD Conceptual Framework for Testing and Assessment of Endocrine (OECD, 2018b). Changes in hormone levels are considered in vivo mechanistic parameters that substantiate evidence for endocrine activity (ECHA and EFSA, 2018).

UBERON:0001969

UBERON blood plasma

2025-01-03T16:02:51

Estradiol availability, increased

Increased E2 availability

Tissue

Currently, there is an existing key event in the AOP Wiki (event number 1973) entitled Increased, estrogens which has a role of KE in one AOP (under development) (number 440 - Hypothalamic estrogen receptors inhibition leading to ovarian cancer). Some sections of the KE description were adapted from that event. In the current AOP, “increased E2 availability” is intended as the increased availability of E2 to its intended biological destination, that is the estrogenic signalling pathways in target tissues (estrogen-sensitive tissues). Biological state The three major forms of endogenous estrogens are estrone (E1), oestradiol (E2, or 17β-oestradiol), and estriol (E3).  Estrogen metabolism is complex and multifactorial (Fig.6). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2024/08/09/7zjvp3k5u5_KE2251_Figure_6.jpg" /> Figure 6. Schematic representation of estrogen metabolism. Dotted circles represent the enzyme system that would influence the bioavailability of oestradiol (Wikoff et al., 2016) Although circulating estrogens exist in a dynamic equilibrium of metabolic interconversions, E2 is the principal intracellular human estrogen and is substantially more potent than its metabolites, E1 and E3 at the receptor level (NCI Thesaurus (NCIt) in PubChem, available at: <a href="https://ncit.nci.nih.gov/ncitbrowser/">https://ncit.nci.nih.gov/ncitbrowser/</a>, version 22.11d). Oestradiol (E2) is principally produced in the ovaries by follicular thecal and granulosa cells under the regulation of follicle-stimulating hormone (FSH) in the reproductive phase in women (Simpson 2003). Before puberty and after menopause E2 is mainly produced in extragonadal tissues, including kidney, breast, brain, liver and fat (Secky et al., 2013). In males it is mainly produced by the Leydig cells in the testis. Vehiculated via the circulatory system to estrogen-sensitive tissues (female reproductive organs, breasts, hypothalamus and pituitary), E2 becomes available to specific estrogen receptors (subtypes alpha (ERα) and beta (ERβ), triggering the estrogenic signalling pathway: the receptor-ligand complex enters the nucleus of the target and promotes the gene expression necessary for the maintenance of fertility and secondary sexual characteristics in females and other effects, such as mild anabolic and metabolic properties, and increased blood coagulability.  E2 also exerts potent agonism of G Protein-coupled estrogen receptor (GPER), which is recognized an important regulator of E2 rapid effects.   The three major forms of endogenous estrogens are estrone (E1), oestradiol (E2, or 17β-oestradiol), and estriol (E3). Although circulating estrogens exist in a dynamic equilibrium of metabolic interconversions, oestradiol is the principal intracellular human estrogen and is substantially more potent than its metabolites, estrone and estriol at the receptor level (NCI Thesaurus (NCIt) in PubChem). In estrogen-responsive organs (uterus, breast, prostate), E2 availability to the estrogen signalling pathway is fine-tuned at cellular level; E2 activation/deactivation is controlled by a local machinery composed by enzymes analogous to those in gonadal tissue (Secky et al., 2013). This “intracrinological” pathway actively contributes to the modification of intracellular levels of E2, modulating its local effects and playing a major role in physiological and pathological conditions in premenopausal and menopausal women, in men and in animal models (Konings et al., 2018). Huhtinen et al., 2012 showed that E2 concentration in the human uterus (endometrium) is up to 10-fold higher in the proliferative phase compared with the secretory phase of the menstrual cycle, and this is accompanied by cyclic changes in intracrine enzyme levels, indicating that steroid exposure is locally cyclically regulated to support endometrial physiology. An important reaction in the intracrine steroidogenesis is the interconversion of 17-keto and 17b-hydroxysteroids controlled by HSD17Bs. 17b-hydroxysteroids (testosterone and E2) have higher affinity for the receptors than the keto-steroids (A4 and E1). This balance determines the final androgenic/estrogenic activity at target tissue level (Konings et al., 2018). In addition, of relevance is the intracellular balance between unconjugated (free, active) and the sulfo-conjugated (inactive) E2. This is controlled by the “sulfatase pathway”, which is based on the interplay between SULT1E1 and STS and contributes to the modulation of E2 effects and protection versus its excess in target tissues (Cui et al., 2013; Cornel, 2018). Biological compartment Gonadal E2 is produced under the control of the hypothalamus-pituitary-ovary (HPO) axis and mainly released into the bloodstream to reach target tissues. In the female reproductive years E2 levels are physiologically subject to cyclic variations in the blood, reaching the highest level immediately before ovulation. Levels of circulating E2 during the follicular phase, pre-ovulatory phase, and luteal phase are 19–140 pg/ml, 110–410 pg/ml, and 19–160 pg/ml, respectively. During the menopause transition, E2 and E1 levels decrease by 85–90% and 65–75% respectively as compared to mean pre-menopausal levels, and in postmenopausal women they are below 35 pg/ml. The above described “intracrinological” pathway actively contributes to the modification of intracellular levels of E2, modulating its local effects and playing a major role in physiological and pathological conditions in premenopausal and menopausal women, in men and in animal models (Konings et al., 2018). As a consequence, cellular E2 levels do not reflect the blood levels (Cornel et al., 2018); in the endometrium they can be up to five-times higher than in serum during the proliferative phase and 1.5-fold higher in the luteal period (Huhtinen et al., 2012, 2014). Levels of estrogens (E2) in both the circulatory (plasma) and tissue compartments should both be considered and could offer a complementary information. Among enzymes relevant in intracrinology, the sulfatase and sulfotransferase (SULT1E1), combined as the sulfatase pathway, represent a major route of estrogen supply and removal in endometrial cells. In physiological conditions the balance of the pathway is shifted towards the formation of free estrone, as indicated by the STS activity, that is few magnitudes higher than that of SULT1E1. Other enzymes relevant for the intracrine regulation and ultimately tissue level of E2 include aromatase CYP19A1 (converting testosterone to oestradiol), hydroxysteroid-dehydrogenase-17B (HSD17B, interconverting estrone and oestradiol)) (reviewed in see Wikoff et al 2015). In addition, other hormones can also contribute to the regulation of E2 at endometrial level. For example, progesterone diminishes estrogenic action in the endometrium by stimulating the local synthesis of 17-hydroxysteroid dehydrogenase and estrogen sulfotransferase, with the effect peaking in the luteal phase. The up regulation of these enzymes decreases intra-tissue estrogen levels and is one of the mechanisms of the uterine antiestrogenic effects of progesterone (Huntinhen et al., 2012). General role in biology Endogenous estrogens are largely responsible for the development and maintenance of the female reproductive system and secondary sexual characteristics. E2 is the principal intracellular human estrogen and is substantially more potent than its metabolites, estrone and estriol at the receptor level (HSD17B in PubChem). In the reproductive phase E2 together with progesterone controls the menstrual cycle and the reproductive functions; it induces endometrial cell proliferation in premenopausal women. E2 effects are mediated by a complex estrogenic signalling, mainly via two nuclear estrogen receptors (ERα and β) and one membrane receptor (GPER); activating genomic and non-genomic actions upon ligand binding (Cornel et al., 2017) Availability of E2 to its receptors is key to trigger the estrogenic signalling in estrogen-sensitive cells. It is related to the levels of circulating estrogens and it is locally fine-tuned by a set of intracellular enzymes (intracrinology). Disruption of estrogen homeostasis resulting in prolonged increased E2 levels, associated with relative decreased progesterone (P4) would be among the leading risk factors for the development of pathological conditions such as endometrial cancer. An estrogen imbalance is associated with endometrial carcinomas in rats (Hilliard and Norris, 1979; Fox, 1984), spontaneous endometrial adenocarcinomas in the Donryu rat (Nagaoka et al., 1990), and in F344 rats (Tang et al., 1984). Epidemiological studies showed increased endometrial cancer risks among postmenopausal women who have increased blood levels of oestradiol (reviewed in Kaaks et al., 2002) as well as of steroid precursors of E2 (testosterone, androstenedione, DHEA, DHEA-S, estrone and estrone-S) compared with healthy controls (Cornel et al., 2017). This can be interpreted in the light of the “unopposed estrogen” hypothesis, which proposes that endometrial cancer may develop as a result of the mitogenic effects of estrogens, when these are insufficiently counterbalanced by progesterone.  Since the majority of the endometrial cancer patients are postmenopausal women, local formation of E2 from circulating precursors either from circulating androgens via the aromatase pathway or from E1S via the sulfatase pathway becomes important.

This KE refers to increased E2 availability in the uterus, where availability is intended as the extent E2 becoming completely available to its intended biological destination ER. Circulating E2 levels are relevant in determining E2 levels available to endometrial cells in the uterus. These are measurable by a variety of standardised routine methods in humans and animals. Circulating levels are intracellularly subject to a complex intracrine control mechanism, activating and deactivating E2 available to receptors. The translation of circulating E2 levels into E2 availability in uterus needs further exploration and it is not fully addressed in this Scientific Opinion. It has been acknowledged that availability of E2 in uterus is strongly associated with estrogenic activity. Therefore, E2 availability in target tissue can be measured with standardised methods evaluating estrogenicity, that are listed below. In vivo <ul> <li>OECD TG 440 Uterotrophic bioassay in rodents</li> </ul> Others Overall, no high resolution and/or standardised methods exist to quantify steroids within target tissues. However, in recent decades it has been recognized that steroid concentration within tissues is modulated independently from circulating levels and therefore investigations in this field are initiated (Cobice et al., 2013). Uterine response to estrogens involves the activation of a large pattern of estrogen-sensitive genes: <ul> <li>Expression of Calbindin-D9k (CaBP-9k) gene and protein. The 9 kilodalton vitamin D-dependent calcium binding protein (CaBP9k), calbindin-D9k, is expressed in the intestine and uterus of mammals (bgee.org; L’ Horset et al., 1990) Different studies demonstrated that in the mammals uterus, the expression of CaBP-9k is regulated by hormones such as E2 and P4.  L’Horset et al., 1993; review by Choi et al., 2005).</li> <li>Complement 3 (C3). It has been demonstrated that C3 could be regarded as an estrogen sensitive marker in rat uterus (Diel et al., 2000; Sundstrom et al., 1989)</li> </ul> In addition to this, it has been reported that blood glutamate levels are inversely related to plasma estrogen and progesterone level in plasma (Zlotnik et al., 2011). In the regulatory area standard methods are available for serum estrogen analysis include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and multiplex immunoassay. Liquid chromatography/mass spectrometry (LC/MS)-based methods are also becoming more widely used (as cost and sample size requirements decrease), particularly for measurement of estrogens and estrogen metabolites. For E2, rodent-specific immunoassays are commercially available (Andersson, 2013). In the OECD TG 422 dedicated to repeated dose toxicity and reproduction, sex hormones data are not routine endpoints. In OECD TG 408, measurement of sexual hormones is optional and should be considered on a case-by-case basis. However, it is not recommended to include female reproductive hormonal measurements in first-tier toxicity studies of standard design. Indeed, due to the limited standard number of animals per group the average number of each animal in each stage of the cycle is generally too few to permit conclusions (Stanislaus, 2012). Specifically designed and statistically powered investigative studies (with appropriate animal numbers) are best suited to measure serum hormones in female rodents (Andersson, 2013).

Taxonomic Applicability: mammals. Endocrine systems with respect to hormone structure, receptors, synthesis pathways, hormonal axes and degradation pathways are well conserved across vertebrate taxa especially in the case of estrogen, androgen and thyroid hormones and steroidogenesis (OECD TG 150) Life Stage Applicability: This KE is applicable to adulthood - reproductive and post reproductive (menopausal, aging) phases. Sex Applicability: males, females

UBERON:0000995

UBERON uterus

Not Specified Male Not Specified Female

Not Specified

Adult

Not Specified

Adult, reproductively mature

Not Specified

Old Age

Not Specified

2024-11-21T17:40:26

Persistent vaginal cornification

Tissue

Vaginal cornification refers to the thickening (increased cell layers) and process where vaginal epithelial cells harden by addition of keratin during estrus. Biological state The rodent cycle is subdivided in four subsequent phases, proestrous, estrous, metestrous (or diestrous 1) and diestrous (or dioestrous 2).  It is characterized by hormonal variation and consequent behavioural, morphological and physiological changes to the reproductive tract (ovary, uterus and vagina), describing these four phases.  Proestrous is the period during which pre-ovulatory development of the follicles takes place in the ovary. Estrous is the brief interval during which the female accepts the male and during which ovulation occurs. Next is metoestrous, the early luteal phase, followed by diestrous. The duration of the estrous cycle of rodent strains most commonly used in toxicology studies is typically 4 to 5 days (Goldman, 2007). For each phase of the cycle, the ovary, uterus and vagina have typical morphologic characteristics (Dixon, 2014). Biological role in physiology The keratinization of vaginal epithelial cells (vaginal cornification) that typically characterizes the day of estrous in the cycling rodents, is a consequence of the rising level of estradiol that peaks around midday on proestrous (Goldman, 2007). Repeated failure of ovulation over successive days produces irregular cycles characterized by a persistent vaginal cornification (PVC). This state is called persistent estrous or constant estrous. Females in persistent estrous exhibit constant sexual receptivity (Westwood, 2008). The term persistent estrous (PE) in adult rats denotes failure of at least two consecutive estrous cycles as documented by cornified vaginal smears for 10 or more days (Singh, 2005). Exposure to estrogenic compounds has commonly been reported to result in a persistent vaginal cornification (Goldman, 2007). As such, persistent vaginal cornification represents a good marker of prolonged E2 bioavailability in the uterus in regulatory studies. It is noteworthy that persistent estrous is not equivalent to the true estrous phase. Indeed, at ovary level, estrous stage is characterized by the presence of new basophilic corpora lutea (CP) formed after the current ovulation and an absence of healthy (non-atretic) tertiary follicles. On the other end, a state of persistent estrous is characterized by ovaries with absence of basophilic corpora lutea, increased number of antral follicles and follicular cysts. Persistent estrous is comparable with the anovulatory cycles in women (Horvath 2004; Finch, 2014).

Regular cyclicity is one of the key parameters in assessment of female reproductive function in rodents. Parameters assessed for cyclicity: <ul> <li>Number of cycling females</li> <li>Number of females with regular cycles</li> <li>Number of cycles</li> <li>Estrous cycle length</li> <li>Percentage of time spent in the various estrous cycle stages. Abnormal cycles were defined as one or more estrous cycles in the 21-day period with prolonged estrous (≥3 days) or prolonged diestrous (≥4 days) within a given cycle (Goldman, 2007).</li> </ul> Visual assessment Visual observation of the vagina is the fastest method. It requires no special equipment and is best used when only proestrous or estrous stages need to be identified. It is non-invasive but the findings from this technique are observer-dependent. In the proestrous phase, the vaginal opening appears full, swollen and moist (Ajayi, 2020). The vaginal opening of mice in proestrous is characterized by swollen, moist, pink tissue. The opening is wide and there are often wrinkles or striations along the dorsal and ventral edges. As the mouse enters estrous, the vaginal opening becomes less pink, less moist, and less swollen (Byers, 2012). Vaginal smear Estrous cycle can be monitored in the rat and mouse by observing the changes in the vaginal smear cytology. It seems to be the most common technique used to determine the phases of the estrous cycle. It is non-invasive and relatively inexpensive (Goldman, 2007; Byer, 2012; Ajayi, 2020) and OECD guidelines (<a href="http://www.oecd.org">www.oecd.org</a>). The estrous phase shows abundant non-nucleate cornified epithelial cells. The cytoplasm is granular, and the cells are irregular in shape. Persistent estrous is characterized by cornified vaginal smears for 10 or more days. Monitoring of estrous cyclicity is included in OECD test guidelines (TG 421: Reproduction/Developmental Toxicity Screening Test, 2016; TG 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 2016; TG 416: Two-Generation Reproduction Toxicity, 2001; TG 443: Extended One-Generation Reproductive Toxicity Study, 2018; and in USA EPA OCSPP 890.1450. Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female Rats Assay, 2011. Histological examination of the reproductive organs This technique is invasive and does not allow individual estrous cyclicity assessment. The minimal requirement for estrous staging evaluation by histology is the complete longitudinal sections of the vagina and cervix, transverse sections of the mid-portion of both uterine horns, and middle sections of both ovaries (Goldman 2007; Byers, 2012). Persistent estrous is characterized in ovary by the presence of numbers of ovarian follicular cysts (due to pre-ovulatory follicles losing the capacity to ovulate) absence of basophilic corpora lutea and decreased corpora lutea while uterine epithelium becomes tall columnar and vagina may show cornification (Westwood, 2008, Dixon, 2014, Shirai 2017, see also Fig. 16). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/13/whddpve6k_Figure_16.png" /> In OECD TG 443: For adult P and F1 females, a vaginal smear is examined on the day of necropsy to determine the stage of the estrous cycle and allow correlation with histopathology in reproductive organs.  Vaginal smear at necropsy is also required in OECD TG 408, OECD TG 421, OECD TG 422 while it is optional in OECD TG 407. Other methods Vaginal wall impedance and urine biochemistry are alternative methods. However, no standard values are available for commonly used laboratory animals (Ajayi, 2020).

The majority of the information comes from in vivo studies with rodents. Regulatory Significance of the KE While an evaluation of the estrous cycle in laboratory rodents can be a useful measure of the integrity of the hypothalamic-pituitary-ovarian reproductive axis, it can also serve as a way of ensuring that animals exhibiting abnormal cycling patterns are excluded from a study prior to exposure to a test compound. When incorporated as an adjunct to other endpoint measures, a determination of a female's cycling status can contribute important information about the nature of a toxicant insult to the reproductive system. In doing so, it can help to integrate the data into a more comprehensive mechanistic portrait of the effect, and in terms of risk assessment, may provide some indication of a toxicant's impact on human reproductive physiology. Significant evidence that the estrous cycle (or menstrual cycle in primates) has been disrupted should be considered an adverse effect (OECD, 2008). Included should be evidence of abnormal cycle length or pattern, ovulation failure, or abnormal menstruation (AOP 7).

UBERON:0001344

UBERON epithelium of vagina

Not Specified Female

Not Specified

Adult, reproductively mature

Not Specified

Ajayi AF and Akhigbe RE, 2020. Staging of the estrous cycle and induction of estrus in experimental rodents: an update. Fertil Res Pract, 6:5. doi: 10.1186/s40738-020-00074-3 Byers SL, Wiles MV, Dunn SL and Taft RA, 2012. Mouse estrous cycle identification tool and images. PLoS One, 7:e35538. doi: 10.1371/journal.pone.0035538 Dixon D, Alison R, Bach U, Colman K, Foley GL, Harleman JH, Haworth R, Herbert R, Heuser A, Long G, Mirsky M, Regan K, Van Esch E, Westwood FR, Vidal J and Yoshida M, 2014. Nonproliferative and proliferative lesions of the rat and mouse female reproductive system. J Toxicol Pathol, 27:1s-107s. doi: 10.1293/tox.27.1S Finch CE, 2014. The menopause and aging, a comparative perspective. J Steroid Biochem Mol Biol, 142:132-141. doi: 10.1016/j.jsbmb.2013.03.010 Goldman JM, Murr AS and Cooper RL, 2007. The rodent estrous cycle: characterization of vaginal cytology and its utility in toxicological studies. Birth Defects Res B Dev Reprod Toxicol, 80:84-97. doi: 10.1002/bdrb.20106 Horvath JE, Toller GL, Schally AV, Bajo AM and Groot K, 2004. Effect of long-term treatment with low doses of the LHRH antagonist Cetrorelix on pituitary receptors for LHRH and gonadal axis in male and female rats. Proc Natl Acad Sci U S A, 101:4996-5001. doi: 10.1073/pnas.0400605101 OECD, 2007. Test No. 440: Uterotrophic Bioassay in Rodents : A short-term screening test for oestrogenic properties. OECD, 2009. Environment Directorate, Series on testing and assessment number 106. Guidance document for histologic evaluation of endocrine and reproductive tests in rodents. Part 3. Section 2. ENDOCRINE CONTROL OF THE OESTROUS CYCLE. In: OECD series on testing and assessment. . Paris, OECD Publishing. OECD, 2018a. Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption. OECD, 2018b. Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents. Organisation for Economic Co-operation and Development.  2001. Test No. 416: Two-Generation Reproduction Toxicity, OECD Guidelines for the Testing of Chemicals, Section 4.  https://www.oecd.org/content/dam/oecd/en/publications/reports/2001/01/test-no-416-two-generation-reproduction-toxicity_g1gh2941/9789264070868-en.pdf (retrieved 15 July 2026) Shirai N, Houle C and Mirsky ML, 2015. Using Histopathologic Evidence to Differentiate Reproductive Senescence from Xenobiotic Effects in Middle-aged Female Sprague-Dawley Rats. Toxicol Pathol, 43:1158-1161. doi: 10.1177/0192623315595137 Singh KB, 2005. Persistent estrus rat models of polycystic ovary disease: an update. Fertil Steril, 84 Suppl 2:1228-1234. doi: 10.1016/j.fertnstert.2005.06.013 US Environmental Protection Agency (EPA).   1998.  Health Effects Test Guidelines OPPTS 870.3800 Reproduction and Fertility Effects https://ntp.niehs.nih.gov/sites/default/files/iccvam/suppdocs/feddocs/epa/epa_870_3800.pdf (retrieved 19 Jan 2026) Westwood FR, 2008. The female rat reproductive cycle: a practical histological guide to staging. Toxicol Pathol, 36:375-384. doi: 10.1177/0192623308315665 NOTE: Italics indicate edits from John Frisch January 2026.  A full list of updates can be found in the Change Log on the View History page. AOPWiki 2025-01-13T15:29:37

2026-08-25T10:11:31

<upstream-id>eb7d0a39-4b57-4db8-adbb-e91e85790d6f</upstream-id> <downstream-id>ae5e9774-5d4b-4759-b0d7-fa3a143b5673</downstream-id>

The development of the KER is based on structured literature review of records. Description for KER is based on reviews and books on the topic. The method used are described in <a href="https://efsa.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.2903%2Fj.efsa.2023.7744&file=efs27744-sup-0006-Annex-B.1.docx">Annex B.1</a>.

The major role of the GnRH is to interact with its receptor expressed by gonadotropic cells in the anterior pituitary inducing the release of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The biological plausibility to the key event relationship is supported by the physiological role of GnRH surge in evoking LH surge.

In vivo Atrazine The effect of atrazine (showing aromatase inducing properties) in lowering LH surge was demonstrated in several papers (Foradori 2009, 2011, 2013, 2014). In Foradori et al., 2009, ovariectomized adult Wistar rats were administered atrazine (50, 100, or 200 mg/kg of body weight daily by gavage) or vehicle for 4 days. Animals were primed with estrogen and progesterone to induce an evening LH surge. Blood samples were obtained over the afternoon and evening and plasma was assayed for LH and FSH. Another cohort of animals was transcardially perfused in the afternoon to examine GnRH activation using FOS immunoreactivity. 4-day treatment with atrazine resulted in a significant reduction in the magnitude of the LH and FSH surges, and this corresponds to a decrease in GnRH neurons expressing FOS immunoreactivity. Examining LH levels and GnRH activation 2 days and 4 days after atrazine withdrawal, within 4 days (but not 2 days) after cessation of atrazine treatment, measures of hypothalamic-pituitary-gonadal (HPG) activation returned to normal (Fig. 4 and Fig. 5). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/6ui5xvnot3_Figure_4.png" style="height:297px; width:881px" /> Figure 4. Photomicrographs of eGFP-GnRH (green), FOS (red), and overlay of control animals and animals treated with 200 mg/kg of atrazine; bar 1&frasl;4 50 lm. Histogram of the mean ± SEM percentage of GnRH neurons immunoreactive for FOS. *Significant difference from control and 50 mg/kg of atrazine (P < 0.05). GnRH-ir, GnRH immunoreactive. From Foradori et al., 2009   <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/4gpww0eq1w_Figure_5.png" style="height:264px; width:685px" /> Figure 5. Histograms depicting the mean ± SEM peak LH levels (A) and the percentage of GnRH cells immunoreactive for FOS (B) on the final day of atrazine treatment (Day 0) and 2 days (Day 2) and 4 days (Day 4) after final atrazine treatment. *Significant difference from control group (p<0.05). #Significant difference between atrazine-treated groups (p<0.05). GnRH-ir, GnRH immunoreactive.  From Foradori et al., 2009. In Foradori et al., (2013) ATR treatment (200 mg/kg or vehicle daily for 4 days via gavage to ovariectomized adult female Wistar rats were) resulted in no changes to GnRH gene expression, peptide levels, or immunoreactivity but a reduction in GnRH pulse frequency and an increased pulse amplitude. These findings suggest that ATR acts to inhibit the secretory dynamics of GnRH pulses without interfering with GnRH mRNA and protein synthesis. Atrazine failed also to alter pituitary sensitivity to the GnRH at any dose used. Tributyltin (TBT) Ovariectomized Wistar rats exposed to 100 ng TBT/kg bw/d for 15 days had reduced hypothalamic GnRH mRNA expression and exogenous KISS responsiveness, decreased basal and surge LH levels, reduced exogenous GnRH responsiveness, as well as decreased expression of both estrogen receptor (ER) (ERα and ERβ) in the pituitary (Fig. 6, Sena et al., 2017). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/91oc3ev3dg_Figure_6.png" style="height:303px; width:414px" /> Figure 6. Tributyltin (TBT) rats have an abnormal estrous cycle. Proestrus (P), estrus (E), metestrus-diestrus (M-D). (C) Baseline and surge serum LH levels (n = 5–6). (D) qPCR analysis of GnRH mRNA extracted from female rat hypothalamic tissue (n = 4). (E) GnRH stimulation test. Assessment of serum LH levels 10 min after injection of GnRH agonist (0.1 nmol/kg via ip). Increased serum LH levels in rats treated with a GnRH agonist (indicated as GnRH +) were identified in both control and TBT rats (n = 5–6). (F) Kisspeptin stimulation test. Assessment of serum LH levels 10 min after injection of kisspeptin-10 (10 nmol ip). Increased serum LH levels in rats treated with kisspeptin (indicated as Kiss +) were identified in both control and TBT rats (n = 5–6). Significant differences compared **p ≤ 0.01 and ***p ≤ 0.01 vs saline-treated control groups (indicated as GnRH − or Kiss −). Two-way ANOVA indicated an interaction between groups and treatment (Tukey's multiple comparison test). From Sena et al., 2017. As reported by Sena et al., (2017), TBT leads to a decreased LH and FSH secretion as result of a lower stimulatory effect induced by reduced GnRH expression levels. A reduction in the hypothalamic GnRH mRNA expression, the impairment of Kisspeptin-stimulated GnRH release was also observed. As a consequence: <ul> <li>a reduction in the serum E2 levels and an increase in the serum T levels, leading to abnormal regulation of feedback mechanisms.</li> <li>increased leptin secretion may also associate with the impairment of Kisspeptin-stimulated GnRH release.</li> </ul> Endopeptidases The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. In recent studies peptidases acting on GnRH at the level of the median eminence have been isolated, the most important appearing to be the zinc metalloendopeptidase-24.15 (EP 24.15; 119 –121). These observations suggest that endopeptidases exert a physiological role in shaping the GnRH signal directed at the pituitary gonadotrophs at the time of the LH surge and that this may be regulated by gonadal steroids including estrogen. Ladsun et al., (1989) concluded that EP24.15 is the dominant factor determining the in vivo LHRH (former name of GnRH) degradation, suggesting a role of the enzyme in the regulation of the amount LHRH reaching pituitary gonadotropes (see also Molineaux et al., 1988). EP 24.15 cleaves the central Tyr5-G1y6 bond in GnRH (Orlowski et al., 1983; Chu and Orlowski, 1985), and it is the primary responsible of the degradation of LHRH in hypothalamic and pituitary membrane preparations and in intact AtT20 cells (an anterior pituitary tumor cell line) (Molineaux, et at., 1988, see Table 1). After intracerebroventricular (icv). administration of GnRH, only about 1% of this peptide was recovered from brain after 1 hr. Concurrent administration of GnRH and N- [1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB, a specific inhibitor of EP 24.15), led to a more than 10-fold increase in GnRH recovery. Administration of N-[1- (RS)-carboxy-3-phenylpropyl]-Phe-pAB(cFP-F-pAB) or captopril, inhibitors of other endopeptidases as “enkephalinase” (EP 24.1 1) and angiotensin converting enzyme respectively, did not significantly increase LH recovery. Intravenous administration of GnRH and either cFP-F-pAB or cFP-AAF-pAB but not captopril, led to an increase in the half-life of GnRH from 10 min to 15 and 20 min, respectively. Concurrent administration of both inhibitors resulted in a dramatic 8-fold increase in the half-life of GnRH Concentrations of plasma GnRH 65 to 80 min after administration of inhibitors were 100- to 200-fold than those in controls. Table 1.  Inhibition of LHRH (GnRH) breakdown by peptidase inhibitors in rats. From Molineaux, 1988. <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/6gy0304b8m_Table_1.png" style="height:350px; width:729px" /> Wu et al., in 1997 (Fig. 7) demonstrated that Endopeptidase EC 3.4.24.15 in the Rat Median Eminence and Hypophysial Portal Blood modulates the LH surge (an inhibition of its activity with a specific inhibitor augmented the steroid-induced LH increase in ovariectomized rats). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/8sb3v9jy1r_Figure_7.png" style="height:231px; width:606px" /> Figure 7. shows the mean (±SE) serum LH concentration (ng/ml) in rats treated with saline only (dashed line), saline plus the EP24.11 inhibitor (dotted line), or saline plus the EP24.11 inhibitor plus the EP24.15 inhibitor (solid line). (B) shows the mean (±SE) integrated area under the curve of serum LH concentration in rats administered saline alone, saline+EP24.11 inhibitor, and saline+EP24.11 inhibitor+EP24.15 inhibitor (from Wu et al., 1997). GABA modulators Muscimol (a selective agonist of GABAAR) significantly decreased GnRH biosynthesis in in the preoptic area (POA, anterior (AH) and ventromedial (VMH) hypothalamus, stalk/median eminence (SME), and led to analogous changes in plasma LH concentration (Ciechanowska et al., 2019, see Fig. 8 and 9). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/2zulusktds_Figure_8.png" style="height:270px; width:465px" /> Figure 8. Effect of muscimol and bicuculline on the level of GnRH in the preoptic area (POA), anterior hypothalamus (AH), ventromedial hypothalamus (VM) and the stalk median eminence (SME) of follicular-phase ewes, *p < 0.05, **p < 0.01, ***p < 0.001. Asterisks indicate values that differ significantly from the control group animals. Data are the mean ± SEM, n=6 animals per group (one-way ANOVA) (from Ciechanowska et al., 2019). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/9f1ni7h3tm_Figure_9.png" style="height:517px; width:239px" /> Figure 9. Effect of muscimol on the level of LH (A) and LH pulse frequency (B) in blood plasma of follicular-phase ewes, *p<0.05. Asterisks indicate values that differ significantly from the control group animals. Data are the mean SEM, n =6 animals per group (one way ANOVA, Wilcoxon test) (from Ciechanowska et al., 2019). Light stimulation In a proposed frequency-dependent control of GnRH neuron firing by RP3V neurons (Piet et al., 2018), neurons using GABA alone (green), kisspeptin alone (blue) and both transmitters innervate GnRH neuron cell bodies and proximal dendrites. At low firing frequencies (2 Hz), RP3V neuronal populations only release GABA that generates small transient increases in GnRH neuron firing (green) and has no substantial impact upon LH secretion (red). At higher firing frequencies (10 Hz), proposed to occur on the afternoon of proestrus, RP3V inputs to GnRH neurons now release kisspeptin (blue) to generate the preovulatory LH surge (red) (see also Fig. 10). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/03/7pdcxvy495_Figure_10.jpg" style="height:427px; width:568px" /> Figure 10. Schematic diagram showing proposed frequency-dependent control of GnRH neuron firing by RP3V neurons. Neurons using GABA alone (green), kisspeptin alone (blue) and both transmitters innervate GnRH neuron cell bodies and proximal dendrites (from Piet et al., 2018).   Dose concordance and temporal concordance See <a href="https://efsa.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.2903%2Fj.efsa.2023.7744&file=efs27744-sup-0008-Annex-B.3.xlsx">Annex B.3</a>.

Atrazine <ul> <li>the effective doses are about 150 times the NOAEL of atrazine (WHO, 2010)</li> </ul> Muscimol, TBT, Atrazine, endopeptidase The Risk of Bias (RoB) of the primary research study was not evaluated. For muscimol opposite effect reported when tested in vitro or in other in vivo models.

Not applicable/not investigated in detail.

At present we are unaware of any well-established quantitative relationships between Kea and KEb.

Not Specified Unspecific

Not Specified

All life stages

Not Specified

Chu TG and Orlowski M, 1985. Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides. Endocrinology, 116:1418-1425. doi: 10.1210/endo-116-4-1418 Ciechanowska MO, Łapot M, Kowalczyk M, Malewski T, Brytan M, Antkowiak B and Przekop F, 2019. Does kisspeptin participate in GABA-mediated modulation of GnRH and GnRH receptor biosynthesis in the hypothalamic-pituitary unit of follicular-phase ewes? Pharmacol Rep, 71:636-643. doi: 10.1016/j.pharep.2019.02.019 Foradori CD, Hinds LR, Hanneman WH and Handa RJ, 2009. Effects of atrazine and its withdrawal on gonadotropin-releasing hormone neuroendocrine function in the adult female Wistar rat. Biol Reprod, 81:1099-1105. doi: 10.1095/biolreprod.109.077453 Foradori CD, Hinds LR, Quihuis AM, Lacagnina AF, Breckenridge CB and Handa RJ, 2011. The differential effect of atrazine on luteinizing hormone release in adrenalectomized adult female Wistar rats. Biol Reprod, 85:684-689. doi: 10.1095/biolreprod.111.092452 Foradori CD, Sawhney Coder P, Tisdel M, Yi KD, Simpkins JW, Handa RJ and Breckenridge CB, 2014. The effect of atrazine administered by gavage or in diet on the LH surge and reproductive performance in intact female Sprague-Dawley and Long Evans rats. Birth Defects Res B Dev Reprod Toxicol, 101:262-275. doi: 10.1002/bdrb.21109 Foradori CD, Zimmerman AD, Hinds LR, Zuloaga KL, Breckenridge CB and Handa RJ, 2013. Atrazine inhibits pulsatile gonadotropin-releasing hormone (GnRH) release without altering GnRH messenger RNA or protein levels in the female rat. Biol Reprod, 88:9. doi: 10.1095/biolreprod.112.102277 Ladsun A, Reznik S, Molineaux CJ and Orlowski M, 1989. Inhibition of endopeptidase 24.15 slows the in vivo degradation of luteinizing hormone-releasing hormone. J Pharmacol Exp Ther, 251:439-447 Molineaux CJ, Lasdun A, Michaud C and Orlowski M, 1988. Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo. J Neurochem, 51:624-633. doi: 10.1111/j.1471-4159.1988.tb01084.x Orlowski M, Michaud C and Chu TG, 1983. A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides. Eur J Biochem, 135:81-88. doi: 10.1111/j.1432-1033.1983.tb07620.x Piet R, Kalil B, McLennan T, Porteous R, Czieselsky K and Herbison AE, 2018. Dominant Neuropeptide Cotransmission in Kisspeptin-GABA Regulation of GnRH Neuron Firing Driving Ovulation. The Journal of neuroscience : the official journal of the Society for Neuroscience, 38:6310-6322. doi: 10.1523/jneurosci.0658-18.2018 Sena GC, Freitas-Lima LC, Merlo E, Podratz PL, de Araújo JFP, Brandão PAA, Carneiro MTWD, Zicker MC, Ferreira AVM, Takiya CM, de Lemos Barbosa CM, Morales MM, Santos-Silva AP, Miranda-Alves L, Silva IV and Graceli JB, 2017. Environmental obesogen tributyltin chloride leads to abnormal hypothalamic-pituitary-gonadal axis function by disruption in kisspeptin/leptin signaling in female rats. Toxicology and Applied Pharmacology, 319:22-38. doi: <a href="https://doi.org/10.1016/j.taap.2017.01.021">https://doi.org/10.1016/j.taap.2017.01.021</a> World Health Organization. Pesticide Residues in Food 2007: Toxicological Evaluations. Vol. 23. World Health Organization, 2010. Wu TJ, Pierotti AR, Jakubowski M, Sheward WJ, Glucksman MJ, Smith AI, King JC, Fink G and Roberts JL, 1997. Endopeptidase EC 3.4.24.15 presence in the rat median eminence and hypophysial portal blood and its modulation of the luteinizing hormone surge. J Neuroendocrinol, 9:813-822. doi: 10.1046/j.1365-2826.1997.00637.x AOPWiki 2025-01-03T12:58:28

2025-01-03T14:46:27

<upstream-id>ae5e9774-5d4b-4759-b0d7-fa3a143b5673</upstream-id> <downstream-id>7cff47f7-d02f-4906-9b98-d1fc33729c7f</downstream-id> Luteinizing hormone (LH) is a gonadotropin that is necessary for sexual maturation, ovulation, and therefore fertility. It is part of the glycoprotein hormone family and is organized as a heterodimer with a common α-subunit and a specific β-subunit (Padmanabhan et al., 2018). An LH surge is needed and responsible for the downstream pathways that induce ovulation; this includes resumption of meiosis in the oocyte and cellular changes that allow rupture of the follicle to release the egg for fertilization. It increases intrafollicular proteolytic enzymes, weakening the wall of the ovary and allowing for passage of the mature follicle (Robker et al., 2018). As follicles grow, estrogen synthesis increases in the female ovary. This in turn promotes GnRH pulses in the hypothalamus and increases the levels of luteinizing hormone (LH) released from the anterior pituitary. The circulating LH can then interact with its receptor (LHCGR) in antral follicles and stimulate ovulation (Duffy et al., 2019). In addition to LH, which is eliminated from the serum quickly, human chorionic gonadotropin (hCG), usually secreted during pregnancy, has a higher affinity with the receptor LHCGR and found longer in the serum than LH. Therefore, hCG is preferentially used for ovulation stimulation in fertility treatments for women and in animal studies (Russell and Robker, 2007). Without the LH surge, the downstream pathways are not able to function and as a result, ovulation does not occur. If the LH surge is delayed, then ovulation may be delayed as well and fails to occur within the correct time window. This can have a negative impact on the reproductive health of females and perturb the estrous cycle.

The LH surge is a tightly controlled phenomenon in female reproductive cycles. Ovulation must occur within a specific window in the estrous cycle as the body prepares for a potential pregnancy. Physiologically, the LH surge induces the activation of many signalling cascades that are needed to provoke ovulation. LH through its receptor, LHCGR, increases the intracellular cAMP that activates the PKA pathway. This is considered the canonical pathway activated by LH. PKA then can activate CREB that is then translocated to the nucleus for transcription of target genes needed for ovulation. In addition, the LH surge is also necessary for the activation of Erk 1/2. The activation of the Erk1/2 and MAPK pathway allows activation of EGF-like ligands important for cumulus oocyte complex (COC) expansion as well as transcription factors such as C/EBPα/β that induce expression of genes essential for follicular rupture, both being important steps for correct ovulation. An increase in progesterone receptor (PGR) expression, due to the LH surge, is observed in granulosa cells of preovulatory follicles in most species examined, including humans, rodents and monkeys, leading to the increase in progesterone locally. Several PGR regulated genes have been demonstrated to play critical roles in ovulation, including proteases ADAMST1 and CTSL that break down the follicular wall at the time of ovulation. Following the LH surge, the granulosa cells have an increase in inflammatory genes (such as COX2) in addition to the proteases (Duffy et al., 2019; Robker et al., 2018). Issues can arise in one of these pathways leading to subsequent problems in ovulation. In addition, it has been shown in many studies that without this surge or with a delayed surge, ovulation is perturbed.

Evidence in Rodents: A few knockout mouse strains have been studied extensively to investigate ovulation and hormonal changes in female mice. For example, a study generated LHCGR knockouts and characterized their phenotype. As expected, these female mice were sterile with delayed ovulation and decreased level of serum and ovarian estradiol and progesterone with an increase in serum LH but no difference in pituitary LH levels (Lei et al., 2001; Zhang et al., 2001). Another study focused directly on LH action and created a LHβ knockout and found that the female mice were sterile. Although theca cell morphology looked normal, oocytes degenerated, serum levels of progesterone and estradiol reduced and steroidogenic enzyme expression (Cyp11a1, Cyp19a1, Cyp17a1) decreased. In addition, estrous cycles were abnormal, and no ovulation was observed (Ma et al., 2004, see also Table 2). Evidence in humans: Although mutations in LH or its receptor are rare, in certain families, these mutations were found. Individuals with mutations in the LH receptor were considered “LH resistant” as they do not respond to LH. The women from the different families had similar phenotypes and were shown to exhibit normal puberty but found to have amenorrhea and were not fertile (Latronico et al., 1996, 1998; Prado Arnhold et al., 1997; Stavrou et al., 1998; Toledo et al., 1996, see also Table 3) . Table 2. Evidence in rodents <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; margin-left:48px; width:593px"> <tbody> <tr> <td style="background-color:#d9e2f3; border-bottom:3px solid black; border-left:3px solid black; border-right:1px solid black; border-top:3px solid black; vertical-align:top; width:168px"> Mouse models </td> <td style="background-color:#d9e2f3; border-bottom:3px solid black; border-left:none; border-right:1px solid black; border-top:3px solid black; vertical-align:top; width:247px"> Observations </td> <td style="background-color:#d9e2f3; border-bottom:3px solid black; border-left:none; border-right:3px solid black; border-top:3px solid black; vertical-align:top; width:178px"> Reference </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:168px"> LHβ KO </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:247px"> Infertility was observed in both sexes as well as perturbed gonadal growth. Estradiol and progesterone level were decreased compared to control and degenerating antral follicles were found. In addition, genes coding for steroid biosynthesis enzymes demonstrated a decreased expression and the expression of COX2, (a marker for ovulation) was also reduced. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px"> (Ma et al., 2004) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:168px"> LH/hCG receptor KO (Deletion of promotor region and most of exon 1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:247px"> Male and female mice were infertile. Females contained smaller gonads with underdeveloped genitalia and no preovulatory follicles were observed. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px"> (Lei et al., 2001) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:168px"> LuRKO (Deletion of exon 11) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:247px"> The female Kos ovaries weighed less than control and had delayed vaginal opening to 35-38 days compared to 30-32 days in WT mice. No preovulatory follicles were found. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:178px"> (Zhang et al., 2001)   </td> </tr> </tbody> </table> Table 3. Evidence in humans <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none; margin-left:48px; width:594px"> <tbody> <tr> <td style="background-color:#d9e2f3; border-bottom:3px solid black; border-left:3px solid black; border-right:1px solid black; border-top:3px solid black; vertical-align:top; width:169px"> Humans </td> <td style="background-color:#d9e2f3; border-bottom:3px solid black; border-left:none; border-right:1px solid black; border-top:3px solid black; vertical-align:top; width:246px"> Observations (case studies) </td> <td style="background-color:#d9e2f3; border-bottom:3px solid black; border-left:none; border-right:3px solid black; border-top:3px solid black; vertical-align:top; width:180px"> Reference </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px"> LH receptor mutation c.1345G>A (p.Ala449Thr) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:246px"> The patient had compromised ovulation with normal LH levels, but with LH resistance in a 27-year-old in China from a non-consanguineous family. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> (Yuan et al., 2017) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px"> LHCGR heterozygous mutation (inactivating mutation on exon1) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:246px"> The 33-year-old woman presented ovarian cysts and amenorrhea and low oocyte yield. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> (Bentov et al., 2012) </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px"> LH receptor mutation (Substitution of K354 into E) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:246px"> A 46, XX sibling displayed normal external genitalia and breast development but has primary amenorrhea. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> (Stavrou et al., 1998)   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px"> LH receptor mutation (ΔL608, V609) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:246px"> The 46, XX woman exhibited oligomenorrhea. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> (Latronico et al., 1998)   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px"> LH receptor abnormalities </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:246px"> This study shows a woman with normal pubertal development, rare menses, infertility, polycystic ovaries, and no ovulation (lack of corpus luteum). They believe the mutation lies with the receptor due to an absence of response to LH and phenotype of However, they were not able to locate a mutation on the LH receptor. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> (Prado Arnhold et al., 1997)   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px"> LH receptor mutation (Substitution of Arg554 into a STOP codon) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:246px"> The 46, XX woman in this family had normal external genitalia but had cystic ovaries of unequal size and amenorrhea. Another woman from a different family also presented this mutation with secondary amenorrhea and elevated levels of LH, anovulation and no corpus luteum formation. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> (Latronico et al., 1996; Tsigos et al., 1997)   </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:169px"> LH receptor mutation (Substitution of A593 to P) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:246px"> The female sibling exhibited primary amenorrhea, a smaller uterus, abnormal menses, no preovulatory follicles and infertility. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:180px"> (Toledo et al., 1996)   </td> </tr> </tbody> </table> Evidence through downstream targets of LH: Erk1/2 mouse Kos (conditional in granulosa cells): The mice were sterile with lack of meiotic resumption of the oocytes, absence of luteinization as well as inhibition of ovulation (Fan et al., 2009). C/EBP mouse Kos (conditional in granulosa cells): The mice exhibited a similar phenotype to the Erk1/2 knockouts as expected, since C/EBP is downstream to Erk1/2. However, although there was lack of ovulation, the oocytes were able to resume meiosis and mature but were trapped and could not be released from the follicle (Fan et al., 2011; Sterneck et al., 1997). PGR KOs; In this case, follicle growth was normal and luteinization was completed, but no ovulation occurred with no corpus luteum because follicle rupture was inhibited and therefore there was no COC release (Kim et al., 2009; Lydon et al., 1995). Evidence through other factors affecting LH: Pituitary-specific p62−/− mice: p26 is an important protein in metabolic processes. Lack of p26 in pituitary induces fertility issues. The authors investigated the role of p26 in female reproduction. They found that p26 is important for LH expression and release through mitochondrial oxidative phosphorylation, and without p26, LH levels are very low leading to a decrease in ovulation, lack of corpus luteum and less pups (X. Li et al., 2021). microRNAs KOs: miR-29a/b1 KO mice were generated to observe its role in reproduction. MicroRNAs regulate gene expression by inhibiting expression of mRNAs. Part of the miR29 family of mature miRNAs, this KO had ovulatory problems with a strong decrease of observable corpus lutea thought to be due to low LH serum levels. Although the molecular mechanism of miR29 is still unknown, this study shows again a clear correlation between issues with LH levels and anovulation (Guo et al., 2021). Two other microRNAs also seem to play an essential role in female fertility in mice. miR-200b and miR-429 double KO mice had low levels of LHβ and therefore anovulation. The authors hypothesize that this is due to higher expression of one of their targets, ZEB1, however, the exact mechanism is still unknown (Hasuwa et al., 2013). Evidence through stressors: A variety of stressors have been shown to affect the LH surge which in turn disrupts ovulation. Atrazine: Foradori et al., exposed SD and LE rats to atrazine through gavage for 4 days. No significant effect was observed for LE rats. However, starting from 50mg/kg of atrazine, there was a significant decrease in LH surge as well as a significant decrease in corpus lutea observed and ova shed (Foradori et al., 2014). A different study using human cumulus granulosa cells showed that exposure to 20µM of atrazine for 48h leads to a decrease of LHCGR mRNA expression as well as ovulatory markers EREG and AREG (Pogrmic-Majkic et al., 2018). TCDD: One study showed that TCDD exposure leads to issues with ovulation that could be due to a decrease in LH levels. The SD rats were first stimulated with eCG and exposed to TCDD (0.3 - 60 µg/kg of TCDD). 72 hrs after the eCG stimulation, the authors observed the decrease of FSH and LH levels and a decrease in the number of rats that ovulated as well as a decrease of ova shed (ED50: 3-10 µg/kg) (X. L. Li et al., 1995). Similarly, another study examined the effect of TCDD exposure on SD rats and compared it to PCDD exposure. They were exposed to 2-32 µg/kg of TCDD following this time PMSG stimulation. After 72 hours of the highest exposure, there was a decrease in LH levels, as well as FSH. They also noted an effect starting from 4 µg/kg on ovulation and ovarian weight (Gao et al., 1999). PFOS: Using ICR mice, Wang et al., exposed the mice to PFOS (10mg/kg) for 30 days. Within 7 days, they observed a reduction of ovulation and a decrease of P4, LH, and GnRH (Wang et al., 2018). In another study, ICR mice were exposed to 0.1mg/kg of PFOS for 6months. After 4 months of exposure there was a reduction of E2, P4, FSH, LH, and GnRH. Less antral and preovulatory follicles were found in these mice as well as less corpus lutea. In this study, they observe loss of body weight after 4 months, so in order to avoid this having an effect on reproductive function, they use mice exposed for 4months and not more. This seems to be an interesting point that most other studies do not underline or pay particular attention to (Feng et al., 2015). GnRH antagonists: SD rats were exposed to Cetrorelix, a GnRH antagonist, for 30 days. LH levels were measured on day 0, 4, 10, 20, and 30 (before sacrifice). The levels of LH measured were lower on days 4 and 10 compared to control rats but interestingly increased back to a similar level to controls on day 20. The estrous cycle resembled that of anovulatory women. In addition, no corpus lutea were observed at sacrifice and no progesterone increase was measured, indicating a lack of ovulation in SD rats exposed to Cetrorelix (Horvath et al., 2004). Dose and temporal concordance See <a href="https://efsa.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.2903%2Fj.efsa.2023.7744&file=efs27744-sup-0008-Annex-B.3.xlsx">Annex B.3</a>.

Although the phenotypes of the different knockout models in mice resemble that of the women that exhibit mutations in either LH itself or its receptor, these studies investigate the absence of LH or LHCGR and not the surge itself. Animal models would be needed to directly investigate the surge by inhibiting the moment of the LH surge to confirm the direct relationship between this surge and ovulation regulation. In addition, gain of function mutants may make these conclusions more complex. Transgenic mice were created expressing bovine LHβ tagged with a carboxyl terminal peptide of hCG that extends the half-life of LHβ (LHβ-CTP). When LHβ is highly secreted in pre-pubertal mice, several consequences arise. The authors observed either anovulation or infrequent ovulation as well as enlarged ovaries, cysts, and tumours. In addition, levels of ovarian hormones were affected, with higher levels of estrogen and testosterone (Risma et al., 1995, Risma et al., 1997). This indicates that all changes in LH dynamics may have an adverse effect on ovulation, and not just negative regulation.  Interestingly, a study using gain of function for LHCGR (KiLHR (D582G)) in mice shows that some clear difference between species can occur. Unlike women with “activating” LHCGR mutations which have a normal phenotype, mice have irregular estrous cycles and anovulation. This shows that, at least in mice, having an increase in activity of LH receptor can have a negative impact on female fertility, but also shows the care that has to be taken to extrapolate data on rodents to humans (Hai et al., 2015). Uncertainties and inconsistencies should be further explored.

Not Specified Female

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Adult, reproductively mature

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Arnhold IJ, Latronico AC, Batista MC, Carvalho FM, Chrousos GP and Mendonça BB, 1997. Ovarian resistance to luteinizing hormone: a novel cause of amenorrhea and infertility. Fertil Steril, 67:394-397. doi: 10.1016/s0015-0282(97)81929-2 Bentov Y, Kenigsberg S and Casper RF, 2012. A novel luteinizing hormone/chorionic gonadotropin receptor mutation associated with amenorrhea, low oocyte yield, and recurrent pregnancy loss. Fertil Steril, 97:1165-1168. doi: 10.1016/j.fertnstert.2012.02.002 Duffy DM, Ko C, Jo M, Brannstrom M and Curry TE, 2019. Ovulation: Parallels With Inflammatory Processes. Endocr Rev, 40:369-416. doi: 10.1210/er.2018-00075 Fan HY, Liu Z, Johnson PF and Richards JS, 2011. CCAAT/enhancer-binding proteins (C/EBP)-α and -β are essential for ovulation, luteinization, and the expression of key target genes. Mol Endocrinol, 25:253-268. doi: 10.1210/me.2010-0318 Fan HY, Liu Z, Shimada M, Sterneck E, Johnson PF, Hedrick SM and Richards JS, 2009. MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science, 324:938-941. doi: 10.1126/science.1171396 Feng X, Wang X, Cao X, Xia Y, Zhou R and 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. Toxicol Sci, 148:368-379. doi: 10.1093/toxsci/kfv197 Foradori CD, Sawhney Coder P, Tisdel M, Yi KD, Simpkins JW, Handa RJ and Breckenridge CB, 2014. The effect of atrazine administered by gavage or in diet on the LH surge and reproductive performance in intact female Sprague-Dawley and Long Evans rats. Birth Defects Res B Dev Reprod Toxicol, 101:262-275. doi: 10.1002/bdrb.21109 Gao X, Son D-S, Terranova PF and Rozman KK, 1999. Toxic Equivalency Factors of Polychlorinated Dibenzo-p-dioxins in an Ovulation Model: Validation of the Toxic Equivalency Concept for One Aspect of Endocrine Disruption. Toxicology and Applied Pharmacology, 157:107-116. doi: <a href="https://doi.org/10.1006/taap.1999.8649">https://doi.org/10.1006/taap.1999.8649</a> Guo Y, Wu Y, Shi J, Zhuang H, Ci L, Huang Q, Wan Z, Yang H, Zhang M, Tan Y, Sun R, Xu L, Wang Z, Shen R and Fei J, 2021. miR-29a/b(1) Regulates the Luteinizing Hormone Secretion and Affects Mouse Ovulation. Front Endocrinol (Lausanne), 12:636220. doi: 10.3389/fendo.2021.636220 Hai L, McGee SR, Rabideau AC, Paquet M and Narayan P, 2015. Infertility in Female Mice with a Gain-of-Function Mutation in the Luteinizing Hormone Receptor Is Due to Irregular Estrous Cyclicity, Anovulation, Hormonal Alterations, and Polycystic Ovaries. Biol Reprod, 93:16. doi: 10.1095/biolreprod.115.129072 Hasuwa H, Ueda J, Ikawa M and Okabe M, 2013. miR-200b and miR-429 function in mouse ovulation and are essential for female fertility. Science, 341:71-73. doi: 10.1126/science.1237999 Horvath JE, Toller GL, Schally AV, Bajo AM and Groot K, 2004. Effect of long-term treatment with low doses of the LHRH antagonist Cetrorelix on pituitary receptors for LHRH and gonadal axis in male and female rats. Proc Natl Acad Sci U S A, 101:4996-5001. doi: 10.1073/pnas.0400605101 Kim J, Bagchi IC and Bagchi MK, 2009. Control of ovulation in mice by progesterone receptor-regulated gene networks. Mol Hum Reprod, 15:821-828. doi: 10.1093/molehr/gap082 Latronico AC, Anasti J, Arnhold I, Rapaport R, Mendonca B, Bloise W, Castro M, Tsigos C and Chrousos G, 1996. Testicular and Ovarian Resistance to Luteinizing Hormone Caused by Inactivating Mutations of the Luteinizing Hormone–Receptor Gene. New England Journal of Medicine - N ENGL J MED, 334:507-512. doi: 10.1056/NEJM199602223340805 Latronico AC, Chai Y, Arnhold IJ, Liu X, Mendonca BB and Segaloff DL, 1998. A homozygous microdeletion in helix 7 of the luteinizing hormone receptor associated with familial testicular and ovarian resistance is due to both decreased cell surface expression and impaired effector activation by the cell surface receptor. Mol Endocrinol, 12:442-450. doi: 10.1210/mend.12.3.0077 Lei ZM, Mishra S, Zou W, Xu B, Foltz M, Li X and Rao CV, 2001. Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Mol Endocrinol, 15:184-200. doi: 10.1210/mend.15.1.0586 Li X, Johnson DC and Rozman KK, 1995. Reproductive effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation, hormonal regulation, and possible mechanism(s). Toxicol Appl Pharmacol, 133:321-327. doi: 10.1006/taap.1995.1157 Li X, Zhou L, Peng G, Liao M, Zhang L, Hu H, Long L, Tang X, Qu H, Shao J, Zheng H and Long M, 2021. Pituitary P62 deficiency leads to female infertility by impairing luteinizing hormone production. Exp Mol Med, 53:1238-1249. doi: 10.1038/s12276-021-00661-4 Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery CA, Jr., Shyamala G, Conneely OM and O'Malley BW, 1995. Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev, 9:2266-2278. doi: 10.1101/gad.9.18.2266 Ma X, Dong Y, Matzuk MM and Kumar TR, 2004. Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc Natl Acad Sci U S A, 101:17294-17299. doi: 10.1073/pnas.0404743101 Padmanabhan V, Puttabyatappa M and Cardoso R, 2018. Hypothalamus–Pituitary–Ovary Axis. Pogrmic-Majkic K, Samardzija D, Stojkov-Mimic N, Vukosavljevic J, Trninic-Pjevic A, Kopitovic V and Andric N, 2018. Atrazine suppresses FSH-induced steroidogenesis and LH-dependent expression of ovulatory genes through PDE-cAMP signaling pathway in human cumulus granulosa cells. Mol Cell Endocrinol, 461:79-88. doi: 10.1016/j.mce.2017.08.015 Risma KA, Clay CM, Nett TM, Wagner T, Yun J and Nilson JH, 1995. Targeted overexpression of luteinizing hormone in transgenic mice leads to infertility, polycystic ovaries, and ovarian tumors. Proc Natl Acad Sci U S A, 92:1322-1326. doi: 10.1073/pnas.92.5.1322 Risma KA, Hirshfield AN and Nilson JH, 1997. Elevated luteinizing hormone in prepubertal transgenic mice causes hyperandrogenemia, precocious puberty, and substantial ovarian pathology. Endocrinology, 138:3540-3547. doi: 10.1210/endo.138.8.5313 Robker RL, Hennebold JD and Russell DL, 2018. Coordination of Ovulation and Oocyte Maturation: A Good Egg at the Right Time. Endocrinology, 159:3209-3218. doi: 10.1210/en.2018-00485 Russell DL and Robker RL, 2007. Molecular mechanisms of ovulation: co-ordination through the cumulus complex. Hum Reprod Update, 13:289-312. doi: 10.1093/humupd/dml062 Stavrou SS, Zhu YS, Cai LQ, Katz MD, Herrera C, Defillo-Ricart M and Imperato-McGinley J, 1998. A novel mutation of the human luteinizing hormone receptor in 46XY and 46XX sisters. J Clin Endocrinol Metab, 83:2091-2098. doi: 10.1210/jcem.83.6.4855 Sterneck E, Tessarollo L and Johnson PF, 1997. An essential role for C/EBPbeta in female reproduction. Genes Dev, 11:2153-2162. doi: 10.1101/gad.11.17.2153 Toledo SP, Brunner HG, Kraaij R, Post M, Dahia PL, Hayashida CY and Kremer HTAP, 1996. An inactivating mutation of the luteinizing hormone receptor causes amenorrhea in a 46,XX female. J Clin Endocrinol Metab, 81:3850-3854. doi: 10.1210/jcem.81.11.8923827 Tsigos C, Latronico C and Chrousos GP, 1997. Luteinizing hormone resistance syndromes. Ann N Y Acad Sci, 816:263-273. doi: 10.1111/j.1749-6632.1997.tb52150.x Wang X, Bai Y, Tang C, Cao X, Chang F and Chen L, 2018. Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences, 165:475-486. doi: 10.1093/toxsci/kfy167 Yuan P, He Z, Zheng L, Wang W, Li Y, Zhao H, Zhang VW, Zhang Q and Yang D, 2017. Genetic evidence of 'genuine' empty follicle syndrome: a novel effective mutation in the LHCGR gene and review of the literature. Hum Reprod, 32:944-953. doi: 10.1093/humrep/dex015 Zhang FP, Poutanen M, Wilbertz J and Huhtaniemi I, 2001. Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol Endocrinol, 15:172-183. doi: 10.1210/mend.15.1.0582   AOPWiki 2025-01-10T14:05:54

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<upstream-id>7cff47f7-d02f-4906-9b98-d1fc33729c7f</upstream-id> <downstream-id>4e76928e-9ef2-4d24-9ba5-488cccdd66c1</downstream-id> The LH surge induces different pathways and provokes ovulation that leads to an overall higher level of progesterone (P4) compared to estradiol (E2). The cycle in rodents is divided into four phases: metestrous, proestrous, estrous, diestrous. During the estrous phase, a peak of estrogen is reached prior to ovulation. Once ovulation occurs, through the complex reactions downstream of the LH surge, progesterone increases and activates the progesterone receptor (PGR) and its downstream targets. This level of progesterone is needed for corpus luteum formation. At this stage, there is a higher concentration of progesterone compared to estradiol which is essential for the correct continuation of the female cycle (Andersson et al., 2013; Duffy et al., 2019; Mittelman-Smith et al., 2017). In instances where ovulation may be disturbed, the rest of the cycle may be affected as well as the steroidogenic profile. In most cases, if ovulation is blocked or delayed, the ratio of progesterone/estradiol (P4/E2) remains low due to lack of progesterone increase that is initiated after ovulation. As a result, ovarian and circulating steroid hormone levels remain in the “pre-ovulatory” state, i.e., high estradiol, and low progesterone. In addition, with ovulation disruption, formation of corpus lutea is delayed or inhibited. This overall disrupts the cycle and can lead to persistent estrous. Persistent estrous is characterized by the lack of corpus lutea formation, and observation of cysts and antral follicles. Morphologically, it is demonstrated by persistent vaginal cornification (PVC). It is considered persistent if at least two cycles were perturbed with the appearance of PVC (Finch, 2014; Stewart et al., 2022). The apparition of PVC in mice and rats occurs spontaneously with age as ovulation ceased. In ageing mice, levels of progesterone are low while estrogen remains in a high-level state. This leads to a higher estradiol/progesterone ratio which also results in an abnormal estrous cycle with persistent estrous observed (Finch, 2014; Lu et al.,1979; Nelson et al., 1981; Westwood, 2008).

LH action via LHCGRs initiates or enhances granulosa cell expression of CYP11A1 and HSD3B1 (Fig. 13). Granulosa cells express only low levels of CYP17A1, so conversion of progesterone to androgens and estrogens is severely limited. This pattern of expression and activity of enzymes results in synthesis of progesterone as the major steroid hormone after the LH surge in primates. Changes in enzyme expression and activity effectively shift the balance of steroid hormone synthesis from primarily estrogens before the LH surge to primarily progesterone after the LH surge (Duffy et al., 2019). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/10/8gccboi8wq_Figure_13.jpg" /> Figure 13. Granulosa and theca cells cooperate to produce steroid hormones. Before the LH surge (i) theca cells produce predominantly androgens in response to LH (ii) androgens diffuse to granulosa cells, and (iii) granulosa cells convert androgens to estrogens in response to FSH. After the LH surge, (i) decreased CYP17A1 expression increases progesterone synthesis and decreases androgen synthesis in theca cells, and (ii) increased HSD3B1 increases progesterone synthesis and declining CYP19A1 decreases estrogen synthesis by granulosa cells. The LH surge also increases HSD11B1 and decreases HSD11B2 in granulosa cells to increase synthesis of cortisol from circulating cortisone. Enzymes shown in green increase after the LH surge. Enzymes shown in red decrease after the LH surge. (Duffy et al., 2019).

A clear example of persistent estrous is the phenotype observed in ageing mice or rats. At older ages, these rodents have less ovulation and irregular cycles that are characterized by longer periods of estrous (persistent estrous) (Mills et al., 2002; Nelson et al., 1981). This can also be observed in women of older reproductive ages. The menstrual blood loss was analyzed in women going through the transition into menopause. In these women with irregular cycles, abnormally high levels of estradiol were measured. This is an example of a link between anovulation and estradiol levels in women of pre-menopausal age (Hale et al., 2010). When LH surge is disturbed in both types of mouse KOs (LHβ or LHCGR KOs) as well as women with LH related mutations, serum levels of both estrogen and progesterone tend to decrease compared to the usual levels expected. According to the measurements displayed in the paper, the ratio of estradiol to progesterone does not increase in the KO mice. This may be because LHβ or its receptor, LHCGR, in these models is never present. It could influence the overall feedback loops necessary and observed in normal estrous cycles leading mainly to a decrease in most steroid hormones. However, overexpression of LHβ also affects ovulation and the estrous cycle. Risma et al., created a transgenic mouse that overexpresses LHβ due to a longer half-life (these mice express a bovine LHβ tagged with a carboxyl terminal peptide of hCG). These mice can have rare ovulations or no ovulation and have morphological issues such as cysts and bigger ovaries. They also were found to have higher levels of estrogen and testosterone compared to their WT counterparts. Progesterone levels were also higher throughout the estrous cycle in comparison to control mice in the Risma et al., 1995 study. However, calculating the ratio isn’t feasible with the data they provided. Their second publication does not include any progesterone measurements. Therefore, it is difficult to say if the ratio of E2/P4 increases in these mutants, but what does seem clear is that the levels of estradiol and progesterone increase in contrast to the LH or LHCGR KOs (Risma et al., 1995, 1997). Another study shows a similar phenotype. Gain of function of LHCGR in mice leads to anovulation and irregular estrous cyclicity with longer periods in estrous phase. However, both estradiol and progesterone increase in levels. According to the data of this study, the ratio of E2/P4 does increase from weeks 2 to 6 but decreases at weeks 12 and 24 (Hai et al., 2015). The role of another essential hormone, GnRH, was investigated using a conditional knockout model of GnRH (GnrhrE90K). The females with no GnRH presented persistent estrous. This was due to the lack of LH surge resulting in anovulation and continuous estradiol synthesis compared to progesterone. Without the LH peak and inhibition of ovulation, corpus luteum is lacking which explains the low levels of progesterone observed. These females were infertile and had issues with the development of uterine glands (Stewart et al., 2022). Without GnRH, there is no LH surge, and estrogen levels are not negatively regulated staying at the high level it reaches prior to the LH peak. Therefore, with lacking LH surge, progesterone levels do not increase giving an advantage to estradiol in the E2/P4 ratio. Another transgenic mouse also exhibited a similar phenotype. This study used mice that overexpress human nerve growth factor (hNGF) in theca cells. NGF was measured in PCOS (polycystic ovary syndrome) patients, and their levels are increased compared to control patients. To test the effect of this increase, the authors overexpressed NGF in mice and observed its consequence. The mice spent more time in estrous compared to the control mice, with lower levels of progesterone after PMSG stimulation and a significant increase of estradiol. The mice overexpressing NGF had significantly lower progesterone levels after injection of the smallest dose of hCG (1 IU) compared to control mice. After determining the ratio from graphs in this study, the ratio of E2/P4 increases after PMSG injection. They had less pups and more time interval between litters pointing at issues with ovulation and fertility (Dissen et al., 2009). Data on rat models for PCOS show that disruption of ovulation leads to persistent estrous. These models are created with the use of different parameters that may stress the rats, such as continuous illumination, or stressors like RU486. Although the goal of PCOS models do not include all the necessary phenotypes observed in humans, many of them do show issues with ovulation leading to less corpus lutea, therefore less progesterone synthesis, a dominance of estradiol resulting in persistent estrous (Prata Lima et al., 2004; Priyadarshani, 2009; Ryu et al., 2019; Sánchez-Criado et al., 1992, 1993; Takeo, 1984). Stressors: Two studies have shown the adverse effect of GnRH antagonists on ovulation and the estrous cycle. One of them exposed SD rats to Cetrorelix for 30 days. At sacrifice, the rats had no corpus lutea showing disruption in ovulation. In addition, the rats were in persistent estrous from day 6 to 20, with a decrease of progesterone from day 4, and a temporary decrease of estradiol (30%) on day 4 but returns to control levels. This indicates that for these rats, exposure to certrorelix leads to an increase in E2/P4 ratio (Horvath et al., 2004). Atrazine exposure studies do not persistently check for possible estrous cycle abnormalities. However, a few have shown that with atrazine gavage, there can be persistent estrous depending on the dosage and period of treatment as well as the type of animal model used. Atrazine was used at different doses and changes in ovulation and the estrous cycle were recorded at 3 and 9 months. With both 70 and 400 rpm, a reduction of corpus lutea was measured. According to the values provided, the ratio of E2/P4 was also increased with atrazine exposure at both doses (Wetzel et al., 1994). Another study shows that atrazine exposure does affect estrous cyclicity. They show a correlation between high doses (200mg/kg) and persistent estrous. Adding atrazine to the diet (400ppm) for 6 months also increase % of rats with persistent estrous. However, they did not investigate rates of ovulation (Eldridgea et al., 1999). According to Foradori et al., administration of atrazine through high doses bolus (≥50mg/kg) and not in the diet leads to diminished ovulation and disrupted estrous cycle. (Foradori et al., 2014). TCDD as a stressor also displays a correlation between deficiencies in ovulation with a higher E2/P4 ratio and therefore persistent estrous. Treatment of TCDD in SD rats leads to a decrease in ovulation rates as well as a peak of estradiol that persists compared to controls. (Gao et al., 1999; X. L. Li et al., 1995; Ushinohama et al., 2001) A study focusing on PFOS exposed mice to PFOS (10mg/kg) for one week and observed a few effects on the reproductive cycle. The exposure led to a prolonged diestrous and a decrease of progesterone. According to the tables provided Wang et al., 2018, there is a slight increase of the E2/P4 ratio after one week (values: 4.2 control, 5.7 PFOS), but almost no difference after day 14 (values: 4.7 control, 4.8 PFOS) (Wang et al., 2018). Several studies have found other stressors that also disrupt ovulation resulting in persistent estrous. Exposure to 17α-ethynylestradiol (EE) neonatally on rats has clear adverse effects on the pups. As they reach reproductive maturity, anovulation is observed, with a decrease in progesterone, an increase of estrogen, and therefore persistent estrous (Sawaki, 2003; Shiorta et al., 2012; Takahashi et al., 2013). Another stressor studied is the environmental pollutant p-tert-octylphenol (OP). Its exposure on both adult and neonatal female rats leads to lack of ovulation and disrupted estrous cycle with persistent estrogen (Blake and Ashiru, 1997; Katsuda et al., 2000; Willoughby et al. 2005). Prepubertal and neonatal exposure of zearalenone, a mycotoxin, was also found to induce anovulation as well as persistent estrous in SD rats (Kumagai and Shimizu, 1982; Nikaido et al., 2003). Neonatal exposure of SD rats to insecticides such as Kepone and DDT also led to anovulation and persistent estrous (Gellert, 1978; Heinrichs et al., 1971). Phenobarbital also has this same effect on SD rats through the blocking of the LH surge, leading to anovulation and persistent estrous due to hypothyroidism (Y. Li et al., 2011). Other stressors used in different studies, such as octamethylcyclotetrasiloxane (D4), coumestrol, and danazol, also induces anovulation or delayed ovulation and an increase in estradiol/progesterone ratio and persistent estrous showing the link between these two factors (Dekant et al., 2017; Kouki et al., 2005; Raj et al., 1981). Dose and temporal concordance See <a href="https://efsa.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.2903%2Fj.efsa.2023.7744&file=efs27744-sup-0008-Annex-B.3.xlsx">Annex B.3</a>.

Regarding the data of atrazine as a stressor, there is one caveat that must be taken into consideration. Atrazine is known to be an aromatase inducer. Therefore, its role in upregulating the E2/P4 ratio doesn’t necessarily pass through the disruption of ovulation. This must be taken into account when observing its effect as a stressor (Gammon et al., 2005). In the knockout strains of LHβ or its receptor LHCGR, both estradiol and progesterone are decreased in the serum or ovary. In these knockouts, the ratio of estradiol to progesterone doesn’t have any clear increase. The mutations in LH or its receptor found in women also provoke an overall reduction of both estradiol and progesterone serum levels. Although it has been proven that the LH surge is essential for ovulation and subsequent higher progesterone synthesis leading to correct estrous cycle, a direct link between a delayed or perturbed LH surge and persistent estrous is difficult to demonstrate. Another factor should also be taken into account when analysing these studies and their data: the reproductive cycle can differ between rodents and in comparison, to humans. This is summarized in the table below. It shows the differences between SD rats, F-344 rats and women when it comes to the parameters of reproductive senescence, showing an important variation of LH surge capability, cycle patterns and estrogen/progesterone ratios (Chapin, 1996, see also Table 4). This would suggest that responses to stressors or mutations could vary according to species and background strains. Table 4. Comparison of Reproductive Senescence in Female Rodent Strains and Human (Chapin, 1996) <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/10/9ov7np0lrz_Table_4.png" />

Not Specified Female

Not Specified

Adult, reproductively mature

Not Specified

Andersson H, Rehm S, Stanislaus D and Wood CE, 2013. Scientific and Regulatory Policy Committee (SRPC) Paper:Assessment of Circulating Hormones in Nonclinical Toxicity Studies III. Female Reproductive Hormones. Toxicologic Pathology, 41:921-934. doi: 10.1177/0192623312466959 Blake CA and Ashiru OA, 1997. Disruption of rat estrous cyclicity by the environmental estrogen 4-tert-octylphenol. Proc Soc Exp Biol Med, 216:446-451. doi: 10.3181/00379727-216-44195 Chapin RE, Stevens JT, Hughes CL, Kelce WR, Hess RA and Daston GP, 1996. Endocrine modulation of reproduction. Fundam Appl Toxicol, 29:1-17. doi: 10.1006/faat.1996.0001 Dekant W, Scialli AR, Plotzke K and Klaunig JE, 2017. Biological relevance of effects following chronic administration of octamethylcyclotetrasiloxane (D4) in Fischer 344 rats. Toxicol Lett, 279 Suppl 1:42-53. doi: 10.1016/j.toxlet.2017.01.010 Dissen GA, Garcia-Rudaz C, Paredes A, Mayer C, Mayerhofer A and Ojeda SR, 2009. 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Endocrinology, 138:3540-3547. doi: 10.1210/endo.138.8.5313 Ryu Y, Kim SW, Kim YY and Ku SY, 2019. Animal Models for Human Polycystic Ovary Syndrome (PCOS) Focused on the Use of Indirect Hormonal Perturbations: A Review of the Literature. Int J Mol Sci, 20. doi: 10.3390/ijms20112720 Stewart CA, Stewart MD, Wang Y, Mullen RD, Kircher BK, Liang R, Liu Y and Behringer RR, 2022. Chronic Estrus Disrupts Uterine Gland Development and Homeostasis. Endocrinology, 163. doi: 10.1210/endocr/bqac011 Ushinohama K, Son D, Roby KF, Rozman KK and Terranova PF, 2001. Impaired ovulation by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) in immature rats treated with equine chorionic gonadotropin. Reproductive toxicology (Elmsford, N.Y.), 15:275-280. doi: 10.1016/s0890-6238(01)00128-9 Wang X, Bai Y, Tang C, Cao X, Chang F and Chen L, 2018. Impact of Perfluorooctane Sulfonate on Reproductive Ability of Female Mice through Suppression of Estrogen Receptor α-Activated Kisspeptin Neurons. Toxicological Sciences, 165:475-486. doi: 10.1093/toxsci/kfy167 Westwood FR, 2008. The female rat reproductive cycle: a practical histological guide to staging. Toxicol Pathol, 36:375-384. doi: 10.1177/0192623308315665 Wetzel LT, Luempert LG, 3rd, Breckenridge CB, Tisdel MO, Stevens JT, Thakur AK, Extrom PJ and Eldridge JC, 1994. Chronic effects of atrazine on estrus and mammary tumor formation in female Sprague-Dawley and Fischer 344 rats. J Toxicol Environ Health, 43:169-182. doi: 10.1080/15287399409531913 AOPWiki 2025-01-10T14:52:44

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<upstream-id>4e76928e-9ef2-4d24-9ba5-488cccdd66c1</upstream-id> <downstream-id>2b4b7c6e-1939-4c30-a8f5-f1eaf01c3aaa</downstream-id> In both rodents and human, estrogens produced from the developing follicles stimulate endometrial growth, and progesterone is responsible for converting the estrogen primed endometrium into a receptive state. In rodents, if pregnancy does not occur, diestrous (secretory phase in humans, cycle days 15–28) terminates with regression of the corpus luteum, and the endometrium is resorbed (menstruation in humans, cycle days 1–5). During proestrous (proliferative phase in humans, cycle days 6–14) follicles develop and start to produce estrogens that stimulate endometrial growth. During estrous (peri-ovulatory period in humans, cycle days 13–15) ovarian follicles mature. The magnitude of uterine growth stimulation is largely dependent upon the duration of bioavailable E2 and receptor interaction (Groothius et al., 2007).

The development of the KER is based on structured literature review of records. Description for KER is based on reviews and books on the topic. The method used are described in <a href="https://efsa.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.2903%2Fj.efsa.2023.7744&file=efs27744-sup-0006-Annex-B.1.docx">Annex B.1</a>.

The biological relationship between these two KEs is considered strong. There is no doubt that a prolonged increased circulating E2/P4 ratio leads to an increase of E2 bioavailability in a variety of estrogenic-responsive organs, including uterus due to insufficient counterbalance by progesterone. However, compensatory mechanisms (e.g., intracrine networks) may differ across different tissues.  The degree to which E2/P4 ratio should increase to overwhelm these compensatory responses has not been established.

Dose and temporal concordance See <a href="https://efsa.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.2903%2Fj.efsa.2023.7744&file=efs27744-sup-0008-Annex-B.3.xlsx">Annex B.3</a>.

Not Specified Female

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Adults

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AOPWiki 2025-01-10T16:07:32

2025-01-14T17:29:45

<upstream-id>4e76928e-9ef2-4d24-9ba5-488cccdd66c1</upstream-id> <downstream-id>7b79584f-8d69-4ed7-8e42-554c9ff3b35e</downstream-id>

The biological plausibility of the key event relationship is supported by aging rodent females. Reproductive senescence in rodents results from initial centrally mediated changes with alterations in hypothalamic function (Gore, 2000; Kermath, 2012). Female rats and mice proceed through sequential reproductive stages. Female rats undergo a transition from regular estrous cycles to irregular cycles (up to the age of 3- to 7-month-old), followed by persistent estrus (characterized by PVC), then repetitive pseudopregnancy (also called persistent diestrus) and finally anestrus (Finch, 2014; Shirai, 2015). However, there are species and strain differences in the pattern of changes and timing in the onset of reproductive senescence (Vidal, 2017). The tree different pathways of reproductive senescence are represented in Fig. 17 (Finch, 2014) and the pattern and the age at cycle cessation in different strains of rats and mice are available in Table 5 (Nelson, 1982). PE begins to be observed in Sprague-Dawley rats by 6 to 7 months of age (Vidal, 2017) or even earlier (Elridge, 1999). In contrast, Han Wistar rats are reported to have cycle irregularities after 6 months of age and move mainly into persistent diestrus (Mitchard and Klein 2016).  <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/13/6ih8tb88g_Figure_17.png" style="height:145px; width:750px" /> Figure 17. Alternate trajectories of rodent reproductive senescence from (Finch, 2014) <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/13/4o0lfm08ay_Table_5.png" style="height:424px; width:956px" /> Table 5. Incidence and vaginal cytological status of prolonged cycles in different strains of mouse and rat ranked by age (Nelson, 1982) In aged rodents showing persistent vaginal cornification, the hormonal profile is characterized by sustained E2 and low P (Finch 2014). This hormonal change is explained by the histopathology of ovary showing numerous follicular cysts (producing estradiol) and lack of corpora lutea (producing progesterone). The link between increased E2/P4 ratio and PVC has been demonstrated in different strains of both species. In a comparative study between Donryu and Fischer-344 rats, Nagaoka et al., sequentially followed estrous cycles by vaginal smear as well as E2 and P plasma levels, up to the age of 15 months. The E2/P4 ratio was higher in Donryu rats than in F-344 rats from 8 month of age and was about 7 times higher (p < 0.01) at 12-month. In Donryu rats persistent estrous (measured as PVC) appeared in 17% of the 5-month animals, the prevalence increased with age (90% of the 10-month animals while almost all F-344 rats showed a normal estrous cycle up to 8 months, and only a few cases showed persistent estrous thereafter). The authors concluded that the sustained increased E2/P4 ratio observed in Donryu rats may explained the high spontaneous occurrence of uterine endometrial adenocarcinoma in Donryu rats compared to F-344 rats (Nagaoka, 1994). The link between increased E2/P4 and PVC was also showed in aging Long Evans rats (Lu, 1979 and C578L/6J mice (Nelson, 1981) in which increased E2/P4 ratio was observed in PVC rats and mice compared to younger cycling animals.

Atrazine Several studies in Sprague-Dawley (SD) female rats have shown that prolonged administration of atrazine by diet induced earlier increase of the number of animals displaying PVC. However, only one publication reports the investigation of both the KE upstream and KE downstream events SD rats and Fischer-344 rats. In a 6-month repeated dose toxicity studies (0, 25, 50 or 400 ppm), the number of animals with persistent estrous at 400 ppm was increased compared to controls from three months onwards (20/90 vs 10/90 at 3 months; 50/90 vs 26/90 at 6 months). A significant increase in percent of total days with estrous was noted in the 400ppm group after three months of treatment (Eldridge, 1999, Simpkins 2011, see also Fig. 18). In a 24-month chronic toxicity studies with interims kills in SD rats (0, 70, 400 ppm with interim kills every 3 months) and Fischer-344 rats (0, 10, 70, 200 or 400 ppm) a dose-dependent increased in the percent days in estrous stage was noted from the 70 ppm SD group after nine months of treatment reaching statistically significance only at time point 9-month). There was no effect of 400ppm atrazine on cyclicity in female Fischer-344 rats. Plasma E2 concentrations were significantly increased from 70 ppm onwards at 3 months in SD rats but not in Fischer rats. No other significant effects on E2 and progesterone were seen in both species (Wetzel, 1994; US EPA 2000; USEPA, 2018). However, when calculating the E2/P4 ratio from the data reported in Wetzel, 1994, a dose-dependent increased of E2/P4 ratio is noted at 3- and 9-month time points in SD female rats while no effect was observed in female Fischer-344 rats (Table 6). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/13/2leb607s40_Figure_18.png" style="height:317px; width:750px" /> Figure 18.  (b) Dose- and time-dependent effect of 0, 25, 50, or 400 ppm atrazine administered in the diet on the percent days in estrous in female SD rat (b) Comparison of the dose- and the time-dependent effects of atrazine administered at dietary concentrations of 0, 70, or 400 ppm on the percent days in estrous in female SD rats (c) Atrazine administered at dietary concentrations of 70 or 400 ppm had no effect on the percent days in estrous in female Fischer 344 rats (Simpkins, 2011) <table align="center" cellspacing="0" class="Table" style="border-collapse:collapse"> <tbody> <tr> <td style="height:20px; vertical-align:top; width:115px">   </td> <td colspan="5" style="height:20px; vertical-align:top; width:400px"> E2/P4 ratio </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> Time (month) </td> <td colspan="3" style="height:20px; vertical-align:top; width:240px"> SD </td> <td colspan="2" style="height:20px; vertical-align:top; width:160px"> Fischer 344 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px">   </td> <td style="height:20px; vertical-align:top; width:80px"> 0 </td> <td style="height:20px; vertical-align:top; width:80px"> 70 </td> <td style="height:20px; vertical-align:top; width:80px"> 400 </td> <td style="height:20px; vertical-align:top; width:80px"> 0 </td> <td style="height:20px; vertical-align:top; width:80px"> 400 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> 1 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.46 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.19 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.57 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.44 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.30 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> 3 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.22 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.68 </td> <td style="height:20px; vertical-align:top; width:80px"> 1.27 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.83 </td> <td style="height:20px; vertical-align:top; width:80px"> 1.24 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> 9 </td> <td style="height:20px; vertical-align:top; width:80px"> 1.97 </td> <td style="height:20px; vertical-align:top; width:80px"> 2.52 </td> <td style="height:20px; vertical-align:top; width:80px"> 4.22 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.94 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.96 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> 12 </td> <td style="height:20px; vertical-align:top; width:80px"> 3.28 </td> <td style="height:20px; vertical-align:top; width:80px"> 1.81 </td> <td style="height:20px; vertical-align:top; width:80px"> 3.66 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.33 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.17 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> 15 </td> <td style="height:20px; vertical-align:top; width:80px"> 1.22 </td> <td style="height:20px; vertical-align:top; width:80px"> 3.85 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.77 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.20 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.15 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> 18 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.19 </td> <td style="height:20px; vertical-align:top; width:80px"> 1.38 </td> <td style="height:20px; vertical-align:top; width:80px"> 1.27 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.10 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.20 </td> </tr> <tr> <td style="height:20px; vertical-align:top; width:115px"> 24 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.75 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.26 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.23 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.02 </td> <td style="height:20px; vertical-align:top; width:80px"> 0.01 </td> </tr> </tbody> </table> Table 6. E2/P4 ratio calculated from Wetzel, 1994 data These different studies are thoroughly assessed in different available documents of US EPA evaluation (Issues Pertaining to Atrazine Cancer Risk Assessment, US EPA 2000 <a href="https://archive.epa.gov/scipoly/sap/meetings/web/html/062700_mtg.html">https://archive.epa.gov/scipoly/sap/meetings/web/html/062700_mtg.html</a>,) GnRH antagonist Cetrorelix After 30-day intramuscularly treatment of Cetrorelix (depot formulation corresponding to 20–24 µg/kg per day) in SD females (proestrous at day 0) Estradiol levels in the group given Cetrorelix were significantly reduced (P < 0.01) by 30% on day 4 of the experiment but returned to control values after day 10. While progesterone levels decreased in the treated rats by more than 50% on day 4 and remained at this significantly lower level (P < 0.01) through the entire experiment, resulting in an increased E2/P4 ratio (Fig. 21).  The treatment induced prolonged (4–6 days) diestrous phase followed from day 6 by persistent estrous smears in most rats (75.5% of the smears examined between days 6 and 30 showed estrous) (Horvath, 2004, see also Fig 19). <img alt="" src="https://aopwiki.org/system/dragonfly/production/2025/01/14/7dftrfe26k_Figure_19.png" style="height:531px; width:510px" /> Figure 19. Effect of Cetrorelix pamoate on serum E2 and P4 in female rats during the treatment (b1 and c1: serum E2 and P in control female rats at different stages of estrous cycle (Horvath, 2004) Dose and temporal concordance <ul> <li>Atrazine: The dose and temporal concordance is well demonstrated in the only study available investigating both the up and downstream KEs. However only two dose levels were tested (Table 7).</li> <li>Cetrorelix: only one dose was tested and the experiment lasted only 30 days, which significantly limit the evaluation of the dose and temporal concordance (Table 7).</li> </ul> Table 7. Dose and temporal concordance studies   <table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:none"> <tbody> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:65px; vertical-align:top; width:90px"> Species, life-stage, sex tested </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:65px; vertical-align:top; width:70px"> Stressor(s) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:65px; vertical-align:top; width:76px"> Upstream Effect: plasma E2/P4 ratio (Y/N) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:65px; vertical-align:top; width:76px"> Downstream Effect: on increased E2 availability PVC (Y/N) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:65px; vertical-align:top; width:114px"> Effect on increased  plasma E2/P4 ratio (descriptive) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:65px; vertical-align:top; width:114px"> Effect on increased E2 availability in uterus (descriptive) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:65px; vertical-align:top; width:90px"> Citation </td> </tr> <tr> <td colspan="7" style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:28px; vertical-align:top; width:630px"> In vivo </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:90px"> SD female rats 24 months diet 0, 70 or 400 ppm (0, 4.23 or 26.23 mg/kg bw per day) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:70px"> Atrazine </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:76px"> Y </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:76px"> Y </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:114px"> E2/P4 ratio: dose response increased from 70 ppm at  Month-3 and Month-9. </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:114px"> Month-9: statistically significant ↑ % total days in Estrous 34.3%, 44.8% at 70 and 400 ppm respectively vs 24.2% in controls </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:90px"> Wetzel 1994 Eldridge 1994 Also reported in US EPA 2000 and 2018 </td> </tr> <tr> <td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:90px"> SD female rats 0, 30-day (IM depot formulation) (corresponding to 20–24 µg/kg per day)  </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:70px"> Cetrorelix </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:76px"> Y </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:76px"> Y </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:114px"> E2: 30% ↓ on D4 Then returns to control values  P: ↓50% * of D4 remained low until the end of the experiment.   </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:114px"> Persistent Diestrous up to D6  Persistent Estrous from D6 to D20 (75.5% of vaginal smear smears estrous stage) </td> <td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:65px; vertical-align:top; width:90px"> Horvath, 2004 </td> </tr> </tbody> </table> Dose and temporal concordance See <a href="https://efsa.onlinelibrary.wiley.com/action/downloadSupplement?doi=10.2903%2Fj.efsa.2023.7744&file=efs27744-sup-0008-Annex-B.3.xlsx">Annex B.3</a>.

<ul> <li style="text-align:justify">Only two stressors have been investigated. The dose and temporal concordance should be further substantiated with other stressors.</li> <li>E2 bioavailability in uterus was not measure directly in any of the studies reported: the empirical evidence of the KER relies on the indirect measurement of the downstream KE (i.e., the surrogate event PVC). It should be highlighted that Atrazine was negative in a uterotrophic bioassay performed in ovariectomized SD females which is not considered as an uncertainty but rather supports that atrazine does not exhibit a direct estrogenic activity. An intact HPG axis is necessary which is not the case in ovariectomized females.</li> <li>Sex hormone measurements are rarely carried out in female rodents' studies, which limits the investigation of the empirical evidence of this KER.</li> <li>Characteristics of reproductive aging in the female rats varies among strains (Chapin, 1996; Finch, 2014). This could explain the discrepancies of the results observed between SD and Fischer female rats exposed to atrazine.</li> <li>The internal quality of the primary research study used to substantiate the empirical evidence has not been evaluated (recommendation).</li> </ul>

Eldridge JC, Tennant MK, Wetzel LT, Breckenridge CB and Stevens JT, 1994. Factors affecting mammary tumor incidence in chlorotriazine-treated female rats: hormonal properties, dosage, and animal strain. Environ Health Perspect, 102 Suppl 11:29-36. doi: 10.1289/ehp.94102s1129 Eldridge JC, Wetzel LT and Tyrey L, 1999. Estrous cycle patterns of Sprague-Dawley rats during acute and chronic atrazine administration. Reprod Toxicol, 13:491-499. doi: 10.1016/s0890-6238(99)00056-8 Finch CE, 2014. The menopause and aging, a comparative perspective. J Steroid Biochem Mol Biol, 142:132-141. doi: 10.1016/j.jsbmb.2013.03.010 Gore AC, Oung T, Yung S, Flagg RA and Woller MJ, 2000. Neuroendocrine mechanisms for reproductive senescence in the female rat: gonadotropin-releasing hormone neurons. Endocrine, 13:315-323. doi: 10.1385/endo:13:3:315 Horvath JE, Toller GL, Schally AV, Bajo AM and Groot K, 2004. Effect of long-term treatment with low doses of the LHRH antagonist Cetrorelix on pituitary receptors for LHRH and gonadal axis in male and female rats. Proc Natl Acad Sci U S A, 101:4996-5001. doi: 10.1073/pnas.0400605101 Kermath BA and Gore AC, 2012. Neuroendocrine control of the transition to reproductive senescence: lessons learned from the female rodent model. Neuroendocrinology, 96:1-12. doi: 10.1159/000335994 Mitchard TL and Klein S, 2016. Reproductive senescence, fertility and reproductive tumour profile in ageing female Han Wistar rats. Exp Toxicol Pathol, 68:143-147. doi: 10.1016/j.etp.2015.11.006 Nagaoka T, Takeuchi M, Onodera H, Matsushima Y, Ando-Lu J and Maekawa A, 1994. Sequential observation of spontaneous endometrial adenocarcinoma development in Donryu rats. Toxicol Pathol, 22:261-269. doi: 10.1177/019262339402200304 Shirai N, Houle C and Mirsky ML, 2015. Using Histopathologic Evidence to Differentiate Reproductive Senescence from Xenobiotic Effects in Middle-aged Female Sprague-Dawley Rats. Toxicol Pathol, 43:1158-1161. doi: 10.1177/0192623315595137 Simpkins JW, Swenberg JA, Weiss N, Brusick D, Eldridge JC, Stevens JT, Handa RJ, Hovey RC, Plant TM, Pastoor TP and Breckenridge CB, 2011. Atrazine and breast cancer: a framework assessment of the toxicological and epidemiological evidence. Toxicol Sci, 123:441-459. doi: 10.1093/toxsci/kfr176 USEPA, online. Issues Pertaining to Atrazine Cancer Risk Assessment. Available online: <a href="https://archive.epa.gov/scipoly/sap/meetings/web/html/062700_mtg.html">https://archive.epa.gov/scipoly/sap/meetings/web/html/062700_mtg.html</a> USEPA, online. Atrazine. Draft human health risk assessment for registration review. In atrazine registration review. . Available online: <a href="https://www.govinfo.gov/content/pkg/FR-2018-07-26/pdf/2018-15998.pdf">https://www.govinfo.gov/content/pkg/FR-2018-07-26/pdf/2018-15998.pdf</a> Vidal JD, 2017. The Impact of Age on the Female Reproductive System. Toxicol Pathol, 45:206-215. doi: 10.1177/0192623316673754 Wetzel LT, Luempert LG, 3rd, Breckenridge CB, Tisdel MO, Stevens JT, Thakur AK, Extrom PJ and Eldridge JC, 1994. Chronic effects of atrazine on estrus and mammary tumor formation in female Sprague-Dawley and Fischer 344 rats. J Toxicol Environ Health, 43:169-182. doi: 10.1080/15287399409531913 AOPWiki 2025-01-13T17:48:23

2025-01-14T17:30:59

Decreased, GnRH pulsatility/release leading to estradiol availability, increased via impaired ovulation

Decreased GnRH release leading to increased E2

Martina Panzarea

<div> Anna Lanzoni Martina Panzarea </div>

BY-SA

2.7

Due to the multiplicity of possible MIEs (Kisspeptin decrease, gamma-aminobutyric acid-ergic (GABAergic) modulation, neuropeptides and vasopressin role, etc.), it was decided to develop this AOP starting from the common relevant KE, the reduced availability of GnRH at pituitary level. However, discussion on plausible MIEs is included under Annex <a href="https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2023.7744#efs27744-sec-0053">B.4</a>. Ovarian hormones regulate normal human endometrial cell proliferation, regeneration and function and therefore they are implicated in endometrial carcinogenesis directly or via influencing other hormones and metabolic pathways. The role of unopposed estrogen in the pathogenesis of EC has received considerable attention, together with other hormones, such as androgens and GnRH. One of the key homeostatic hormonal loops in this system is provided by the ovarian hormones, E2 and P4, that modulate the activity of the neuronal network controlling the release of GnRH. The hypothalamic GnRH neurons release GnRH in an episodic manner into the pituitary portal circulation to generate distinct pulses of luteinising hormone (LH) and follicle-stimulating hormone (FSH) throughout the ovarian cycle. Thus, the brain and pituitary produce an on-going pulsatile pattern of gonadotropin secretion that slows on oestrous to allow appropriate follicular development and a surge pattern of secretion at mid-cycle to initiate ovulation. Numerous studies have reported that the oestrous-stage decline in LH pulse frequency results from the post-ovulatory secretion of P4 (Soules et al., 1984; Smith et al., 1989; Goodman 2015) and the administration of P4 was found to dramatically slow GnRH pulse generator activity in the mouse (McQuillan et al., 2019). Thus, it seems very likely that P4 is the key gonadal hormone exerting a negative feedback influence upon the pulse generator during the cycle and does so to bring about the post-ovulatory slowing of pulsatility. As follicles grow, estrogen synthesis increases in the female ovary. This in turn promotes GnRH pulses in the hypothalamus. GnRH binds to its receptor expressed by pituitary gonadotropic cells and induces the release of 2 gonadotropins, LH and FSH. In turn, LH and FSH stimulate gametogenesis and steroidogenesis in the gonads (Duffy et al., 2018). An LH surge is needed and responsible for the downstream pathways that induce ovulation; this includes resumption of meiosis in the oocyte and cellular changes that allow rupture of the follicle to release the egg for fertilisation. It increases intrafollicular proteolytic enzymes, weakening the wall of the ovary and allowing for passage of the mature follicle (Robker et al., 2018). The suppression of GnRH availability, due to the impairment of regulatory systems or destruction of the peptide, results in a failure of response to pre-ovulatory level of estrogen to produce LH surges. Without the LH surge, the downstream pathways are not able to function and as a result ovulation does not occur. If the LH surge is delayed, then ovulation may be delayed as well and fails to occur within the correct time window. This can have a negative impact on the reproductive health of females and perturb the oestrous cycle. In most cases, if ovulation is blocked or delayed, the ratio of estradiol/progesterone (E2/P4) remains high due to lack of P4 increase that is initiated after ovulation. As a result, ovarian and circulating steroid hormone levels remain in the ‘pre-ovulatory’ state, i.e. high E2, and low P4. In addition, with ovulation disruption, formation of corpus lutea is delayed or inhibited. This overall disrupts the cycle and can lead to persistent oestrous. Persistent oestrous is characterised by the lack of corpus lutea formation, and observation of cysts and antral follicles. Morphologically, it is demonstrated by persistent vaginal cornification (PVC). It is considered persistent if at least two cycles were perturbed with the appearance of PVC (Finch, 2014; Stewart et al., 2022). A prolonged increased circulating E2/P4 ratio leads to an increase of E2 bioavailability in a variety of estrogenic-responsive organs, including the uterus due to insufficient counterbalance by P4. However, compensatory mechanisms (e.g. intracrine networks) may differ across different tissues. The degree to which E2/P4 ratio should increase to overwhelm these compensatory responses has not been established.

Please refer to the <a href="http://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2023.7744#efs27744-sec-1004">EFSA Scientific Opinion</a> for an overview of the Context and Strategy of the AOP development.

Persistent vaginal cornification is an adverse outcome monitored in Endocrine Disruptor Screening Program (EDSP) protocol (US EPA 1998; OECD 2001).

adjacent

High

High adjacent

High

High adjacent

Moderate

Moderate adjacent

Not Specified

Not Specified adjacent

Not Specified

Not Specified Male Not Specified Female

Not Specified

Adults

Not Specified

Sex Applicability: Males and Females Taxonomic Applicablity: Restricted to mammals. Life Stages Applicability: Adulthood.

<table cellspacing="0" class="MsoTableGridLight" style="border-collapse:collapse; border:none; width:980px"> <tbody> <tr> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; height:39px; width:93px"> KER title </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; height:39px; width:83px"> Biological Plausibility </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; height:39px; width:87px"> Empirical Support<a class="msocomanchor" href="#_msocom_1" id="_anchor_1" name="_msoanchor_1">[KT1]</a>  </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; height:39px; vertical-align:top; width:94px"> Essentiality </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:1px solid #bfbfbf; height:39px; width:623px"> Brief Explanation (summary) </td> </tr> <tr> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; height:8px; width:93px"> KER 1: KE1 => KE2 reduced GnRH availability pulsatory release leads to decrease/delayed LH surge </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:8px; width:83px"> H </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:8px; width:87px"> H </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:8px; width:94px"> H </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:8px; vertical-align:top; width:623px"> Biological Plausibility. There is an extensive understanding based on extensive previous documentation and broad acceptance that LH surge depends by GnRH release and availability, it is considered a consolidated scientific concept with a well-established mechanistic basis. As consequence, the KER is considered a high biological plausible KER.   Empirical Support. There are multiple studies showing dependent change in both events following exposure to a range of specific stressors i.e., atrazine, tributyltin (TBT), endopeptidases, GABA modulators and light stimulation. Also, the available evidence support a concordance in terms of temporal, dose- response and incidence with no or few critical data gaps or conflicting data (see section “empirical evidence” of this KER). The empirical support was therefore considered high.   Essentiality. There is enough direct evidence supporting the essentiality of the current KE, blocking the GnRH receptor with a competitive antagonist led to a full blockage of the LH surge in ewes (Karsch et al., 1997) and rats (Wu, 1997 and Lasdun, 1089). Moreover, different studies demonstrates that the selective deletion of estrogen receptor alpha from kisspeptin neurons (neurons that innervates the GnRH neurons) as well lesions of the AVPv nucleus, resulted in an abolishment of the LH surge leading to persistent estrous (Dubois, 2015). As a matter of facts, estrogens are acting on the kisspeptin neurons, the GnRh neurons have not the estrogen receptor. Indirect evidence (i.e., Goodman 2015) is also available.   The essentiality is therefore weighted as high.   </td> </tr> <tr> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; height:75px; width:93px"> KER 2: KE2 => KE3:  Reduced LH surge leads to delayed ovulation </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:75px; width:83px"> H </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:75px; width:87px"> H </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:75px; width:94px"> H </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:75px; vertical-align:top; width:623px"> Biological Plausibility. The biological plausibility of this KERs is linked to the physiological role of LH in mammals which is a consolidated scientific concept (dogma). The biological plausibility is therefore high. Empirical support. There are multiple studies showing dependent change in both events following exposure to a range of specific stressors i.e., atrazine, TCDD, PFOS, and GnRH antagonists. Also, the available evidence (see section “empirical evidence” of this KER) supports a concordance in terms of temporal, dose- response and incidence with no or few critical data gaps or conflicting data. The empirical support was therefore considered high.   Essentiality.  KO animal models were used to demonstrate that both LH and the LHCGR is necessary to trigger ovulation. This is also corroborated by the evidence that LH or LHCGR mutations found in women from different families presents lack of ovulation and issue on menstrual cycle.   Therefore, there is enough direct evidence supporting the essentiality of this KEs. The essentiality is considered high.   </td> </tr> <tr> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:1px solid #bfbfbf; border-right:1px solid #bfbfbf; border-top:none; height:93px; width:93px"> KER3 – KE3 => KE4: Delayed ovulation leads to estrogen dominance </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:93px; width:83px"> H </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:93px; width:87px"> M </td> <td style="background-color:#e7e6e6; border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:93px; width:94px"> M </td> <td style="border-bottom:1px solid #bfbfbf; border-left:none; border-right:1px solid #bfbfbf; border-top:none; height:93px; vertical-align:top; width:623px"> Biological Plausibility. The biological plausibility of this KERs is linked to the physiology of reproductive system.  Being a consolidated scientific concept, the biological plausibility is high. Empirical support. There are few studies showing dependent change in both events. Moreover, the analytical methods used to measure the levels of hormones in plasma, make difficult a comparison of the E2/P4 (Estradiol/Progesteron) ratio among different studies. The empirical support was therefore considered moderate.   Essentiality.  From the biological perspective it is well established that a lack of ovulation perturbs the estrous cycle due to the absence of the expected increase in progesterone secretion with the formation of corpus lutea. This alters the balance between estrogens and progesterone expected after ovulation.   However, it is noted that the empirical evidence found in support of the current AOP development, clearly reported that in the knockout strains of LHβ or its receptor LHCGR, where the ovulation was found to be destroyed, both estradiol and progesterone are decreased in the serum or ovary. This evidence is not supporting the essentiality of this KEs and its impact on the later KE in this AOP i.e., estrogen dominance.   The essentiality is therefore weighted as moderate. </td> </tr> </tbody> </table>

<div> <table class="table table-bordered table-fullwidth"> <thead> <tr> <th>Modulating Factor (MF)</th> <th>Influence or Outcome</th> <th>KER(s) involved</th> </tr> </thead> <tbody> <tr> <td> </td> <td> </td> <td> </td> </tr> </tbody> </table> </div>

Dubois SL, Acosta-Martínez M, DeJoseph MR, Wolfe A, Radovick S, Boehm U, Urban JH and Levine JE, 2015. Positive, but not negative feedback actions of estradiol in adult female mice require estrogen receptor α in kisspeptin neurons. Endocrinology, 156:1111-1120. doi: 10.1210/en.2014-1851 Duffy DM, Ko C, Jo M, Brannstrom M and Curry TE, 2019. Ovulation: Parallels With Inflammatory Processes. Endocr Rev, 40:369-416. doi: 10.1210/er.2018-00075 Finch CE, 2014. The menopause and aging, a comparative perspective. J Steroid Biochem Mol Biol, 142:132-141. doi: 10.1016/j.jsbmb.2013.03.010 Karsch FJ, Bowen JM, Caraty A, Evans NP and Moenter SM, 1997. Gonadotropin-releasing hormone requirements for ovulation. Biol Reprod, 56:303-309. doi: 10.1095/biolreprod56.2.303 Lasdun A, Reznik S, Molineaux CJ and Orlowski M, 1989. Inhibition of endopeptidase 24.15 slows the in vivo degradation of luteinizing hormone-releasing hormone. J Pharmacol Exp Ther, 251:439-447 McQuillan HJ, Han SY, Cheong I and Herbison AE, 2019. GnRH Pulse Generator Activity Across the Estrous Cycle of Female Mice. Endocrinology, 160:1480-1491. doi: 10.1210/en.2019-00193 Robker RL, Hennebold JD and Russell DL, 2018. Coordination of Ovulation and Oocyte Maturation: A Good Egg at the Right Time. Endocrinology, 159:3209-3218. doi: 10.1210/en.2018-00485 Stewart CA, Stewart MD, Wang Y, Mullen RD, Kircher BK, Liang R, Liu Y and Behringer RR, 2022. Chronic Estrus Disrupts Uterine Gland Development and Homeostasis. Endocrinology, 163. doi: 10.1210/endocr/bqac011 Wu TJ, Pierotti AR, Jakubowski M, Sheward WJ, Glucksman MJ, Smith AI, King JC, Fink G and Roberts JL, 1997. Endopeptidase EC 3.4.24.15 presence in the rat median eminence and hypophysial portal blood and its modulation of the luteinizing hormone surge. J Neuroendocrinol, 9:813-822. doi: 10.1046/j.1365-2826.1997.00637.x AOPWiki 2025-01-10T14:01:22

2025-04-03T15:58:17