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

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).

Figure 14. Schematic pattern of typical endocrine changes during the rat estrous cycle (Anderson, 2013)

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

• 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 P4 and E2, rodent-specific immunoassays are commercially available (Andersson, 2013). 

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).

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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

Levine J, 2015. Neuroendocrine Control of the Ovarian Cycle of the Rat. pp. 1199-1257.

Lu KH, Hopper BR, Vargo TM and Yen SS, 1979. Chronological changes in sex steroid, gonadotropin and prolactin secretions in aging female rats displaying different reproductive states. Biol Reprod, 21:193-203. doi: 10.1095/biolreprod21.1.193

McKenna NJ, 2015. Chapter 9 – Gonadal Steroid Action. Proceedings of the

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O'Connor KA, Ferrell RJ, Brindle E, Shofer J, Holman DJ, Miller RC, Schechter DE, Singer B and Weinstein M, 2009. Total and Unopposed Estrogen Exposure across Stages of the Transition to Menopause. Cancer Epidemiology, Biomarkers & Prevention, 18:828-836. doi: 10.1158/1055-9965.EPI-08-0996

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, 2018. Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents.

Prior JC and Hitchcock CL, 2011. The endocrinology of perimenopause: need for a paradigm shift. Front Biosci (Schol Ed), 3:474-486. doi: 10.2741/s166

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Westwood FR, 2008. The female rat reproductive cycle: a practical histological guide to staging. Toxicol Pathol, 36:375-384. doi: 10.1177/0192623308315665