Impaired ovulation leads to Plasma estradiol/progesterone ratio, increase

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).

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 Annex B.3.

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)