Decreased Gnrh gene expression as a result of prenatal letrozole treatment in female rats was proved in this study for the first time. In a similar study, pregnant treatment of letrozole in rats caused depression and anxiety- like behaviors due to decreased neurogenesis in hippocampus (17). Letrozole induced mouse model of PCOS showed increased expression of gonadotropin releasing hormone receptor (Gnrhr) in the pituitary, that was not reversible by flutamide treatment (18). Moreover, increased hypothalamic Gnrh and pituitary Gnrhr transcripts were observed in an adult rat model induced by 21 days 0.5 mg letrozole administration (19). In another adult letrozole PCOS model, no changes in Gnrh mRNA expression, but increased pituitary Gnrhr mRNA expression was observed (20). Moreover, an elevated number of AR and GnRH immunoreactive cells and AR mRNA expression were shown due to DHT induced PCOS in adult rats (21). The lack of prenatal androgenization PCOS studies evaluating Gnrh expression, and inconsistencies in studies of PCOS induction with letrozole in adulthood complicate the interpretation of results. However, the mechanism that indicates an increase in Gnrhr expression appears to be more influential in the etiology of PCOS (20).
On the other hand, puberty onset is controlled by high frequency GnRH neurons that affect FSH and LH release to trigger gonads for puberty initiation (22); therefore, the elevated Gnrh expression in our study in untreated group may be due to the normal neuroendocrine changes at puberty onset. At the same time, decreased Gnrh expression in letrozole treated groups led to late puberty. However, it would be better to investigate the direct effect of androgens using flutamide during pregnancy on these neuroendocrine changes, which was not performed in our study due to budget constraints.
In addition, the impact of upstream mechanisms on GnRH control should not be overlooked. In prenatal androgenization mice models, the putative γ-aminobutyric acid GABAergic synaptic connections to GnRH neurons was elevated at adulthood; suggesting an increase in GnRH neurons pulse and frequency due to the effect of GABAergic neurons via mediating the negative feedback of steroids (23–26). In addition, prenatal androgenized female rats by testosterone displayed decreased progesterone receptor (Pgr) mRNA expression in the hypothalamic POA indicating the capability of prenatal androgen in creating alteration in the GnRH neurosecretory system and neuroendocrine dysfunctions at adulthood such as infertility related to PCOS (27). Moreover, some abnormalities in KNDy neuropeptides secretion were shown in various animal models of PCOS; for instance, prenatal testosterone treated ewes showed KNDy expression abnormalities (28). In a study by Caldwell et al. (29), prenatal administration of DHT did not alter the KNDy neuropeptides. Furthermore, adult PCOS models are noteworthy in this field. Letrozole treated adult female mice revealed an increased neuronal activation of Kiss1 (30). A DHT induced PCOS rat model resulted in decreased kiss1 gene expression, but the serum levels of testosterone, estradiol, LH, FSH were unaltered (31). Interestingly, in a letrozole induced PCOS model in adult rats, increased positive-cell number of kisspeptin in the arcuate and decreased number of positive kisspeptin in anteroventral periventricular (AVPV) nucleus were reported (32). Kisspeptin neurons in the arcuate nucleus are involved in the negative feedback of estradiol on the GnRH/LH system. On the other hand, kisspeptin neuropeptides of the AVPV mediate preovulatory LH surge (33). Therefore, increased kiss1 gene expression in the arcuate nucleus can interfere with the PCOS pathology (32). In general, the effect of prenatal and adult administration of androgens on inhibitory and excitatory regulators of GnRH neurons is likely more important than the direct impact of androgens on these neurons. In various models, alterations in Gnrh expression or basal or pulsatile levels of GnRH neuropeptide are different. This might be due to the fact that different neural pathways influence the GnRH neurons (30). In spite of the fact that, there are limitations due to the lack of information on GnRH pulse frequency and amplitude, so the baseline value of serum LH levels cannot be attributed to the pulsatile GnRH secretion and also Gnrh gene expression. Given the fact GnRH secretion is affected by a set of stimulatory and inhibitory factors, including KNDy neuropeptides and gamma-aminobutyric acid (GABA)ergic neurons, further studies are needed to evaluate the changes in upstream GnRH regulators, especially in prenatal PCOS models.
Compared with other groups, increased expression of Rfrp gene in the hypothalamic DMN was observed at 1.25 and 1 mg/kg BW letrozole treatment. Prepubertal letrozole implants releasing 50 µg/day for 16 days before puberty did not impact on Rfrp expression in DMN; however, increased LH levels were found in adult female rats suggesting the role of other endogenous regulators of GnRH such as KNDy neuropeptides or GABAergic inputs rather than RFRP-3 neurons (30). Moreover, in our previous study, constant light induced PCOS rat model decreased Rfrp expression, along with unaltered FSH and LH serum levels in adult female rats (34). Furthermore, in neonatal testosterone treated female rats, decreased Rfrp mRNA expression was reported, without any effect on LH serum level (35). These studies revealed that serum LH concentration did not reflect the effect of Gnrh and Rfrp expression changes directly. In other words, the changes in the expression of these neuropeptides are probably not the main neuroendocrine mechanism for LH increase in PCOS women. On the other hand, it was shown that the intracerebroventricular injection of RFRP-3 decreased Gnrh mRNA expression in female rats (22), demonstrating the inhibitory effect of RFRP-3 on GnRH neurons. These results are consistent with our study. It has also been shown that the number of Rfrp expressing neurons in adulthood is lower than at birth in both sexes of mice (36). Therefore, alteration in Rfrp expression in control groups is normal for the ages after puberty onset, but in treated group, especially at 1.25 and 1 mg/kg BW, enhanced Rfrp expression may be as a result of excessive androgen production during intrauterine life.
Taken together, given that intrauterine hyperandrogenic environment is a critical etiological agent in neurodevelopmental diseases and also hyperandrogenism status in PCOS women, such studies are very important for evaluating PCOS neuroendocrine and neuro-behavior abnormalities (17). Although in our study addressing to main purpose of assessing neuroendocrine changes specific to PCOS was not straightforward, but this study is a background for future assessments.
This work highlighted delayed puberty as a result of 1.25 and 1 mg/kg BW prenatal letrozole treatment. Commonly, vaginal opening in rats occurs at 28–49 postnatal days (PNDs), but the age of puberty onset and beginning of sexual maturation is different according to the species and growth rate (37). Delayed puberty in our study, may be due to increased Rfrp expression in 1.25 and 1 mg/kg BW groups. Similarly, in a study by Han et al (22) it was shown that intracerebroventricular injection of RFRP-3 between days 28 and 36, at the time of puberty onset, delayed vaginal opening in rats. Furthermore, Rfrp expression in DMN decreased during early pre-pubertal stages in the mouse. This reduces its inhibitory effect on GnRH neurons (38). Similarly, GPR147 (the RFRP-3 receptor) knockout on GnRH neurons caused delayed puberty in mice (38). The increased expression of Rfrp may be due to elevated androgen levels during the intrauterine life but this hypothesis should be investigated further by evaluating the AR on RFRP-3 neurons or by blocking the direct effects of androgen by flutamide.
The findings of our research showed markedly elevated testosterone levels and reduced estradiol levels in 1.25 and 1 mg/kg BW groups. Serum FSH and LH levels were also higher in control groups showing that gonadotropins levels were not affected by prenatal androgenization via letrozole. However, progesterone concentration, in proportion to the higher number of corpora luteua in the 0.25 mg/kg BW group, was the highest in this group. Letrozole treatment in adult female rats for 14 days resulted in reduced estradiol levels (39). Increased LH levels, as a main feature of PCOS women, has also been observed in adult rodent letrozole models (20, 40, 41). In the present study, serum LH levels at 1.25 mg/kg BW were increased compared with other groups. In prenatal androgenized female rats, FSH levels were not affected by androgen action (42, 43). In our study, Serum FSH concentration decreased in letrozole treated groups vs control groups. There have been inconsistencies in the baseline and pulse concentration of LH and FSH in various PCOS prenatal and clinical studies of PCOS women (7, 23, 44). These inconsistencies may be due to the different estrous phases of female rats during sampling, animal models and species, time of induction and other experimental conditions. In letrozole treated animals, increased endogenous androgen production by inhibiting aromatase function is unavoidable, and has been reported in different models of PCOS induction with letrozole (19, 45, 46). In our study, consistent with previous findings, considerable increase in testosterone at 1.25 and 1 mg/kg BW was recorded, as well as decreases in estradiol levels. In daughters of women with PCOS, increased testosterone levels were reported during puberty but in general there is little information available (47). Serum estradiol levels did not change in PCOS induced by prenatal testosterone administration (42) and also adult letrozole model (19, 40, 48). But in another studies, reduced estradiol levels were reported in letrozole treated PCOS adult rat model (46, 49). Given that estradiol levels vary during the estrous cycle, and estradiol is at the lowest level in estrous phase, these differences are probably due to measurements taken at different times during the estrous cycle. On the other hand, variations in estradiol levels are the direct effects of letrozole due to reducing the conversion testosterone to estradiol.
In the current study, all letrozole treated groups exhibited acyclicity. Moreover, the delay in initiation of cycles (first proestrus) and reduced number of females that were able to complete one or more cycles were observed in all treated groups. In our study, the total number of growing follicles, atretic follicles, cystic follicles and corpora lutea was higher at 0.25 mg/kg BW letrozole. Increased the total number of follicles and atretic follicles was observed in 1.25 and 1 mg/kg BW respectively. It seems that prenatal administration of letrozole has not been able to develop the morphological characteristics of PCOS as well as the adult PCOS model of letrozole (50). Further, irregular cyclicity was observed as a result of prenatal letrozole administration. Irregular cycles and ovulation dysfunction are the usual effect of exposure to androgen excess during intrauterine life (27, 51). It seems that anovulatory cycles arise from androgen mediated functions, because flutamide as an androgen receptor antagonist could recover ovulatory cycles in prenatal androgenized female mice (23). Irregular cycles, fewer corpora lutea and ovarian cysts formation were shown as a result of prenatal DHT treatment in wild-type mice, but these effects were not observed in AR knockout mice indicating the key role of androgen signaling in creating PCOS-like features (29). Increased number of antral and preantral follicles was observed in prenatal rats exposed to androgen on 16–19 GDs but not on 20 GD (42), indicating that the timing of androgen administration is important. Induction of testosterone and dihydrotestosterone at the end of gestation had no effect on polycystic ovarian development, although it induced irregular and anovulatory cycles (7, 43). These inconsistent results suggest that, first, the prenatal hyperandrogenic letrozole model is unable to produce morphological features of PCOS; and, second, that this trait may not be affected by the intrauterine environment.
The letrozole androgenized female rats showed longer anogenital distance (AGD) at puberty than untreated groups. Furthermore, we observed increased AGD in all treated groups. The anogenital distance (AGD) and anogenital distance index (AGDI) are both main indicators of maternal hyperandrogenism (52). In general, AGD is longer in the male rats than in the female ones, so ADG in female reflects the degree of uterine hyperandrogenism that she experienced during the intrauterine life and the higher doses of androgen exposure during fetal life led to the longer AGD in female offspring. Consistent with our data, longer AGD caused by prenatal testosterone administration on 20 GD was reported (42). In addition, the long AGD in prenatal T and DHT (on 16–19 GDs) female rats was shown (43). Increased AGD reflects the androgenic effect of letrozole during the time of external genitalia differentiation. External genitalia differentiation in female rat occurs during late pregnancy (19–20 GDs), underlying the direct effect of dihydrotestosterone (53). DHT is a non-aromatizable androgen and acts like an aromatase inhibitor, therefore, letrozole and DHT have the same effect on AGD changes mediated by androgen increments. These findings suggest that androgen action during embryonic life, especially at the time of external genitalia differentiation, affects AGD, which is a good indicator of direct effect of androgens.
Prenatal letrozole administration at 1.25, 1.0 and 0.75 mg/kg BW significantly increased the body weight gain compared with other groups. These body weight gain increments were greater than control groups at 6 to 8 postnatal weeks, suggesting the metabolic effects of prenatal letrozole appeared at adulthood. Increased body weight gain as a metabolic feature of PCOS has been confirmed in various adult letrozole induced PCOS models in the rat (8, 48, 49, 54), and also in DHT PCOS induction model in mice (55). Arroyo et al. (56) reported that prepubertal letrozole model caused increased body weight gain in mice which did not improve with letrozole removal, contrary to the reproductive traits induced in this model that were completely recovered. However, in another study, weight gain caused by adult letrozole PCOS model was reversible by flutamide in mice (18). These findings suggested that metabolic alterations such as body weight gain by prenatal or prepubertal origins were more permanent.
Delayed delivery observed in treatments with 1.25 and 1 mg/kg BW letrozole in this study may be due to the reduced level of estrogen induced by letrozole treatment. In fact, high levels of estrogen are necessary for initiation of parturition (39). Letrozole administration at 0.002 or 0.02 mg/kg BW per day during 15–21 GDs delayed parturition in rat (57). Reduced litter size was observed in 1.25 mg/kg group, suggesting that letrozole at high doses has severe feto toxic effects. In a study by Zhang et al (58), co-treatment of pregnant rats with DHT (1.66 mg/kg BW per day from 7.5–13.5 GDs) and insulin resulted in fetal death and subsequently reduced litter size. Treatment of pregnant rats with 0.02 mg/kg letrozole at 15–21 GDs caused fetal mortality (57). Letrozole administration during organogenesis (6–16 GDs) in rats at 0.01, 0.02 and 0.04 mg per kg doses resulted in post implantation loss that included early and late resorption and decreased number of viable fetuses (59). These findings suggested that embryo and feto toxic effects of letrozole were dose-dependent, depending on the time of administration (39). It seems these changes are caused by the letrozole action in reducing estrogen levels because simultaneous estrogen treatment at maximum dose of letrozole (0.04 mg/kg BW) restrained the feto toxic effects of letrozole (60). However, increased fetal mortality may be due to the direct effect of the androgen increment as a result of letrozole, as PCOS women have been reported to have higher perinatal fetal mortality rates than women with normal androgen levels (61). Furthermore, the shorter exposure time and higher letrozole dose was the main difference between our study and these studies; so it seems that the disrupting effects of letrozole are related to the specific (organogenesis or fetal stage) time during pregnancy (39). and the administrated dose. In our study, the moderate effect of higher letrozole dose on fetal mortality could be due to administration at the late pregnancy, that is, letrozole had the greatest effect on fetal viability. Generally, the letrozole mediated embryo or feto toxic effects were dose dependent and the administered dose in pregnant rats in at least 1% of the maximum dose prescribed to humans on a daily basis resulted in fetal or embryonic death (62). Due to the fact that estrogen is greatly increased in the last third of rat pregnancy (62) and rodent ovaries are the prominent site of estrogen biosynthesis throughout pregnancy (39), inhibition of estrogen synthesis in the ovary in late pregnancy causes adverse effects on maternal and fetal aspects of pregnancy in rats as observed in our study with increasing dose.
In summary, the results of our study suggested that prenatal letrozole treatment at doses lower than 1 mg/kg BW is safe and has no detrimental embryotoxic and lethal effects on fetuses and mothers in Sprague-Dawley rats. A comprehensive dose response study was carried out to evaluate the alterations of major genes controlling reproductive phenomena, Gnrh and Rfrp, steroid hormones and gonadotropins and ovarian function in response to prenatal letrozole administration. We recorded irregularities in estrous cycles, follicular development changes, increased testosterone levels, decreased estradiol levels, decreased Gnrh, increased Rfrp expression at 1.25 and 1 mg/kg BW letrozole. Due to the greater effect of 1.25 mg/kg BW treatment on fetal mortality, we finally concluded that prenatal letrozole treatment at 1 mg/kg BW on 16–18 GDs in the rat, is probably optimal for PCOS induction by inhibiting aromatase and indirectly increasing endogenous androgen levels. In terms of future work, it may be best to first confirm the prenatal PCOS letrozole model at a dose of 1 mg/kg BW which was not possible in this study due to lack of budget. Further, it would be interesting to examine the upstream and downstream pathways that control the GnRH and RFRP-3 neurons. Another possible area of future research would be to investigate whether there is a relationship between these neuroendocrine pathways and changes in androgen levels.