Biol Reprod. 2005 Oct;73(4):798-806.
Adverse effects on female development and reproduction in CD-1 mice following neonatal exposure to the phytoestrogen genistein at environmentally relevant doses.
Jefferson WN, Padilla-Banks E, Newbold RR.
Developmental Endocrinology Section, Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
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Outbred female CD-1 mice were treated with genistein (Gen), the primary phytoestrogen in soy, by s.c. injections on Neonatal Days 1-5 at doses of 0.5, 5, or 50 mg/kg per day (Gen-0.5, Gen-5, and Gen-50). The day of vaginal opening was observed in mice treated with Gen and compared with controls, and although there were some differences, they were not statistically significant. Gen-treated mice had prolonged estrous cycles with a dose- and age-related increase in severity of abnormal cycles. Females treated with Gen-0.5 or Gen-5 bred to control males at 2, 4, and 6 mo showed statistically significant decreases in the number of live pups over time with increasing dose; at 6 mo, 60% of the females in the Gen-0.5 group and 40% in the Gen-5 group delivered live pups compared with 100% of controls. Mice treated with Gen-50 did not deliver live pups. At 2 mo, >60% of the mice treated with Gen-50 were fertile as determined by uterine implantation sites, but pregnancy was not maintained; pregnancy loss was characterized by fewer, smaller implantation sites and increased reabsorptions. Mice treated with lower doses of Gen had increased numbers of corpora lutea compared with controls, while mice treated with the highest dose had decreased numbers; however, superovulation with eCG/hCG yielded similar numbers of oocytes as controls. Serum levels of progesterone, estradiol, and testosterone were similar between Gen-treated and control mice when measured before puberty and during pregnancy. In summary, neonatal treatment with Gen caused abnormal estrous cycles, altered ovarian function, early reproductive senescence, and subfertility/infertility at environmentally relevant doses.

From the full text article:

Exposure to estrogenic substances during critical periods of development can have adverse consequences on differentiating reproductive systems of rodents and humans [1]. The most well-known example is the synthetic estrogen, diethylstilbestrol (DES), which has been documented to cause benign and malignant reproductive tract abnormalities in prenatally exposed males and females of several species [1]. In particular, malformations of the female reproductive tract, alterations in the onset of puberty, alterations in estrous cyclicity, subfertility/infertility, and reproductive tract lesions have been reported in experimental animals and humans [1–4]. Because numerous chemicals in our environment possess estrogenic activity [5–7], the possibility exists that some of these chemicals may disrupt normal processes of development, differentiation, and subsequent function of the reproductive system similar to the adverse effects caused by DES [8].

Phytoestrogens are a group of naturally occurring compounds that have been reported to cause fertility problems in animals [9–13]. Of particular concern is genistein (Gen), the major phytoestrogen in soy products [14], which has potent estrogenic activity both in vitro and in vivo [15–18]. Human fetuses and neonates can be exposed to high levels of Gen if their mothers consume excessive amounts of soy [19] or if they are given soy-based formulas and other soy products marketed specifically for children [14, 20, 21]. The concentrations of Gen and other isoflavones found in some of these soy-based products far exceeds the amount found in an adult diet; one study estimates that infants fed soy-based formulas consume approximately 6–9 mg/kg per day of Gen compared with 1 mg kg day for an adult vegetarian [14]. Soybeans also have an extremely variable isoflavone content depending on variety and environmental conditions such as growing season and location [22], and the U.S. Department of Agriculture reports variable amounts of Gen in various soy products [23].

Over the last few years, public and scientific interest in phytoestrogens such as Gen has increased because of its proposed beneficial effects. Currently, there are mixed results on developmental exposure to Gen, suggesting some beneficial effects, but also adverse effects depending on the timing of exposure, dose level, and end points examined. For example, two studies report that prenatal exposure to Gen prevents carcinogen-induced mammary gland cancer in rats [24, 25], whereas another study shows an increase in mammary gland cancer if the developmental window of exposure is shifted to neonatal life [26]. Other investigations report improved cholesterol synthesis rates of human infants consuming soy-based formulas [27]. Vegetarian diets containing high levels of soy during pregnancy have been associated with increased incidence of hypospadias in the male offspring [28]. Further, an epidemiology of health outcomes in young adults who were fed soy-based infant formulas reported an increase in more frequent use of allergy medicines in both men and women, and longer menstrual bleeding and more discomfort during the menstrual cycle in women [29, 30]. So the adverse effects of developmental exposure to Gen remain of concern.

A recent study from our laboratory has shown that neonatal exposure to Gen at a dose of 50 mg kg day on Days 1–5 leads to an increased incidence of uterine adenocarcinoma in mice later in life; the incidence of uterine tumors in Gen-treated mice (35%) was similar to the incidence found in mice treated with an equal estrogenic dose of DES (0.001 mg/kg per day; 31%) [31]. Although Gen was administered as s.c. injections, the levels of Gen used in our studies produced circulating serum levels similar to the range of those found in infants consuming soy-based formulas [32]. Therefore, the dose of Gen to the target tissue was comparable between s.c. injections and oral exposures. Similar findings have also been reported by Lewis et al. [33] in neonatal rats using a dose of 40 mg/kg per day. Further studies have shown adverse effects on the developing rat following Gen exposure, including altered brain function, estrous cyclicity, and reproductive behavior [34, 35]. Studies using other phytoestrogens including coumestrol [9, 36], daidzein [13, 35], and red clover [10, 12] have also demonstrated disruptions in reproduction and/or reproductive end point, supporting the concept that phytoestrogens, although weaker than DES or 17ß-estradiol, can cause adverse effects on the developing reproductive tract [7]. Also, some of these effects may not be apparent until later in life, similar to those caused by DES [1].

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This study found that neonatal exposure of mice to Gen at environmentally relevant doses caused abnormal estrous cyclicity, altered ovarian function, early reproductive senescence, and subfertility/infertility. The relevance of our findings to human health with these environmental exposure levels is a matter of concern. It is well known that humans are exposed in utero to varying levels of phytoestrogens with higher levels in vegetarian mothers who eat soy foods [19, 28]; human infants are also exposed to phytoestrogens in breast milk from vegetarian mothers [20, 39]. However, infants exposed to high levels of phytoestrogens (6–9 mg/kg per day) such as Gen in soy-based infant formulas and other soy products [14, 39, 40] are of most concern.

... Data from another laboratory using orally dosed neonatal rat pups with Gen at similar doses used in our study showed similar adverse effects, including MOFs in the ovary and reduced female fertility [48]. Another study exposing Sprague-Dawley rats during pregnancy and lactation to Gen in the diet showed a reduction in the percentage of female mice delivering live pups (controls, 9/ 10; Gen 1250 ppm, 5/10) [49]. All of these data taken together strongly suggest that Gen exhibits similar results on the female reproductive system regardless of the route of exposure (oral or s.c.) or the species examined (rat or mouse).

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The current study indicates that there are problems with female reproductive development and function in mice exposed neonatally to Gen. There were no apparent differences in serum hormone levels in immature Gen-treated mice compared with controls, indicating no inherent differences in circulating levels of progesterone or estradiol between the treatment groups before puberty. While there were slight alterations in the onset of puberty, with lower doses advancing vaginal opening and higher doses delaying it, the mean day of vaginal opening was not statistically different between the groups. Although we did not show a significant difference in the mean age of vaginal opening in the current study, others have shown differences, particularly in the rat model, supporting the idea that puberty may be altered following developmental exposure to Gen depending on the time of exposure and the species examined. For example, Nikaido et al. [51] showed earlier onset of puberty in mice following prenatal exposure to several environmental estrogens including Gen at a low dose of 0.5 mg/kg [51]. Levy et al. [52] showed a delay in vaginal opening following prenatal exposure of rats to Gen at a dose of 5 mg/kg, and Kouki et al. [35] showed an advanced time of vaginal opening in rats treated neonatally with Gen at a dose of 1 mg/kg.

Data from the current study also showed alterations in the estrous cycle of mice following neonatal exposure to Gen at all doses examined with extended cycle length being the most common finding. Others have shown similar estrous cycle alterations in other model systems, including the study by Nikaido et al. showing several environmental estrogens including Gen, resveratrol, zearalenone, and bisphenol A given during pregnancy caused extended estrous cycles [51]. Kouki et al. also showed irregular estrous cycles following neonatal exposure of rats to Gen with prolonged periods in estrus [51]. Alterations in estrous cyclicity were exacerbated over time, with more mice exhibiting persistent estrus at 6 mo compared with 2 mo of age; interestingly, not only at the high dose, but at the lower doses as well. This altered estrous cyclicity at the lower doses may in part explain the early reproductive senescence observed in these mice, particularly at 6 mo of age.

Our current study also clearly demonstrates that the ovary is adversely affected by neonatal exposure to Gen. Although all Gen-treated mice ovulate under exogenous hormonal influence, the ovulation rate was much lower in the highest dose of Gen under their own hormonal cues as evidenced by fewer CLs at 2 mo and during pregnancy, and by the absence of CLs at 4 mo of age. In addition, the lower doses of Gen treatment also resulted in alterations in ovarian function with more CLs than their control counterparts at 4 mo of age. This enhanced ovulation rate is similar to what was observed at 26 days of age following superovulation in a previous study in our laboratory [37]. This may also in part explain the early reproductive senescence observed in the two lower doses of Gen treatment at 6 mo of age. Because more oocytes are ovulated earlier, there may be a decrease in the number of oocytes available for fertilization at later time points. This effect is not unique to Gen, because earlier studies in our laboratory using DES showed similar effects; low doses of prenatal exposure, neonatal exposure, or both to DES causes enhanced ovulation rates and early reproductive senescence, further suggesting that ovulation of too many oocytes early in life may lead to lower fertility rates later in life [2]. It has been reported that aged mice do not typically exhaust their total complement of oocytes; however, they exhibit characteristics of reproductive senescence, including lowered responsiveness of the pituitary to estradiol, gradual loss of ovulatory function, decreased fertility, and smaller litter sizes [53–57], but this occurs much later in life than 6 mo of age, which was observed following neonatal Gen exposure. Early reproductive senescence could be important for human reproductive health because more and more women are waiting longer to become pregnant [58].

One possible explanation of enhanced ovulation rates observed in lower doses was proposed by Faber and Hughes [59]. That study showed that neonatal exposure of rats to low doses of Gen (0.01 mg/kg) was associated with an increased pituitary response to GnRH, producing higher levels of LH [59]. Mice treated with lower doses of Gen used in this study may be hyperresponsive to GnRH stimulation leading to enhanced ovulation rates, which we have shown previously in younger mice [37] and again in older mice in this study. In addition, Faber and Hughes showed that higher doses of Gen were associated with decreased pituitary responsiveness [59], which may explain the lower number of CLs at 2 mo of age and the lack of CLs at 4 mo of age observed herein. Altered pituitary responsiveness later in life could also account for the early reproductive senescence observed in mice treated with lower doses used in our study. Further investigation of the effects of Gen on the hypothalamic-gonadal axis is currently underway in our laboratory.

The current study also shows that Gen adversely affects pregnancy outcome, as mice exposed to the highest dose of Gen did not deliver live pups, and mice exposed to lower doses of Gen showed signs of reduced fertility with age. While some of the mice treated with Gen-50 were able to become pregnant at 2 mo of age, they were unable to carry these litters to term. The Gen-50-treated mice that became pregnant had fewer implantation sites, which may be explained, in part, by the lower ovulation rate exhibited by these mice under their own hormonal cues. In addition to having fewer implantation sites, the implantation sites were much smaller than the implantation sites from controls at the same gestational age. There are several possibilities why this may have occurred. One explanation is that the lower number of CLs in the Gen-50-treated mice could have led to inability to support pregnancy due to lower levels of circulating progesterone, because progesterone is essential for maintenance of pregnancy. However, we have shown that serum circulating levels of progesterone, as well as estradiol and testosterone, were similar in Gen-treated and control pregnant mice. Another possibility is that the uterus was unable to support pregnancy. The observation that the embryos implanted suggests that there was some capacity of the uterus to function properly. Another possibility is that the oocyte itself is of poor quality. We have shown previously that the development of the ovary and ovarian follicle were altered following neonatal Gen treatment [37]. Ovaries of Gen-treated mice contained MOFs at 19 days of age, a phenotype not often observed in control CD-1 mice. This phenotype may be a marker for altered development of the ovary, leading to oocytes of poor quality. In fact, a paper by Iguchi et al. using neonatal DES treatment showed that oocytes derived from single oocyte follicles were far more likely to be fertilized in vitro than oocytes derived from MOFs, suggesting that these oocytes are less competent [60]. Because neonatal Gen treatment causes an increase in MOFs [37], perhaps fewer ovulated oocytes are capable of being fertilized. We are currently investigating specific uterine and ovarian defects using embryo transplantation experiments.

In summary, our data demonstrate that neonatal exposure to Gen has deleterious effects on the developing murine reproductive system and can have long-term consequences on fertility at environmentally relevant doses. While there are certainly adverse effects on the reproductive system, this appears to be a multifaceted problem, because these mice have altered estrous cycles, altered ovarian function, and lower pregnancy rates. While the most severe effects were observed at a dose of 50 mg/kg with lack of ovarian function and inability to carry pups to term, there were also adverse consequences to reproduction observed at the Gen 0.5 and 5 mg/kg treatment groups with altered ovarian function, extended estrous cycles, and early reproductive senescence. Additional studies are warranted in human infants who are exposed to high levels of Gen during development before concluding that such exposure is safe.

Categories: 2005, Soy, Phytoestrogens, Endocrine, Sexual development, Nutrition and diet