Female reproductive disorders: the roles of endocrine-disrupting compounds and developmental timing

Experimental data from animal models of human reproduction have shown that numerous ubiquitous environmental chemicals (including banned or highly regulated chemicals such as DES, DDT, and PCBs, as well as contemporary-use chemicals such as BPA, atrazine, and phthalates) have detrimental effects on female reproduction. Given that the reproductive physiology of humans and other mammals is remarkably similar, it is reasonable to predict that human female reproductive disruption can occur after exposure to these compounds. A number of human epidemiology studies support these predictions. However, there remains uncertainty as to the nature and scope of the influence of EDCs on human reproductive health. We suggest several specific research directions that can elucidate the relative impact of EDC exposure on female reproductive disorders ( Table 6 ) and emphasize three larger-scale needs: [1] studies of early life exposures with sufficient follow-up tounderstand links to adult onset of disease, [2] national and international coordination of samples and data, and [3] establishment of an interdisciplinary consortium to improve research, policies, and education. First, the major factor limiting the certainty of EDC disruption on human female reproduction is the paucity of data linking human fetal exposures to adult-onset reproductive disorders, and this should be a focus of future studies. The data from DES exposure are compelling; however, EDCs in lower concentrations than DES exposure or with comparably lower, similar, or higher affinity for ER- a and ER- b should be investigated. Human exposure data concerning the reproductive system are essential for risk assessment, and although serum and follicular fluid have been analyzed for many environmental contaminants, few studies have examined associations between measured physiologic EDC concentrations or history of environmental exposure and particular reproductive disorders. Studies of cohorts with strong exposure assessment data during prenatal, neonatal, and pubertal development are needed. However, these types of epidemiologic studies take much time and resources, and we need also to pursue both model animal studies and development of new techniques to clarify the links between early perturbations and future female reproductive disorders. Second, although we are certain that fetal and neonatal exposures to particular EDCs contribute to specific reproductive disorders in adults, the links among the various female reproductive disruptions are less clear. If, as we hypothesize, adult female reproductive disruptions result from similar windows of exposure for EDCs (see Fig. 3 ), then multiple outcomes and endpoints could share etiologies (although time of onset likely will differ for different disorders). This would be analogous to the role of EDCs during embryogenesis in males, which is hypothesized to result in a number of different adverse outcomes (hypospadias, cryptorchidism, reduced spermatogenesis, and testicular cancer) collectively referred to as the “testicular dysgenesis syndrome” ( 267 ). This hypothesis can be tested most effectively through national and international coordination of research laboratories that can share samples through a tissue bank repository and share results through centralization of a database. Currently, most studies (and research laboratories) focus on a single disorder (e.g., endometriosis) as an endpoint of interest and a single biomarker for disorder detection. Although this focus improves diagnosis and understanding of a particular disorder, it fails to address the larger issue of total female reproductive system disruption. Establishment of a “laboratory without walls” would maximize knowledge gained about EDC-associated reproductive disorders by increasing the efficiency of both time and money used in such studies. Third, a multidisciplinary, coordinated approach is needed to reduce the detrimental impact of EDCs on reproductive health. Numerous private, governmental, and academic organizations currently work to clarify and reduce the detrimental effects of EDCs, but no coordinated consortium exists to organize and focus such efforts. We suggest the establishment of an interdisciplinary consortium that can coordinate research, policy, and education pertaining to the role of EDCs on reproductive health. Collectively, the data reviewed in this article illustrate the role of EDCs in numerous human female reproductive disorders and emphasize just how sensitive the embryo, fetus, neonate, and adolescent are to EDCs. To reduce the risk of reproductive disorders in the next generation of women, we as a society must commit to reducing EDC contamination of air, water, and land by the products we use. We also must focus on collaborative research and solution-based efforts as we have described and provide easily accessible information about EDCs to the public. It is of concern that the endocrine-disrupting potential of the majority of chemicals in production, including those that are found in common consumer products, has not been assessed systematically for their effects on reproduction. Although there is much information on EDCs available from the US Food and Drug Administration ( http://edkb.fda.gov ), US Environmental Protection Agency ( http://www.epa.gov/endo ), and private organizations (e.g., http://www.ourstolenfuture.org , http://www.silentspring.org , http://www.nrdc.org , http://www.healthandenvironment.org ), there is not yet an easily accessible format for centralized public information about how to reduce EDC exposure, why it is important, and when it is most critical. The combination of regulatory effortsto reduce exposures to EDCs and increased public education about EDCs is likely to improve the reproductive health of women and future generations.

Obesity, diet, and physical activity are external factors relevant to multiple female reproductive endpoints, and they may modify or exacerbate the effects of EDCs. New studies are finding links between obesity and incidence of hysterectomy ( 252 ) and stillbirths ( 253 , 254 ), and some of the specific reproductive disorders mentioned in this article show an increased incidence in obese and physically inactive individuals. For instance, higher body mass index is associated with earlier puberty in girls ( 255 ), and low physical activity in working women has a significant influence on incidence of both deep endometriotic nodules (OR 4.58, 95% CI 1.80-11.62) and peritoneal endometriosis (OR 5.61, 95% CI 1.90-16.60) ( 256 ). One explanation is that higher physical activity reduces circulating E 2 in females ( 257 ); therefore, the reduced physical activity associated with modern society could increase E exposure. Diet is also a contributor to the progression of endometriosis, primarily through the action of phytoestrogens and/or dietary suppression of enzymes (cyclooxygenase-2, matrix metalloproteinase-2) or growth factors (tumor necrosis factor- a , interleukin-6, interleukin-8) that promote endometriosis ( 258 ). These associations may be linked to increased “estrogenicity” in adults, as adipose tissue has high aromatase activity that produces more Es( 259 ), but the complexity of reproductive system organogenesis suggests that early exposure to EDCs could be associated simultaneously with obesity and adult reproductive problems. Instead of obesity being a causative factor in reproductive disorders, it may be the result of early developmental channeling, just as is the reproductive disorder. The observed correlation between EDC exposure, obesity, and female reproductive failure should be examined and clarified. We believe that clarity for disease onset amidst developmental complexity is possible if associations among diseases are carefully studied and considered. Recent studies in women with PCOS and their offspring illustrate this. When women with PCOS become pregnant, the offspring are more likely to be exposed to elevated prenatal androgen concentrations and also to have an increased likelihood for low birth weight ( 260 , 261 ). This low birth weight is often compensated for with postnatal catch-up growth, which is a risk factor for insulin insensitivity, obesity, and diabetes later in life ( 262 ), particularly in children who are not breastfed or who are weaned from the breast early ( 263 - 265 ). Furthermore, reduced fetal growth appears to be associated with precocious adrenarche, early puberty, poor fertility, and PCOS during later life stages ( 266 ). Clearly, female reproductive problems develop through a complex link between genetics, EDCs, and other factors such as obesity. Much more research is needed to assess the multigenerational effects of early exposure to hormonally active agents. The documented associations among PCOS, early androgen exposure, fetal growth restriction, catch-up growth, and consequent metabolic and reproductive disorders, for example, suggest that early hormonal disruption can manifest in multiple ways.

Puberty is characterized as a cascade of events leading to the attainment of adult reproductive capacity and involves maturation of the hypothalamus, anterior pituitary, ovary, uterus, and breast. The pubertal cascade is initiated by activation of the hypothalamic GnRH pulse generator, which results in anterior pituitary release of FSH and LH. Secretion of these gonadotropins stimulates ovarian E 2 production that initiates breast development (thelarche) and, subsequently, uterine-endometrial maturation. Simultaneous with the pituitary-gonadal activation, the adrenal glands increase the secretion of androgens (adrenarche), which results in pubic hair growth (pubarche) approximately 6 months after breast budding. Approximately 2 years after pubertal onset, first menstruation (menarche) occurs as a late pubertal event. Although there are many associated pubertal events, the three notable characteristics that have been used to indicate puberty onset in human females (which are different from pubertal markers in nonhuman animals) are breast development, pubarche, and menarche ( 209 ). There is considerable variation among human populations in the age at puberty, and this variation has not been sufficiently explained solely by genetic influences. For girls, the age of puberty onset has declined over the last half century in the United States ( 210 ) and several other developed nations( 211 ). Indeed, the majority of an expert panel asked to evaluate secular trends in puberty onset concluded that the weight of evidence supported trends in earlier breast development and earlier menarche ( 212 ). In a recent review of the timing of puberty in populations around the world ( 211 ), mean age at first measured breast development (Tanner stage B2) in 15 developed-nation populations ranged from 8.9 to 11.2 years. Age at menarche in these populations was slightly less variable, ranging from 12.0 to 13.5 years. On average, age at first breast development occurred several years before age at menarche (median 2.5, range 1.7 to 3.3 years). However, in developing countries these pubertal markers occurred considerably later, with menarcheal age ranging from 12.9 to 16.1 years. These data demonstrate the considerable variability in age of breast development compared with age at menarche and suggest a role for environmental factors in the timing of pubertal onset. The occurrence of early puberty can have a detrimental effect on a woman’s health. Precocious puberty has been associated with increased incidence of depression ( 213 , 214 ), sexual victimization ( 215 ), substance abuse ( 213 ), and adult breast cancer ( 216 ). As a result, recent studies have given deserved attention to the environmental factors shown to influence precocious puberty. These factors include obesity, increased psychosocial stressors, and increased exposure to environmental pollutants ( 211 ). The human data linking EDCs to early breast development and early age at menarche are limited; however, data from animal models have lent more certainty to this association. Peripubertal breast development in females progresses through five stages, termed Tanner stages following the criteria outlined in the early 1960s ( 209 ), and the earliest indication of puberty is breast development from Tanner stage B1 (prepubertal) to Tanner B2 (breast budding). There is considerable evidence that the age of Tanner B2 attainment has declined significantly, particularly in the United States. A significant reduction in age of onset for breast development has been shown in US girls ( 210 ), especially African American females ( 210 , 217 ). The well-established contributors to such precocious breast development include childhood obesity ( 218 , 219 ) and obesity-associated hyperinsulinemia and insulin resistance( 220 ). Studies also have suggested links to exposure to estrogenic chemicals. The roles of EDCs and obesity in promoting early breast development are complex, and an emerging hypothesis links EDC exposure to obesity in animal models ( 71 , 221 , 222 ). One way to examine environmental influences on premature breast development is to study populations that have increased incidence of precocious breast development. For the past 25 years, researchers have examined females in Puerto Rico because of an increased incidence of thelarche (breast development before 8 years of age) that is estimated to be 8 in 1,000 live births ( 223 ). This increased rate does not appear to be of genetic origin, because the incidence is independent of ethnic group ( 224 ), and multiple environmental factors have been used to explain the elevated occurrence of thelarche in this population. All such factors are associated with increased cumulative estrogenic exposure: [1] consumption of phytoestrogen-laden soy-based formula ( 224 ), [2] maternal history of ovarian cysts ( 224 ), [3] elevated serum zearalenol, a nonsteroidal E ( 225 ), and [4] elevated serum phthalate concentrations ( 226 ). The association with elevated phthalates is intriguing, as phthalates are EDCs present in plastics and personal care products, and human exposure to phthalates is high and common ( 227 , 228 ). Although di-(2-ethylhexyl) phthalate was not detected in serum from all the girls with thelarche, di-(2-ethylhexyl) phthalate was significantly elevated in cases (average 450 ppb) compared with controls (70 ppb) ( 226 ). These results of Colon et al.( 226 ) have been criticized on the basis that phthalates were measured in serum, and such measures are subject to contamination in the collection process. Moreover, phthalates are rapidly metabolized and excreted from the body, and, thus, any phthalate measured in the serum had no influence on the already existing condition ( 229 ). However, recently similar results were found in girls with precocious puberty in Shanghai ( 230 ). Thus, the hypothesis that phthalate exposureis associated (through either causation or correlation) with early breast development is still viable. Another population experiencing early breast development is children who immigrate from developing to developed countries. A study in Belgium found that 28% of patients with precocious puberty were children who immigrated from developing countries where organochlorine exposure is high, and these children had significantly elevated p,p’ -DDE concentrations ( 231 ). Similarly increased incidence of precocious puberty has been noted in adoptees in Denmark, although the contributions of genetic and environmental influences are unknown ( 232 ). Both in utero and neonatal time periods are sensitive windows of susceptibility for effects on early puberty in humans( 233 ) and rodents (see Table 5 ). In a study examining breast development in girls exposed both in utero and lactationally to differing amounts of the flame retardant polybrominated biphenyl, there was an increased OR (2.2, 95% CI 0.5-9.8) of early breast development in girls exposed to moderate, but not high or low, concentrations of polybrominated biphenyl ( 233 ). Similarly, a study in girls in Sonora, Mexico, indicates that it is possible that fetal, neonatal, or childhood exposure to contemporary pesticides can alter the pattern of breast development ( 208 ). The role of peripubertal EDC exposure on female breast development is less clear, but data from rodents show that mammary gland tissue is altered after acute, peripubertal exposure to DES, genistein, or DDT, with these EDCs acting as potent morphogens ( 234 ). Numerous laboratory animal studies indicate that embryonic and neonatal exposure to natural and synthetic Es can accelerate puberty. In rodents, vaginal opening is the earliest visible sign of puberty occurring in response to elevated Es, being analogous to Tanner B2 breast development in humans. Accelerated, early vaginal opening is seen in rodents exposed in utero or after birth to E 2 , the synthetic E DES, phytoestrogens, and many plasticizers (see Table 5 ). Vaginal opening is not the only indicator of early puberty in rodents; when theyare exposed to environmentally relevant doses of BPA, vaginal opening itself is not altered but the number of days between vaginal opening and first estrus is reduced, indicating an acceleration of postpubertal ovulation ( 235 ). Consistent with the findings that prepubertal exposure to Es can induce early breast development, exposure to compounds that block Es can delay breast development. 2,3,7,8-Tetrachlorodibenzo- p -dioxin has an antiestrogenic effect on breast development, and exposure of girls to PCBs with dioxin-like activity results in retarded breast development ( 236 ). Results from laboratory animals support these studies, because mice exposed in utero to TCDD have delayed puberty ( 237 ) and mammary gland development( 238 ). Age at menarche is much easier to record in large-scale epidemiologic studies when compared with other pubertal indices, but menarcheal age is less likely to be sensitive to EDCs than age at onset of breast development. Menarche occurs only after several years of endogenous E 2 stimulation of the endometrial tissues, whereas breast budding is the result of E stimulation of prepubertal glandular tissues na€ive to Es. Thus, while menarche is clinically easier to measure, early breast development is a more sensitive and earlier pubertal index. Age at menarche is reduced in girls exposed to estrogenic organochlorines, but the exact contribution of organochlorines to precocious menarche is unknown because of the numerous environmental variables influencing menarche (see Fig. 2 ). A study of women exposed to DDE (one breakdown product of DDT) through Great Lakes fish consumption found a 1-year reduction in age at menarche for each increase of 15 m g/L serum DDE ( 239 ), and a study of Chinese textile workers determined that a 10 m g/L serum DDT increase was associated with 0.2-year reduction in menarcheal age ( 74 ). However, predictions of exact age reductions in puberty by specific EDCs are confounded by the fact that other environmental variables (including other EDCs) also contribute to pubertal onset ( Fig. 2 ). Determining the role of particular xenobiotics and phytoestrogens in promoting early menarche (and other reproductive disorders) is complicated by the fact that humans are exposed to numerous xenobiotics, each of which could have a different effect. For instance, what is the result of simultaneous exposure to environmentally relevant concentrations of lead (which delays menarche) and DDE (which accelerates menarche)? Such a question was asked for girls residing in the Akwesasne Mohawk Nation ( 240 ). Girls with elevated circulating estrogenic PCBs were more likely to have reached menarche compared with those with lower PCB concentrations. Conversely, girls with higher blood lead concentrations attained menarche at a later age. Thus, the cumulative effect of all EDCs is what appears important, with compounds likely having additive or subtractive influences. It has been suggested that a reduction in environmental lead levels, one of the environmental success stories of the past 30 years, may contribute to recently documented decreases in menarcheal age in the United States. Peripubertal lead exposure is associated with delayed sexual maturation in rats ( 241 , 242 ) and humans ( 240 , 243 , 244 ). Thus, it is possible that reduced circulating lead concentrations as a result of US regulation ( 245 ) could be a contributing factor to the occurrence of earlier menarche seen in US girls. The lack of knowledge, until recently, about human embryonic EDC exposures has hampered our understanding of the role of contemporary-use EDCs in reducing age at menarche. As illustrated in this review, most EDCs examined for their role in altering age of puberty are the well-characterized organochlorines, phytoestrogens, and pharmaceutical Es. These studies clearly illustrate the potential for fetal and neonatal exposures to influence breast development and menarche, but, in some cases (such as DES exposure), menarche itself may not be altered but menstrual regularization may be delayed ( 77 ). Thus a data gap exists for understanding the effects of contemporary-use EDCs in early menarche. The mechanisms for EDC-accelerated puberty are characterized as either central (with the EDC causing hypothalamic and/or pituitary maturation) or peripheral (with the EDC acting directly on the ovary or breast independent of the hypothalamic-pituitary axis) ( 246 ). Data supporting the central hypothalamic maturation hypothesis come from studies of genetically similar populations exposed to different environmental conditions. For children emigrating from countries having higher EDC exposure, it is proposed that estrogenic EDC exposure accelerates maturation of the hypothalamus at the same time as inhibiting gonadotropin production, and the absence of EDC exposure initiates early puberty ( 211 ). The role of EDCs in initiating hypothalamic maturation is seen in rats, where early postnatal exposure to either E 2 or o,p’ -DDT caused early GnRH secretion and subsequent puberty ( 247 ). Early breast development could be due either to proliferation of ducts and periductal stroma (gynecomastia) or excessive adipose-tissue deposition (lipomastia). Most studies ignore such distinctions, but the few that have considered the tissue-level cause of early breast development have indicated lipomastia. When breast development was compared for two populations—one exposed to contemporary pesticides and the other not—the pesticide-exposed girls exhibited large breast fields with breast size not being related to glandular development, as it was in non-pesticide-exposed girls ( 208 ). Such lipomastia may precede gynecomastia, as mammary gland epithelial cell differentiation is dependent on induction by adipose tissue ( 248 ). Studies in the 1990s led to the hypothesis that occurrence of early breast development in obese girls could be influenced by elevated Es associated with obesity. When pregnant rats are fed high-fat diets, maternal E 2 concentrations are significantly higher and the offspring of these females have puberty onset at a younger age ( 249 ). However, plasma E 2 concentrations are not consistently elevated in obese girls, raising doubts about this hypothesis. Recent studies do show that perinatal E exposure is associated with altered adiposity and weight homeostasis later in life ( 250 ). Thus, the mechanisms through which nutrition and EDCs promote early breast development are likely conserved, both involving elevated exposure to Es. Numerous studies confirm that prenatal and neonatal exposure to xenoestrogens affect breast development, but the amount of exposure to xenoestrogens required to elicit such effects is unknown. Morphometric measurements indicate that embryonic exposure to E 2 results in a nonmonotonic dose-response in the mouse, such that intermediate doses promoted terminal end bud formation and ductal elongation, but low and high doses had an inhibitory effect ( 251 ). Such nonmonotonic dose-response curves are likely due to the interaction of two or more monotonic dose-response curves and make hazard modeling and subsequent risk assessment difficult ( 251 ).

Combined with the limited data on reproductive health trends, there is a very poor understanding of the causes of these disorders and the environmental factors that may influence them. One reason for this uncertainty concerning the etiology of reproductive disorders is the historical genocentric views of both developmental biology and disease predisposition. Only recently have we acknowledged the major role of the environment in dictating phenotype via at least three pathways: [1] a direct induction of gene expression, whereby environmental agents act directly as hormones or disrupt the metabolism or synthesis of endogenous hormones, [2] a neuroendocrine route, whereby the nervous system monitors the environment and sends signals to the endocrine system, and [3] an epigenetic route, whereby environmental agents alter transcriptional capabilities without changing DNA sequence ( 19 ). Human disease researchers now consider these environmentally mediated mechanisms, and recent focus on epigenetic mechanisms has provided insight into disease onset( 18 , 20 , 21 ). In epigenetic disruption, the environmental agent modifies chromatin packaging by either modifying histones (thus altering the DNA-nuclear protein interactions) or by promoting DNA methylation (resulting most often in repressed transcription by inhibiting interaction with specific transcription factors). It has been suggested recently thatthese chromatin modifications acquired from environmental signals during the individual’s development can be passed on to future generations ( 22 ), reminiscent of the idea of inheritance of acquired characteristics promoted by the 16th- and 17th-century biologist Jean-Baptiste Lamarck. Epigenetic modifications help explain how developmental exposure to a toxicant can increase the likelihood of a disease state later in life ( 23 - 26 ) and possibly even promote disease across several generations ( 22 ). Epigenetic-induced changes permit developmental plasticity that is evolutionarily adaptive because it allows the developing fetus to alter the course of organogenesis in anticipation of later life needs. For instance in our hominid ancestors, increased maternal dietary cholesterol would suggest ample nutrients in the environment, and the developing fetus would respond to this information by altering function of pancreatic b cells, hepatocytes, and adipocytes in anticipation. The increased maternal dietary cholesterol often parallels elevated maternal E concentrations and “imprints” greater E sensitivity in female offspring, a condition that would increase reproductive output. If later life demands match those prescribed by the fetus, health is expected. On the contrary, a mismatch would lead to disorders. Today, novel anthropogenic compounds are introduced to the fetal environment, and, although many such compounds do not cause genetic mutations, many likely contribute to adult disease through “misinforming” fetal developmental plasticity. This emerging theory of early origins of adult disease due to the “mismatch” between fetal exposures and adult has received increased attention ( 27 , 28 ). The literature from EDC research supports this mismatch theory of disease. Besides the relatively new understanding of genetic-environmental interactions on adult phenotype and disease, another reason that the etiologies of many reproductive disorders have not been elucidated is due to the complexity associated with ontogeny of reproductive organs. Endocrine-disrupting compounds can have varying effects throughout development because of variations in tissue hormone receptor isoforms and concentrations at different developmental stages. For example, the grape phytoestrogen resveratrol acts as an E agonist in many cell types expressing E receptor (ER)- a or ER- b but also acts as an E antagonist for ER- a with the E response elements (EREs) EREc38 and PR1148 ( 29 ). Thus, in vitro data suggest that resveratrol will have an antiestrogenic effect when organs express ER- a isoforms with EREc38 or PR1148 but an estrogenic effect when other EREs are expressed. This is confirmed in in vivo studies of rats, where resveratrol acts as an E agonist on gonads but an E antagonist in the brain ( 30 , 31 ). Indeed, the effects of many EDCs are dictated by the complement of ER isoforms, coactivators, and corepressors in cells and tissues, and these components vary with developmental stage. A combined understanding of both developmental plasticity and ontogenetic complexity helps clarify the role of EDCs in the onset of reproductive disturbances. To illustrate, we begin with disruptions of ovarian function.