Environmental Factors and Puberty Timing: Expert Panel Research Needs

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1 SUPPLEMENT ARTICLE Environmental Factors and Puberty Timing: Expert Panel Research Needs Germaine M. Buck Louis, PhD a, L. Earl Gray, Jr, PhD b, Michele Marcus, PhD, MPH c, Sergio R. Ojeda, DVM d, Ora H. Pescovitz, MD e, Selma Feldman Witchel, MD f, Wolfgang Sippell, MD, PhD g, David H. Abbott, PhD h, Ana Soto, MD i, Rochelle W. Tyl, PhD j, Jean-Pierre Bourguignon, MD, PhD k, Niels E. Skakkebaek, MD, DMSc l, Shanna H. Swan, PhD m, Mari S. Golub, PhD n, Martin Wabitsch, MD, PhD o, Jorma Toppari, MD, PhD p, Susan Y. Euling, PhD q a Epidemiology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; b Endocrinology Branch, National Health and Environmental Effects Research Laboratory, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, North Carolina; c Department of Epidemiology, Rollins School of Public Health, Emory University, Atlanta, Georgia; d Division of Neuroscience, Oregon National Primate Research Center-Oregon Health and Sciences University, Beaverton, Oregon; e Department of Pediatrics and Cellular and Integrative Physiology, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, Indiana; f Division of Endocrinology, Children s Hospital of Pittsburgh, Pittsburgh, Pennsylvania; g Division of Endocrinology, Department of Pediatrics, University of Kiel, Kiel, Germany; h Department of Obstetrics/Gynecology and Wisconsin National Primate Research Center, University of Wisconsin, Madison, Wisconsin; i Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston, Massachusetts; j Center for Life Sciences and Toxicology, RTI International, Research Triangle Park, North Carolina; k CHU Sart-Tilman, University of Liege, Belgium; l University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark; m Department of Family & Community Medicine, University of Missouri, Columbia, Missouri; n Office of Environmental Health Hazard Assessment, California Environmental Protection Agency, Sacramento, California; o Department of Pediatric and Adolescent Medicine, University of Ulm, Ulm, Germany; p Departments of Physiology and Pediatrics, University of Turku, Turku, Finland; q National Center for Environmental Assessment, Office of Research and Development, US Environmental Protection Agency, Washington, DC The authors have indicated they have no financial relationships relevant to this article to disclose. ABSTRACT Serono Symposia International convened an expert panel to review the impact of environmental influences on the regulation of pubertal onset and progression while identifying critical data gaps and future research priorities. An expert panel reviewed the literature on endocrine-disrupting chemicals, body size, and puberty. The panel concluded that available experimental animal and human data support a possible role of endocrine-disrupting chemicals and body size in relation to alterations in pubertal onset and progression in boys and girls. Critical data gaps prioritized for future research initiatives include (1) etiologic research that focus on environmentally relevant levels of endocrine-disrupting chemicals and body size in relation to normal puberty as well as its variants, (2) exposure assessment of relevant endocrine-disrupting chemicals during critical windows of human development, and (3) basic research to identify the primary signal(s) for the onset of gonadotropinreleasing hormone dependent/central puberty and gonadotropin-releasing hormone independent/peripheral puberty. Prospective studies of couples who are planning pregnancies or pregnant women are needed to capture the continuum of exposures at critical windows while assessing a spectrum of pubertal markers as outcomes. Coupled with comparative species studies, such research may provide insight regarding the causal ordering of events that underlie pubertal onset and progression and their role in the pathway of adult-onset disease. ences. An expert review of available animal, clinical, and epidemiologic data was undertaken and synthesized for identifying critical data gaps that are relevant for prioritizing a multidisciplinary research agenda. For purposes of this article, the environment is defined as the sum of all external conditions that affect life, development, and survival of an organism ( By extension, an environmental factor is defined as a nonwww.pediatrics.org/cgi/doi/ / peds e doi: /peds.1813e The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the US Environmental Protection Agency. Mention of trade names of commercial products does not constitute endorsement or recommendation for use. Drs Buck Louis, Gray, and Marcus contributed equally to this work. Key Words human puberty, puberty timing, breast development, menarche, pubic hair development, genital development, endocrine disruptors, body fat Accepted for publication Sep 5, 2007 Address correspondence to Germaine M. Buck Louis, PhD, Epidemiology Branch, Division of Epidemiology, Statistics and Prevention Research, National Institute of Child Health and Human Development, 6100 Executive Blvd, Room 7B03, Rockville, MD louisg@mail.nih.gov PEDIATRICS (ISSN Numbers: Print, ; Online, ). Copyright 2008 by the American Academy of Pediatrics IN LIGHT OF an increasing body of evidence supporting environmental influences on pubertal onset and progression in contemporary birth cohorts, an expert panel met during and after the Serono Symposia International s The Role of Environmental Factors on the Onset and Progression of Puberty to identify research priorities for delineating the impact of environmental influences on children s pubertal experi- S192 BUCK LOUIS et al

2 FIGURE 1 Regulation of human puberty onset and progression via the HPG and the HPA axes. The key regulatory changes that occur for the initiation and progression of puberty in boys and girls are shown. The HPG activation requires signaling from the central nervous system (CNS) to the hypothalamus. The hypothalamus releases GnRH, which in turn stimulates the production of LH and FSH from the pituitary. LH and FSH activate the gonad to produce sex hormones, which in turn initiates pubertal changes in many of the target organs. The HPA activation requires signaling from the CNS to the hypothalamus. The hypothalamus releases adrenocorticotropin releasing hormone (CRH) that in turn stimulates the production of adrenocortical tropic hormone (ACTH) from the pituitary. ACTH activates the adrenal cortex to produce androstenedione and dehydroepiandrosterone (DHEA) that in turn initiates armpit hair, pubic hair, and skin changes. Androgen action is also considered to be part of the initiation of armpit and pubic hair development. Positive ( ) and negative ( ) indicate stimulatory and inhibitory signals and / in the CNS indicates a combination of excitatory and inhibitory neuronal signals. genetic factor; however, for purposes of brevity and consistent with the weight of evidence regarding environmental influences on puberty, this review focuses on endocrine-disrupting chemicals (EDCs) and body size in relation to puberty timing. An in-depth discussion of the literature cited in this article is beyond the scope of this article, but key study aspects are summarized in accompanying tables. REGULATION OF NORMAL PUBERTY ONSET AND PROGRESSION IN HUMANS AND ANIMALS Puberty is the period of transition from childhood to adolescence and is marked by the development of secondary sexual characteristics, accelerated growth, behavioral changes, and eventual attainment of reproductive capacity. Available approaches for the assessment of pubertal onset and progression have recently been reviewed, 1 including the use of Tanner stages 2,3 for female breast and pubic hair development and male gonadal and pubic hair development. Menarche is also an important marker used for assessing puberty in girls. Biochemical markers for female puberty onset and progression include assaying elevations in reproductive hormones (eg, luteinizing hormone [LH], follicle-stimulating hormone [FSH], 17 -estradiol, inhibin A and B) before the onset of physical signs (eg, Tanner breast development stage 2). Other maturational markers, such as bone age determinations (radiograph of left wrist) and ultrasonographic assessment of ovarian and uterine volume, can be used. Puberty changes occur as a consequence of the activation of the hypothalamic-pituitary-gonadal (HPG) axis and hypothalamic-pituitary-adrenal (HPA) axis. The HPG axis is under the control of both inhibitory and stimulatory mechanisms, as illustrated in Fig 1. In the human, the HPG is active in the midfetal, neonatal, and early infancy periods but becomes relatively quiescent during childhood. Puberty is marked by the reactivation of the HPG axis manifested by an increase in frequency and amplitude of gonadotropin releasing hormone (GnRH) pulses in the hypothalamus, leading to a rise in pulsatile secretion of the gonadotropins LH and FSH from the anterior pituitary. Gonadotropin release stimulates the gonads. In girls, such maturation of reproductive neuroendocrine function eventually leads to the onset of ovulatory menstrual cycles. Appropriate gonadotropin regulation of ovarian function results in ovarian follicle secretion of ovarian androgens from theca cells and estradiol from granulosa cells before ovulation and secretion of progesterone from the corpus luteum and estradiol after ovulation. In boys, LH initiates the secretion of androgens (testosterone and androstenedione) from the testicular Leydig cells. Such onset of gonadal endocrine function is termed gonadarche. The initial molecular triggers of puberty onset remain unknown, although genetic and environmental factors are suspected. Puberty is also associated with an independent physiologic event, adrenarche, or adrenal activation, that typically occurs between 6 and 8 years of age in both genders. Adrenarche leads to pubarche, the secondary sexual changes including pubic hair development, acne, and body odor. Adrenarche is marked by increased 17 hydroxylase (17,20 lyase) activity of the P450c17 enzyme and increased cytochrome b activity resulting in increased dehydroepiandrosterone, dehydroepiandrosterone sulfate, and androstenedione production. These initial hormonal increases rise over time, resulting in a cumulative dosage of androgens. Adrenarche is a strictly primate phenomenon. 4 Although little information is available, there is some evidence that adrenarche occurs before puberty in chimpanzees and during the neonatal period in rhesus macaques and baboons. 5 PEDIATRICS Volume 121, Supplement 3, February 2008 S193

3 Recently, much has been learned about the central mechanisms underlying the initiation of mammalian puberty. Rodents, nonhuman primates, and humans have been and remain the main species used to study the basic components and regulatory hierarchies that control the process of puberty. Traditionally assessed pubertal onset end points in rodents include the age of male preputial separation (PPS), an androgen-mediated event, and, in females, the age of female vaginal opening (VO), an estrogen-mediated event. Age at first estrus is an end point used to assess completion of the pubertal process in females because it occurs the day after the first preovulatory surge of gonadotropins. 6 Some studies have assessed the age of breast cell differentiation events in the mouse and rat. 7,8 Estrogen-mediated pubertal events that occur in nonhuman female primates include menarche, sex skin swelling and reddening, and nipple growth. 9,10 Epiphyseal closure and cessation of long bone growth also have been assessed. 11 In male monkeys, increases in testicular volume indicate onset of male puberty and reflect both gonadotropin- and testosterone-mediated events. 12 Measurement of changes in testicular size seems to be a reliable, noninvasive procedure to detect the initiation of puberty in both human and nonhuman primates. 13 In addition to differences in the time frame, progression, and phenotypic landmarks of puberty, 14 there is a fundamental difference in the neuroendocrine process that controls the initiation of puberty in rodents versus primates. In rodents and sheep, 15 GnRH secretion is maintained at low levels during juvenile development by strong steroid inhibitory control, whereas a similar reduction in GnRH secretion is achieved in primates (including humans) by central mechanisms that operate independent of gonadal regulatory inputs. 16,17 Thus, data indicate that the peripubertal regulation of the GnRHreleasing system is a more centrally controlled process in primates than in rodents that exhibit gonadal control of GnRH secretion. Despite this operational difference, the pubertal activation of GnRH release in rodents and primates is initiated and regulated by similar excitatory and inhibitory pathways, providing input to GnRH neurons (reviewed by Ojeda and Terasawa 18 and Ojeda and Skinner 6 ). These inputs include both trans-synaptic and glia-to-neuron communication pathways. As neuronal networks that use excitatory amino acids and the newly discovered kisspeptin peptide for neurotransmission or neuromodulation become activated, there is a concomitant reduction in the activity of those neurons using the inhibitory neurotransmitters amino butyric acid and opioid peptides for transsynaptic communication. In addition to this neuron-to-neuron flow of information, several trophic molecules have been identified as components of the cell cell signaling process used by glial cells to regulate GnRH secretion. Prominent among these regulatory systems is a signaling complex that uses epidermal growth factor like ligands and members of the epidermal growth factor receptor family for glia-toneuron communication (reviewed by Ojeda et al 19 ). Although some gene products that are involved in glia and neuronal communication to regulate puberty onset have been identified, gene(s) upstream that provide the initial trigger of puberty timing remain largely unknown. The integrated use of genomics and proteomics using transgenic rodent models, nonhuman primates, and humans with alterations in the onset of puberty is beginning to uncover some of the common evolutionary conserved molecular components that are used by the neuroendocrine brain to control GnRH secretion and, in turn, the initiation of puberty in both rodents and higher primates An example is the homeodomain gene TTF1, a transcription factor that was recently shown to be involved in the neuroendocrine control of both primate and rodent puberty. 24 In addition, recent findings suggest that structural remodeling of GnRH neurons may play a key role in the onset of puberty. 25 Although the initiating trigger(s) for pubertal onset is unknown for rodents, nonhuman primates and humans, earlier molecular markers have been identified, such as the newly described kisspeptin-gpr54 regulatory system. Humans who carry mutations of the GPR54 receptor 22,23 and mice that lack this receptor 23,26 each fail to reach puberty, indicative that activation of this signaling complex is essential for pubertal onset. Peptides that are produced by peripheral tissues, such as leptin and insulin-like growth factor 1, seem to be more important in maintaining pubertal progression rather than in its initiation despite that insulin-like growth factor 1 has been reported to accelerate pubertal initiation in rodents and monkeys (reviewed by Ojeda and Terasawa 18 ). Understanding the mechanisms underlying the initiation, progression, and regulation of normal human puberty is essential for assessing the effect(s) of EDCs on human development. With limited empirical population-based data, it is important to consider other sources of data, such as those that arise from clinical populations. Although external validity may be limited from these data sources, useful information about potential cause is possible. CLUES FROM CLINICAL RESEARCH THAT SUPPORT ENVIRONMENTAL INFLUENCES A number of clinical conditions offer clues regarding the influence on environmental factors, such as nutrition and body size, on puberty. In fact, a recent clinical report attributed topical application of lavender and tea tree oils to gynecomastia in prepubescent boys on the basis of in vitro evidence supporting their weak estrogenic and anti-androgenic activities. 27 A brief overview of environmental influences follows in relation to precocious puberty, delayed puberty, and polycystic ovary syndrome (PCOS). Precocious Puberty Precocious and delayed puberty in humans can have a genetic and/or environmental cause. Precocious puberty is defined herein as the development of secondary sexual characteristics before 8 years of age in girls and boys. Precocious puberty can be classified into central, or gonadotropin-dependent, precocious puberty; peripheral, or gonadotropin-independent, precocious puberty; or S194 BUCK LOUIS et al

4 combined. Examples of genetic forms of peripheral precocious puberty are McCune Albright syndrome in girls and testotoxicosis (or familial male precocious puberty) in boys. Premature thelarche and premature adrenarche may be regarded as partial forms of precocious puberty, in which the full spectrum of pubertal changes is absent and only specific changes occur. 28 Premature thelarche is usually slowly progressing or even self-limiting but can occasionally transform into overt central precocious puberty. Although not precocious puberty, per se, premature breast development and gynecomastia after exposure to estrogen-containing products have been reported in girls and boys, respectively, with reversal on cessation of the exposure Pubertal Delay Delayed puberty, defined as the absence of breast development by 13 years of age in girls or testicular volume 4 ml by 14 years of age in boys, can occur as a result of primary hypothalamic-pituitary dysfunction or primary gonadal failure. The most common cause of pubertal delay is constitutional, representing a transient delay in sexual development that is a variant of normal. Permanent hypogonadotropic hypogonadism may occur as an isolated hypothalamic-pituitary deficiency (eg, Kallman s with or without cleft lip and/or palate) and may be associated with a variety of molecular genetic defects. All are congenital anomalies that are associated with multiple pituitary hormone deficiencies, not just those that affect puberty onset. Not all cases of delayed puberty have a known genetic cause, such as those that arise from head trauma or infection. Primary gonadal failure is associated with hypergonadotropism and is caused by numerous congenital or acquired disorders with a genetic (eg, gonadal dysgenesis, Klinefelter syndrome, molecular genetic defects of the steroidogenic pathway, galactosemia) or environmental cause (eg, gonadotoxic chemotherapy [alkylating chemotherapy impairs spermatogenesis but rarely affects Leydig cell function], radiotherapy, surgery). Autoimmune disorders can cause delayed puberty through 2 different mechanisms: hypogonadotropic hypogonadism as a result of hypophysitis or primary gonadal failure as a result of autoimmune gonadal destruction. Polycystic Ovary Syndrome PCOS is a common heterogeneous disorder that affects 5% to 10% of reproductive-aged women. Insulin resistance/hyperinsulinemia occurs in 50% to 60% of PCOSaffected women compared with a prevalence of 10% to 25% in the general population. Criteria for diagnosing PCOS vary and were defined by the 1990 National Institutes of Health National Institute of Child Health and Human Development conference as including chronic anovulation and hyperandrogenism (including acne, hirsutism, and male pattern baldness) after exclusion of other disorders, such as congenital adrenal hyperplasia (CAH), hypercortisolism, and hyperprolactinemia. 34 The more recent Rotterdam criteria for PCOS 35 expanded the National Institutes of Health definition to include 2 of 3 of the following: (1) clinical or biochemical evidence of hyperandrogenism; (2) intermittent or absent menstrual cycles; and (3) polycystic ovary morphology as visualized by ultrasound. Puberty timing is altered in some girls with PCOS, dependent, in part, on androgen secretion/exposure and nutritional status. Premature pubarche has been reported among some but not all girls with PCOS and is believed to arise from increased adrenal androgen secretion secondary to premature adrenal pubertal maturation in the absence of other known causes (eg, CAH, androgen-secreting tumors, Cushing syndrome). Androgen concentrations are elevated for chronologic age but appropriate for the stage of pubic hair development. Skeletal maturation may be normal or slightly advanced. The presence of insulin resistance/hyperinsulinemia, dyslipidemia, exaggerated GnRH-stimulated ovarian thecal cell androgen secretion in the early stages of puberty, and subsequent development of hyperandrogenic chronic anovulation during the later stages of puberty suggest that premature pubarche is related to the development of PCOS. 36 In addition, the age at menarche is typically 6 months earlier in girls with PCOS than in unaffected girls. 37 The relation between PCOS and adult-onset diseases is now recognized. For example, girls and women with PCOS are at increased risk for developing impaired glucose tolerance (IGT) and type 2 diabetes in comparison with unaffected women; however, the clinical manifestations of hyperandrogenemia (anovulation, hirsutism, acne, and androgenic alopecia) differentiate PCOS from type 2 diabetes and obesity. Obese adolescent girls with PCOS have greater insulin resistance than obese girls without PCOS. 38 In PCOS, IGT, and type 2 diabetes, impaired metabolic activity leads to suppression of insulin-mediated glucose uptake and lipolysis, leading to compensatory hyperinsulinemia. Potential mechanisms underlying PCOS include (1) early origins or in utero programming stemming from exposures during critical windows of development, 39 (2) LH hypersecretion, (3) primary ovarian abnormality, (4) dysregulation of fat metabolism and adipogenesis, and/or (5) dysregulation of the inflammatory axis. Data arising from girls who present clinically with CAH 40 and nonhuman female primates that are exposed in utero to androgens 39,41,42 are suggestive of prenatal programming of gonadotropin secretory dynamics and PCOS. Women with CAH and PCOS have accelerated episodic release of LH and increased hypothalamic release of GnRH. 40 The persistence of the hyperandrogenic phenotype in cultured theca cells derived from women with PCOS suggests the possibility of a primary ovarian abnormality in PCOS 43 possibly with an in utero origin. 44 Investigation into the mechanism(s) responsible for insulin resistance in type 2 diabetes has suggested that abnormalities in fatty acid uptake and use lead to increased plasma free fatty acid concentrations followed by ectopic fat storage in skeletal muscle and liver; this aberrant fat storage (lipotoxicity) alters insulin signaling, insulin metabo- PEDIATRICS Volume 121, Supplement 3, February 2008 S195

5 lism, and pancreatic -cell insulin secretion, leading to the development of insulin resistance with compensatory hyperinsulinemia. Oxidative stress may also contribute to the development of insulin resistance. Activation of the NF- B/I B- /IKK- pathway, a major pathway for inflammatory processes, by free fatty acid provides a potential link among coronary artery disease, type 2 diabetes, insulin resistance, and PCOS. 45 Familial clustering of IGT, type 2 diabetes, and PCOS suggests a role for genetic determinants of this metabolic phenotype or in utero events such as decrements in fetal growth as measured by reduced birth size. 46 The heterogeneity of PCOS may account for the lack of candidate genes identified to date. 47 ENVIRONMENTAL INFLUENCES ON PUBERTY Consistent with our definition, environmental factors are defined to include both EDCs and body shape and adiposity. Consistent with the focus of the workshop, this review focuses on single chemicals by mode of action. A more comprehensive review describing the toxicology of puberty in rodents is found elsewhere. 48,49 Last, this review urges caution in assessing an individual pubertal marker given the highly interrelated and timedependent nature of pubertal onset and progression as orchestrated by gonadal hormones under the control of gonadotropins. In the presence of exogenous endocrineactive agents, pubertal markers can be independently induced or blocked so that they are not necessarily in synchrony with centrally regulated pubertal maturation. For example, peripubertal exposure to the gonadotropin agonist Lupron and the estrogenic agent diethylstilbestrol both delayed menarche in monkeys, but other markers of puberty were delayed or accelerated depending on the agent. 9,50 Therefore, the relative timing of all puberty-onset markers is the most informative, which argues for a complete pubertal assessment and related deviations in lieu of extreme categorization such as precocious puberty. Animal Studies A summary of selected examples of single environmental chemicals whose exposure at specific dosages may lead to puberty-timing alterations is presented in Table 1. This table is organized by mode of action (MOA). Estrogen receptor (ER) agonists (also called estrogen agonists or estrogens) and ER antagonists compete with the endogenous estrogen hormones estradiol and estrone for binding to the classical ER. Such agonists and antagonists bind to the ER and activate or repress, respectively, transcription of ER-dependent genes and thereby enhance or diminish estrogenic activity in vivo (eg, induce uterine weight increase/decrease, accelerate/ delay puberty). After in utero exposure to a potent estrogen agonist, gross reproductive malformations may result in female rats, presumably by altering the development of reproductive organs that are responsive to estrogen action. Examples of estrogen agonists that are present as environmental contaminants include 2,2- bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), a metabolite of the pesticide chemical methoxychlor, and some natural products such as isoflavonoids, flavonoids, stilbenes, lignans, equol, and phytoestrogens (eg, genistein, daidzein, coumestrol, 8-prenylnaringenin, resveratrol). Gestational and lactational maternal oral exposure to dosages as low as 25 mg/kg per day methoxychlor resulted in an earlier age of VO and first estrus in the female rat. 51 This was found to be pseudoprecocious rather than true precocious puberty because VO was accelerated but the onset of estrous cyclicity was not affected. Similarly, prenatal subcutaneous injections of BPA, 52,53 genistein, 53 resveratrol, 53 zearalenone, 53 or DES 53 accelerated VO in mice. Although prenatal oral exposure to BPA did not significantly accelerate the age at VO or first estrus in either the rat or mouse, prenatal oral treatment of mice with BPA significantly reduced the number of days between VO and first vaginal estrus. 54 When female rats were exposed after weaning throughout puberty to xenoestrogens like methoxychlor 51 or 17 beta estradiol, 56 early puberty onset was induced as measured by age at VO in the female rat. 55 In addition, prenatal BPA exposure resulted in altered maturation of the mammary gland at puberty and increased sensitivity to endogenous estrogens. 7 Androgen receptor (AR) antagonists (also called androgen antagonists) compete with natural hormones testosterone and dihydrotestosterone for binding to the AR. Thus, a decrease in binding between the natural ligand and receptor occurs, which in turn inhibits ARdependent gene expression in vivo. AR antagonist exposure in vivo can induce malformations in male reproductive tract and delay puberty in the male rat. AR antagonists include insecticides, herbicides, fungicides, toxic chemicals, and combustion byproducts. HPTE has also been shown to act as an AR antagonist. 57 Peripubertal exposure to the AR antagonists methoxychlor or vinclozolin led to a delayed puberty in males as measured by the age of PPS in the rat. 51,58 Conversely, AR agonists (also called androgen agonists) compete with the endogenous hormones testosterone and dihydrotestosterone for AR, leading to an increase in AR-DNA binding in vitro and AR-dependent gene expression in vivo. Such environmental androgen contaminants induce malformations in the female reproductive tract 59,60 ; accelerate male puberty in the rat 61 ; and have been associated with the masculinization of female fish in the United States, Sweden, and New Zealand. 62 Other receptors, including orphan receptors, are the targets of certain environmental agents. The environmental chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has 1 of the best-characterized MOAs, binding to the aryl hydrocarbon (Ah) receptor that in turn initiates Ah-dependent gene expression changes. Even low dosages of TCDD in utero can lead to gene expression changes and malformations in the reproductive tract in rodents Ah receptor ligands other than TCDD include other polychlorinated dibenzodioxins and some polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers, and pesticide contaminants as well as the pesticide methoprene, a juvenile hormone agonist. Exposure to TCDD in utero caused delayed puberty in male and female rats as measured by an older age at acquisition of PPS and VO, S196 BUCK LOUIS et al

6 TABLE 1 Examples of Published Studies of Chemical Exposures With Puberty-Timing Effects in Animals MOA Chemical Species, Treatment Interval Effects (at Dosages) Reference(s) Gender ER agonist E2 Rat, female Peripubertal Early VO (2 and 4 mg/kg per d) Marty et al 56 (1999) ER agonist DES Rhesus monkey, Peripubertal Early sex skin swelling and reddening; Golub et al 50 (2003) female delayed nipple growth; completely suppressed menarche; increased serum estrogen activity (0.5 mg/kg per d) ER agonist BPA Mice, female In utero Increased lateral branching at 3 mo (25 Munoz de Toro 124 (2005) and 250 ng/kg per d) ER agonist BPA Mice, female In utero Pregnancy stage like mammary gland Markey et al 7 (2001) differentiation (25 and 250 ng/kg) at PND 10, 1 mo, 6 mo ER agonist BPA Mice, female In utero Reduced interval between VO and first vaginal estrus (dose) Howdeshell et al 54 (1999) ER agonist and AR antagonist ER agonist and AR antagonist Methoxychlor Methoxychlor Rat, female and male Rhesus monkey, female Peripubertal Female: early VO and first estrus (25, 50, 100, 200 mg/kg per d); VO was accelerated, but first estrus was not; F1 exposed in utero and lactation but not directly; VO acceleration noted Male: delayed PPS (100 and 200 mg/kg per d) Peripubertal Early sex skin swelling and reddening; delayed nipple growth and menarche; increased serum estrogen activity (25 or 50 mg/kg per d) Gray et al 51 (1989) Golub et al 50 (2003) AR antagonist Vinclozolin Rat, male Peripubertal Delayed PPS (30 and 100 mg/kg per d) Monosson et al 58 (1999) AR antagonist p,p -DDE Rat, male Peripubertal (PND 22 55) Delayed PPS at 100 mg/kg per d Ashby and Lefevre 118 (2000) AR antagonist p,p -DDE Rat, male Peripubertal (PND 22 55) Delayed PPS at 30 and 100 mg/kg per d Kelce et al 116 (1995) Ah agonist TCDD Rat, female in utero Delayed VO and decreased/incomplete mammary epithelial differentiation ( g/kg per d) Gray and Ostby 66 (1995); Gray et al 64 (1997); Fenton et al 8 (2002) Aromatase inhibitor Fadrozole Rat, female Peripubertal Delayed VO (0.6, 1.2, and 6.0 mg/kg per d) Marty et al 56 (1999) Steroidogenesis inhibitor Ketoconazole Rat, female Peripubertal Delayed VO (100 mg/kg per d) Marty et al 56 (1999) Steroidogenesis inhibitor Prochloraz Rat, male Peripubertal Delayed PPS (125 mg/kg per d) Blystone et al 73 (2007) CNS-HPG; reduces LH Atrazine Rat, male Peripubertal Delayed PPS (12.5, 50, 100, 150, 200 mg/ Stoker et al 48,68 (2000) and prolactin kg per d) CNS-HPG; reduces LH and prolactin Atrazine Rat, female Peripubertal Delayed VO (50, 100 and 200 mg/kg per d) Laws et al 69 (2000) GnRH agonist Leuprolide acetate Rhesus monkey, female Prepubertal E2 indicates 17 -estradiol; DES, diethylstilbestrol; CNS, central nervous system; PND, postnatal day; IGF-1, insulin-like growth factor 1. Blocked menarche; delayed body growth (weight and height); decreased muscle mass; increased serum IGF-1 (0.75 mg/ kg per month) Golub et al 9 (1997) respectively, and a delayed or incomplete differentiation of mammary epithelium. 8 Some environmental chemicals affect puberty via MOAs that are involved in the central nervous system/ HPG axis, altering brain, hypothalamic, and/or pituitary function via direct interactions with the central nervous system neuroendocrine function. These include the pesticides thiram, molinate, metam sodium, chlordimeform, amitraz, triazoles, dichloroacetic acid, atrazine, propazine, simazine, methanol, and linuron. Prepubertal exposure to atrazine causes delayed puberty in the male and the female, as evidenced by an older age at acquisition of PPS 68 and VO, 69 respectively. The modes of action for atrazine s reproductive effects include puberty timing and a reduction in circulating LH and prolactin. 70 Perinatal exposure of mice to BPA results in a decrease of tyrosine hydroxylase neurons in the rostral periventricular preoptic area at 22 to 24 days of age (prepubertal), an important brain region that regulates estrous cyclicity and estrogen-positive feedback. 71 Chemicals that inhibit the synthesis of endogenous hormones (eg, testosterone, 17 -estradiol, adrenal steroids) typically do so by competitively inhibiting the activity of 1 P450 steroidogenic enzymes. The predominant effects are mediated through the steroidogenic enzymes cholesterol side chain cleavage enzyme, steroid 17,20 lyase, and aromatase (which converts androgens to estrogens). Some affect estrogen synthesis preferentially over testosterone or adrenal hormones, and others affect adrenal and liver function. Examples of environmental and pharmaceutical aromatase inhibitors include conazole and imadazole fungicides (eg, prochloraz, fenarimol, ketoconazole, fadrozole). Peripubertal exposure to the aromatase inhibitor fadrozole at dosages as PEDIATRICS Volume 121, Supplement 3, February 2008 S197

7 low as 0.6 mg/kg per day led to a delayed VO in female rats. 56 Peripubertal exposure to 100 mg/kg per day of the steroidogenesis inhibitor ketoconazole also led to a delayed VO. 56 The MOA of prochloraz is the inhibition of aromatase 72 and 17,20 lyase 73,74 apparently directly inhibiting steroidogenesis of estrogens and androgens. Prochloraz also delays puberty in male rats. 73 The MOA of the fungicide chemical ketoconazole is inhibition of sidechain cleavage enzyme (SCC), progesterone, and adrenal steroid synthesis. The MOA of many phthalate esters, including diethylhexyl phthalate, dibutyl phthalate, and benzyl butyl phthalate, is to inhibit fetal testis Leydig cell steroidogenesis and insl3 synthesis, leading to male developmental and reproductive system malformations (eg, retained nipples, reduced or absent reproductive organs, undescended testes, hypospadias, delayed PPS) with in utero exposure and delays in puberty with peripubertal exposure. 75 Administration of phthalates during puberty to marmosets induced ovarian and uterine alterations in females and reduced peripubertal testosterone levels in males 76 ; however, the effect on androgen levels was not significant because of the extreme variability in this measure in this species. Human Studies Many human studies have shown a positive relation between body fat, primarily measured by BMI or skinfold thickness, and onset of puberty, as determined by the onset of the growth spurt, breast development, or menarche Similar findings are observed in boys as measured by growth spurt or genital and pubic hair maturation. 81,85 87 Body size measures also have been associated with early puberty, 82,86,88 as have dietary and physical activity exposures and pubertal timing. 79,89 94 Environmental exposure to persistent halogenated organic chemicals such as PCBs, dichlorodiphenyltrichloroethane/dichlorodiphenyldichloroethylene (DDT/DDE), and brominated flame retardants have been associated with pubertal alterations, as have dioxin, hexachlorobenzene (HCB), endosulfan, and heavy metals, but to a lesser extent (Table 2). The first study to report that timing of puberty was associated with developmental exposure to persistent organic pollutants was conducted among a cohort of individuals who were exposed to brominated flame retardants. 95 An industrial accident resulted in the contamination of cattle feed with polybrominated biphenyls and widespread human exposure through consumption of meat and dairy products. Daughters of women who consumed contaminated farm products were interviewed or completed a self-administered mailed questionnaire. Age at menarche and Tanner stages (for those who were younger than 18 years) were assessed. Maternal serum levels of polybrominated biphenyl at the time of conception were estimated from a decay model using serum concentrations that were measured before and/or after the pregnancy. 96 Girls who were exposed in utero to high concentrations ( 7 ppb) and who were breastfed reported menarche 1 full year earlier than unexposed girls ( 1 ppb, the limit of detection) or girls who were exposed in utero but not breastfed. Breastfed girls with high in utero exposure also reported more advanced pubic hair development compared with unexposed girls or girls who were not breastfed. There was no association found with Tanner stage of breast development. Various studies have evaluated pubertal development among children who were exposed to DDT and its primary metabolite DDE. Two studies specifically assessed developmental exposure (in utero and/or postnatal via lactation) within a cohort. Gladen et al 97 conducted a prospective cohort study of boys and girls who resided in North Carolina in relation to DDE concentrations that were previously measured in the mothers serum, cord serum, and the placenta (averaged for in utero exposure). Concentrations in breast milk also were determined (lactational exposure). Puberty-timing measures, Tanner stages (boys and girls), and menarche were assessed by annual questionnaires. For boys, no association was observed for either in utero or lactational DDE exposure. Among girls, there was no association with age at menarche; however, there was a suggestion of an association of higher in utero or lactational exposure and earlier breast and pubic hair development that was not statistically significant. Vasiliu et al 98 also evaluated in utero exposure to DDT/DDE that was estimated using a decay model based on maternal measurements among daughters of a Michigan angler cohort. The authors observed a significantly earlier menarche among girls with an increased in utero exposure. Specifically, menarche was 1 year earlier for every 15- g/l increase in in utero exposure. No association was seen with breastfeeding and age at menarche. Several additional studies evaluated DDT/DDE exposure in childhood using various designs. Krstevska-Konstantinova et al 99 measured DDE concentration among girls who were born in Belgium compared with those who were foreign-born. The foreign-born girls had significantly higher levels of DDE that were correlated with age at immigration and time since immigration than native-born girls. Other migratory effects have been seen in other populations. 100 They also estimated the incidence of precocious puberty among native Belgium girls and foreign adopted girls from vital records and adoption registries and found the rate of precocious puberty to be 80 times higher than that for native-born Belgium girls. Although suggestive, this preliminary study was limited in size and scope, especially with regard to other differences unaccounted for between the 2 groups of children. Ouyang et al 101 studied newly married female textile workers in China by measuring serum DDT/DDE concentration. Women were asked to recall age at menarche. A significant dose-response relation was found between serum DDT/DDE concentrations and earlier menarche. A 10 ng/g increase in exposure was associated with 0.2 years earlier onset of menarche. Because exposure occurred after pubertal development was complete, the causal order of exposure-puberty cannot be established because pubertal development may have affected the metabolism/distribution of DDT or other behaviors that influenced DDT exposure. Denham et al 102 conducted a cross-sectional study among girls who were aged 10 to 17 years and resided in the Mohawk Nation S198 BUCK LOUIS et al

8 TABLE 2 Human Studies That Assessed Associations Between Environmental Chemical Exposures and Puberty Timing Chemical Exposure (Biospecimen) Brominated flame retardant (estimated maternal serum PBB at conception) DDE (maternal blood, cord blood, and placenta averaged; breast milk) DDE (serum) DDE (estimated maternal serum DDE at conception) DDT/DDE (serum in adulthood) DDE (serum) Dioxin-like activity (serum; CALUX) Dioxin (serum) Endosulfan HCB (serum) Lead (serum) Lead (serum) Lead (serum) Population (Sample Size) Study Design Findings Reference US, Michigan girls aged 5 24, accidental in utero and/or lactational exposure (327) US, North Carolina boys and girls (594) Girls with precocious puberty: foreign-born girls (26); Belgium girls (15) US, Michigan daughters of women who consumed Great Lakes fish (151) Chinese female textile workers, newly married (466) US/Canada Mohawk Nation, girls (138) 17-y-old Belgium boys (78) and girls (120) from polluted and nonpolluted areas Seveso, Italy, women exposed postnatally and prepuberty to dioxin industrial accident (282) India, boys aged in exposed (117) and unexposed (90) areas US/Canada Mohawk Nation, girls (138) US 8- to 16-y-old girls NHANES III (2186) US 8- to 16-y-old girls NHANES III (1706) US/Canada Mohawk Nation, girls (138) CS/C; assessment of Tanner stages in female offspring; recalled age at menarche in postmenarchal offspring C; assessed menarche, Tanner stages by annual questionnaire CS; serum DDE compared between foreign-born and Belgium girls; all had precocious puberty Earlier menarche and pubic hair development among girls highly exposed in utero and breastfed; no association with breast development Boys: no association; girls: suggestion of earlier breast and pubic hair development with high transplacental and with high lactational exposure Foreign-born girls with precocious puberty had significantly higher DDE concentration than native Belgium girls with precocious puberty. C; recalled age at menarche Higher DDE associated with earlier menarche. CS; recalled age at menarche Higher DDT/DDE associated with earlier menarche Blanck et al 95 (2000) Gladen et al 97 (2000) Krstevska-Konstantinova et al 99 (2001) Vasiliu et al 98 (2004) Ouyang et al 101 (2005) CS; menarche (yes/no) No association Denham et al 102 (2005) CS; assessment of Tanner stage by examination (boys and girls), testicular volume (boys), recalled age at menarche (girls). Boys: no association; girls: girls with high exposure were less likely to have reached the highest stage of breast development; no association with pubic hair development or age at menarche. C; recalled age at menarche No association; suggestion of earlier menarche among girls exposed before the age of 5 C/CS; Tanner stage by physical examination; serum hormones and endosulfan levels for 70 exposed, 45 unexposed. Exposed boys were less mature and had lower testosterone and higher LH than unexposed boys Den Hond et al 106 (2002) Warner et al 108 (2004) Saiyed et al 111 (2003) CS; menarche (yes/no) No association Denham et al 102 (2005) CS, assessed age at menarche and Tanner stages; analyses stratified by ethnic groups CS, assessed age at menarche and Tanner stages CS; menarche (yes/no) Later pubic hair and breast development associated with higher lead among blacks and Mexican Americans, suggested association among non- Hispanic whites; later menarche associated with higher lead among blacks, suggested association among Mexican Americans and non-hispanic whites Later menarche and pubic hair development with higher lead exposure; no association with breast development Exposure to lead associated with lower likelihood of menarche Selevan et al 112 (2003) Wu et al 113 (2003) Denham et al 102 (2005) PEDIATRICS Volume 121, Supplement 3, February 2008 S199

9 TABLE 2 Continued Chemical Exposure (Biospecimen) Mercury (serum) Mirex (serum) PCBs (serum) PCBs (maternal blood, cord blood, and placenta averaged; breast milk) Population (Sample Size) Study Design Findings Reference US/Canada Mohawk Nation, girls (138) US/Canada Mohawk Nation, girls (138) US/Canada Mohawk Nation, girls (138) US, North Carolina boys and girls (594) PCBs (maternal serum) US, Michigan girls aged 5 24 (256) CS; menarche (yes/no) Suggestion of earlier menarche Denham et al 102 (2005) with mercury exposure CS; menarche (yes/no) No association Denham et al 102 (2005) CS; menarche (yes/no) C; assessed menarche, Tanner stages by annual questionnaire CS; assessment of Tanner stages; recall of age at menarche in postmenarchal girls PCBs (cord blood) Faroe Islands boys (175) C/CS; genital anomalies at birth, examination at age 14 for Tanner stage, testicular size, spermaturia, and serum hormones PCBs (serum; congeners 138, 153, and 180) 17-y-old Belgium boys (78) and girls (120) from polluted and nonpolluted areas PCBs/PCDFs Taiwan, girls aged exposed to contaminated oil (Yucheng) in utero (27) and controls (21) PCBs/PCDFs PCBs (estimated maternal serum PCBs at conception) PCBs (serum congeners) Taiwan, boys (mean age: 12.3) exposed to contaminated oil (Yucheng) in utero (61) and controls (60) US, Michigan daughters of women who consumed Great Lakes fish (151) US/Canada Mohawk Nation, girls (138) Phthalates (serum) Puerto Rico (41 PT cases, 35 controls) CS; assessment of Tanner stage by examination and testicular volume C; recalled age at menarche, menstrual cycle characteristics, serum hormones C/CS; Tanner stages by exam, testicular size, serum hormones 4 potentially estrogenic PCB Denham et al 102 (2005) congeners (52, 70, , and 187) significantly associated with a greater probability of having reached menarche Boys: no association; girls: Gladen et al 97 (2000) suggestion of earlier breast and pubic hair development with high transplacental, suggestion of earlier pubic hair development with lactational exposure No association Blanck et al 95 (2000) No significant associations; suggestion of higher testosterone with higher PCB exposure Boys: high exposure to PCB 138 less likely to have reached highest stage of genital development, high PCB 153 less likely to have reached highest stage of pubic hair; girls: no associations No association with age at menarche; exposed girls reported shorter cycle length and more irregular cycles, exposed girls had higher levels of estradiol and FSH No association with Tanner stage or testicular size; some differences in hormone levels in boys 13 years Mol et al 103 (2002) Den Hond et al 106 (2002) Yang et al 104 (2005) Hsu et al 105 (2005) C; recalled age at menarche No association Vasiliu et al 98 (2004) CS; menarche (yes/no) Case control Exposure to estrogenic congeners (52, 70, 90, 101, 187, geometric mean) associated with greater likelihood of menarche; no association with other congener groups Serum phthalate (primarily DEHP) significantly higher in cases than controls Denham et al 102 (2005) Colon et al 109 (2000) CS, cross-sectional study; C, cohort study; CS/C cross-sectional assessment within a cohort; CALUX, chemically activated luciferase gene expression; PBB, polybrominated biphenyl; NHANES III, Third National Health and Nutrition Examination Survey; PCDFs, polychlorinated dibenzo-furans; PT, premature thelarche. along the United States/Canadian border to assess serum concentrations and self-reported menarchal status (yes/ no). No association was observed between DDT exposure and menarche despite the authors assumption that current serum concentrations were indicative of in utero and lactational exposures, given the long-standing con- S200 BUCK LOUIS et al

10 cern regarding consumption of contaminated fish among this population. Five studies evaluated developmental PCB exposures in relation to pubertal development. Gladen et al 97 reported no relation between in utero or lactational PCB exposure and age at menarche among girls. Higher lactational exposure was associated with earlier Tanner staging in boys and girls, although the results did not achieve statistical significance. Mol et al 103 established a prospective cohort study of boys in the Faroe Islands, where PCB levels are known to be relatively high. PCB levels were measured in cord serum and tissue. Boys were examined at 14 years of age for Tanner staging, testicular size, spermaturia (sperm in urine), and serum hormones. No statistically significant associations were found between PCB concentrations and measures of pubertal development. A suggestion of a positive association between testosterone and PCB exposure was reported. Two cohort studies previously described 95,98 also evaluated in utero exposure to PCBs among daughters of women who were exposed. Neither study found an association between PCB exposure and age at menarche. Last, Yang et al 104 and Hsu et al 105 described no pubertal developmental differences among boys or girls with and without exposure to PCB- and polychlorinated dibenzofuran contaminated cooking oil. Two cross-sectional studies of PCB exposure have been conducted. Den Hond et al 106 measured PCB congeners in a sample of boys and girls who were aged 17 years and recruited from 1 of 2 exposed areas in Belgium: residence near a lead smelter or waste incinerator or a referent rural area. Puberty timing was measured using the Tanner staging as a part of a physical examination. Odds ratios were estimated for PCB concentration and odds for being in a puberty stage that indicated that puberty was not yet complete (ie, lower than stage 5). Exposed boys had completed pubertal development later, because odds ratios for higher PCB exposures (for both specific congeners and total PCBs) were associated with stage 5 for genital and pubic hair development. Because this study evaluated 17- year-olds, it is not clear whether puberty began later or the peripubertal duration was longer. No pubertal delays were seen in girls with PCB exposure. Among Akwesasne Mohawk Nation Girls, exposure to a group of 4 potentially estrogenic PCB congeners (52, 70, , and 187) was associated with a significantly greater likelihood of having reached menarche. 102 Two studies assessed childhood exposure to dioxins and puberty. Den Hond et al 106 measured dioxin-like activity with the chemically activated luciferase gene expression assay 107 and reported an association with Tanner stage 5 breast development; however, pubic hair stage in boys and girls and genital stage in boys was not significantly associated with dioxin-like activity. Warner et al 108 examined pubertal development among girls who were exposed postnatally or during childhood to dioxin in Seveso, Italy. TCDD was not associated with age of menarche (self-reported at interview). None of the women was exposed prenatally. Colon et al 109 studied phthalate esters exposure and premature thelarche. Forty-one girls with thelarche were compared with 35 control girls with regard to serum pesticides and phthalate esters. Significantly higher phthalate levels were found among the girls with thelarche than comparison girls. The authors concluded that the findings were suggestive of a possible association between exposures and premature breast development in girls. Interpretation of these findings may be limited by residual confounding and concerns about the analytical method of measuring the phthalate parent compound instead of the metabolites. 110 Saiyed et al 111 found that boys who were exposed to endosulfan in India were less mature and had lower testosterone and higher LH than unexposed boys. Three cross-sectional published articles assessed the relation between lead exposure and pubertal measures using either the Third National Health and Nutrition Examination Survey 112,113 or the Mohawk Nation Study. 102 In the Third National Health and Nutrition Examination Survey analyses, blood lead levels were analyzed in relation to age at onset for Tanner stage 2 for breast and pubic hair and menarche among US girls aged 8 to 18 years. 113 Selevan et al 112 estimated odds ratios for lead levels in relation to progression of puberty (stages 2 5) among girls. Combined, these studies suggested an inverse relation between blood lead levels and the onset and progression of puberty in girls, especially for subgroups within the population. An association also was found between higher blood lead level and delayed menarche, but this association was statistically significant only for black girls. The cross-sectional analysis among Mohawk nation girls also found later puberty among girls with higher lead exposures than girls with lower exposure. Cross-species Concordance of Pubertal Timing Effects Most chemicals have not been consistently studied in both animals and humans, with a few notable exceptions. Even when such research is available for a particular chemical, interpretation is exceedingly challenging given issues pertaining to dosage and timing of exposure and limited measurement of other relevant covariates. When such data are available (eg, TCDD, lead, DDE, PCBs, estrogens), similar findings tend to be observed across species. For example, pharmaceutical estrogens (eg, diethylstilbestrol, ethinyl estradiol) are the best examples of accelerated female breast development in humans, but the effect tends to be reversible and isolated from other pubertal effects. 29,30 33 This is similar to the effects of estrogen exposure in the female rat 56 and the monkey, which cause pseudoprecocious or dysregulated puberty markers. 50 In male humans, pharmaceutical agents that are used to halt early puberty also cause similar effects in male rats. In the rat, in utero TCDD exposure resulted in delayed puberty as measured by a late age of VO and a delayed or incomplete differentiation of mammary epithelium. 8 Similarly, Den Hond et al 106 reported an association between high dioxin blood levels and later completion of breast development. This cross-species concordance may indicate that the Ah receptor agonism is active across species. For developmental lead exposure, animal and human studies both show a delay in PEDIATRICS Volume 121, Supplement 3, February 2008 S201

11 TABLE 3 Research Recommendations for Focusing on Environmental Influences on Puberty Priority Research Questions Recommended Studies Recommended Species Etiologic Research 1. Are environmental levels of EDCs affecting onset and progression of puberty and aberrant pubertal development such as delayed or precocious puberty in children? 2. Do body shape and adiposity affect onset and progression of puberty and aberrant pubertal development such as delayed or precocious puberty in children? Critical windows of exposure 3. What are the critical windows, ranging from periconception through adolescence, for human pubertal development in relation to environmental exposures? Mechanistic 4. What is the primary signal(s) for the onset of GnRH-dependent (central) puberty? 5. What is the primary signal(s) for the onset of GnRH-independent (peripheral) puberty? 6. What is the molecular basis for sexual dimorphism in puberty onset and progression? 7. How is the tempo of puberty progression including the temporal relationship among the different markers regulated? Prospective epidemiologic research inclusive of existing cohort currently being followed (eg, Seveso, Yucheng, Yusho, PBB, DES grandchildren) or newly designed epidemiologic research capturing environmentally relevant exposures (single chemical and mixtures) Cross-sectional studies for monitoring population changes particularly across geographic areas and inclusive of high-risk subgroups of the population Prospective epidemiologic research inclusive of existing cohort currently being followed (eg, Seveso, Yucheng, Yusho, PBB, DES grandchildren) or newly designed epidemiologic research capturing environmentally relevant exposures (single chemical and mixtures) Cross-sectional studies for monitoring population changes particularly across geographic areas and inclusive of high-risk subgroups of the population Prospective research comprising couples attempting pregnancy to capture parentally mediated exposures from periconception window to prospective pregnancy cohorts Research focusing on the early origins of human health and disease Genomics and proteomic studies using nonhuman primates and humans with alterations in the onset of puberty Genomic and proteomic studies using transgenic rodent model. Identifying the similarities and differences in molecules involved from puberty initiation to final outcome (described in 4 and 5) in males and females Environmental exposure and puberty-timing studies that measure 1 puberty-timing end point/outcome. Humans Humans Humans and nonhuman primates Nonhuman primates and humans Rodents Rodents, nonhuman primates Rodents, nonhuman primates, humans pubertal development, suggesting a similar mechanism for rodents and humans. P,p -DDE exposure in foreign-born girls was associated with earlier puberty relative to native-born Belgians 99 and, possibly, increasing DDE in utero exposure with earlier puberty in girls 97,98 ; however, no animal studies of DDE exposure and female puberty timing were identified, limiting additional interpretation of the human data. In male rats, peripubertal DDE exposure delays PPS, although no relation to lower exposures was found in boys after in utero DDE exposure. 97 The weight of animal and human data, although suggestive, remains inconclusive for establishing a causal relation between EDCs and human pubertal disturbances. As reviewed, much of the available literature focuses on a single chemical or class, ignoring the mixture of compounds to which humans are exposed. Much of the data are derived from cross-sectional studies, which are unable to establish the causal ordering of environment in relation to puberty. To this end, timing and dose at critical windows of human development have not been purposefully assessed. Other relevant covariates such as lifestyle, genetic determinants, and other xenobiotics in the chemical mixture have not been well studied, if at all, in the context of EDCs. RESEARCH RECOMMENDATIONS The panel was asked to make research recommendations that are responsive to critical data gaps regarding purported environmental factors that may adversely affect puberty, to prioritize research initiatives including meth- S202 BUCK LOUIS et al

12 ods development, and to identify avenues for research collaboration. Environmental Factors Identified as Important for Human Puberty After reviewing the weight of evidence described herein, the panel agreed that EDCs and body size or adiposity may be associated with pubertal disturbances in humans. The panel further recognizes that the 2 broad exposures may be interrelated in that exposure to EDCs can affect fat metabolism (eg, organotin compounds and diethyl hexyl phthalate act as peroxisome proliferator activated receptor agonists in adipose tissue). Although humans can do little to minimize exposure to ubiquitous EDCs other than change dietary practices, particularly with regard to fish consumption and preparation, body size is potentially amenable to behavioral intervention. Carefully designed epidemiologic research, particularly that with longitudinal capture of exposures and relevant covariates at critical windows and the sensitive and specific measurement of markers of pubertal onset, progression, or deviations from expected normative standards, is needed to answer remaining questions about the role of environmental influences on developmental milestones. Such studies are rightfully complex and require a priori consideration and capture of other environmental determinants of puberty such as those that are believed to be attributed to body size and composition and possible genotypes and phenotypes that may emerge. Further complicating future research will be the challenges afforded by environmental mixtures, including those that arise from natural sources such as phytoestrogens. Research Priorities and Future Directions The panel identified 7 priority research questions reflecting 3 broad research domains: etiologic, exposure assessment, and mechanistic (Table 3). The panel strongly supported additional etiologic work aimed at answering lingering questions about the impact of EDCs and body size and adiposity on the onset and progression of puberty and aberrant clinical manifestations (ie, delayed or precocious puberty). The natural history of puberty requires delineation and ordering of a multitude of factors such as lipids, body shape, and diet in relation to pubertal onset and progression. With regard to exposure assessment, continued work is needed to capture the timing and dosage of maternal, paternal, or parental exposures across sensitive and critical windows of human development to identify agents that may disrupt normal embryonic or fetal development resulting in fetal (re)programming with lifetime effects. 119,120 Last, the panel continued to support basic research to identify the primary signal(s) for the onset of GnRH-dependent and GnRH-independent puberty in humans, nonhuman primates, and other animals. The increasing methodologic complexities that are associated with capturing exposures, including environmental mixtures, in the context of lifestyle and behavior across critical windows of human development argue for prospective epidemiologic studies whose utility and feasibility have been demonstrated. 121,122 Ideally, such studies would incorporate longitudinal biospecimen collection at timed intervals for the quantification of chemical mixtures and lifestyle exposures that may affect growth and development inclusive of pubertal milestones. Efforts to develop a suite of sensitive pubertal markers, rather than a sole marker, that are consistent with the manner in which humans are exposed are needed. Future research needs are discussed elsewhere in this issue but underscore the need to develop behavioral, psychosocial, and neurologic end points along with the social and psychological consequences of precocious or delayed puberty. 11 Last, regardless of the research question, newly designed animal and human research must use the best available methods. With regard to animal research, careful attention to choice of the most appropriate animal model, relevant critical or sensitive windows of exposure, route and duration of exposure, timing and relevant dosages, assumptions regarding the dose-response curve, and an analytic statistical plan that is responsive to the study design, particularly assessment of mixtures and litter effects, is needed. Epidemiologic research also needs to address these same issues while recognizing the highly correlated nature of reproductive outcomes and the need to model appropriately previous history of adverse reproductive outcomes, a strong predictor, in the context of more subtle environmental effects. 123 CONCLUSIONS A concerted body of multidisciplinary research is needed to answer lingering questions about the impact of EDCs, body size, and adiposity on human puberty. This research can be grounded within a broader research base inclusive of human reproduction given the highly interrelated and timed series of critical events that are characteristic of human growth and development, of which puberty is 1 of many key milestones. Basic research targeting the primary signals for onset is critical so that epidemiologic investigators can design research that is sensitive to the capture and measurement of key biomarkers of pubertal onset. Additional basic research is needed regarding comparative species physiology of puberty, the primary signal of puberty onset for central versus peripheral signaling, and the basis for sexual dimorphism. ACKNOWLEDGMENTS Support for The Role of Environmental Factors on the Onset and Progression of Puberty workshop was provided by US Environmental Protection Agency cooperative agreement , the National Institute of Environmental Health Sciences, and Serono Inc. We gratefully acknowledge Serono Symposia International and David Schlumper for organizing and holding The Role of Environmental Factors on the Onset and Progression of Puberty Expert Panel Workshop. In addition, we thank the other members of the Expert Panel for participating in the discussions: Carlos Bourdony, Farid Chehab, Gwen Collman, Ralph Cooper, Leo Dunkel, Paul Foster, Beth Gladen, Mari Golub, Mel PEDIATRICS Volume 121, Supplement 3, February 2008 S203

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17 Environmental Factors and Puberty Timing: Expert Panel Research Needs Germaine M. Buck Louis, L. Earl Gray, Jr, Michele Marcus, Sergio R. Ojeda, Ora H. Pescovitz, Selma Feldman Witchel, Wolfgang Sippell, David H. Abbott, Ana Soto, Rochelle W. Tyl, Jean-Pierre Bourguignon, Niels E. Skakkebaek, Shanna H. Swan, Mari S. Golub, Martin Wabitsch, Jorma Toppari and Susan Y. Euling Pediatrics 2008;121;S192 DOI: /peds.1813E Updated Information & Services Citations Subspecialty Collections Permissions & Licensing Reprints including high resolution figures, can be found at: /content/121/supplement_3/s192.full.html This article has been cited by 10 HighWire-hosted articles: /content/121/supplement_3/s192.full.html#related-urls This article, along with others on similar topics, appears in the following collection(s): Endocrinology /cgi/collection/endocrinology_sub Puberty /cgi/collection/puberty_sub Environmental Health /cgi/collection/environmental_health_sub Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: /site/misc/permissions.xhtml Information about ordering reprints can be found online: /site/misc/reprints.xhtml PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, Copyright 2008 by the American Academy of Pediatrics. All rights reserved. Print ISSN: Online ISSN:

18 Environmental Factors and Puberty Timing: Expert Panel Research Needs Germaine M. Buck Louis, L. Earl Gray, Jr, Michele Marcus, Sergio R. Ojeda, Ora H. Pescovitz, Selma Feldman Witchel, Wolfgang Sippell, David H. Abbott, Ana Soto, Rochelle W. Tyl, Jean-Pierre Bourguignon, Niels E. Skakkebaek, Shanna H. Swan, Mari S. Golub, Martin Wabitsch, Jorma Toppari and Susan Y. Euling Pediatrics 2008;121;S192 DOI: /peds.1813E The online version of this article, along with updated information and services, is located on the World Wide Web at: /content/121/supplement_3/s192.full.html PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, Copyright 2008 by the American Academy of Pediatrics. All rights reserved. Print ISSN: Online ISSN: