Wang et al. disorders that may be apparent at birth or not apparent until adulthood. Some notable exam- ples of this include diethylstilbestrol (DES) and thalidomide. DES was used in the early 1940s and 1950s for the treatment of irregular vagi- nal bleeding and threatened abortions. DES is associated with the occurrence of cervical or vaginal clear-cell adenocarcinoma in the F1 generation when exposure was before 18 weeks of gestation (Hatch et al. 1998). Thalidomide was used in the late 1950s to treat nausea and vomiting associated with pregnancy, causing severe limb malformations in the newborn when exposure occurred during days 34–50 of gestation (Smithells and Newman 1992). The in utero exposure to various stimuli leading to altered pubertal development was demon- strated in animal experiments using intra- uterine artery ligation and chemical treatments. Intrauterine ligation during gestation was used to simulate malnutrition in a rat model and was shown to cause a delay in the onset of puberty in males and females (Engelbregt et al. 2000). Bisphenol A (Howdeshell et al. 1999) and octylphenol (Bogh et al. 2001; Wright et al. 2002) treatments in animals were demon- strated to advance puberty by shortening the time to either the ﬁrst estrus or its equivalent. For example, the gilts of sows treated with octylphenol from days 23–85 of gestation were observed to have a shorter period to first estrous compared with controls (mean ± SD, 211.5 ± 14.5 days vs. 246.0 ± 36.7 days) (Bogh et al. 2001). However, octylphenol did not alter testicular size in boars that were exposed to octylphenol as fetuses (Bogh et al. 2001). When ewes were treated with octyl- phenol from day 70 of gestation to birth, the times to the dates of ﬁrst estrous and ﬁrst prog- esterone rise in the lambs were decreased com- pared with controls by an average of about 20 days and 44 days, respectively (Wright et al. 2002). In the same study the lambs of ewes treated through birth and to weanling had shorter times to puberty compared with controls as well, but they were not different from lambs from ewes treated to birth. Atrazine delayed mammary gland development in female rats treated either as fetuses during gestational days 15–19 or during lactation (Rayner et al. 2004). In the same model, vaginal opening was delayed only in rat pups that were exposed to atrazine by lactation. Phytoestrogens, such as genistein (Casanova et al. 1999) and the lignan-containing ﬂaxseed (Tou et al. 1998), advanced the time of onset of puberty in female rats when high doses were initiated during the gestational period and continued postnatally. Of note, when ﬂaxseed was administered at a lower dose, it delayed the onset of puberty (Tou et al. 1998). No observed pubertal effects (i.e., time of onset, tes- ticular weight) were found in males in these phytoestrogen models. The effects of in utero exposure to environmental chemicals on puber- tal development were evaluated in several epi- demiologic studies. For PCBs and p,p´-DDE this route of exposure was compared with that from breast-feeding in one study (Table 1). The signiﬁcant difference between these two expo- sure periods is that the amount of chemical delivered to the infant from lactation is usually greater than the amount of chemical delivered to the fetus by the placenta (Jacobson and Jacobson 1996). The importance of these experimental ﬁndings in animals and data on human exposure to chemicals is that they lend support to the biologic plausibility or likelihood that a chemical can either cause or modify the outcome of the purported health effect. It will be important to consider mechanisms of action, timing of exposure, and existing chemi- cal levels in the general population from back- ground exposure when candidate chemicals are reviewed for this hypothesis. During the in utero period, the fetus should be assessed for exposures that coincide with the developmental windows of the pri- mary and secondary sexual organs (Pryor et al. 2000) (Table 2). For example, mammary Table 2. Sampling periods for the assessment of exposure to persistent and nonpersistent chemicals. Preconceptiona In utero Perinatal M/F M/F M/F M (10–15 weekse) M (10–15 weekse) M (10–15 weekse) Persistent chemicals Urineb,c,d Serumb,f Whole bloodb,f Hairb Human milkb Cord serumb Cord whole bloodb Meconiumb Dietary assessmentg Home air sampleb,g Home composite dust sampleb,g Other environmental samplesb,g Questionnaireg Ecologic analysis (e.g., GIS)g Nonpersistent chemicals Urineb,c,d Serumb,f Human milkb Cord serumb Meconiumb Dietary assessmentg Home air sampleb,g Home composite dust sampleb,g Other environmental samplesb,g Questionnaireg Ecologic analysis (e.g., GIS)g x x x Special studies x x M/F M/F x x x Special studies x x M M M M M M x x x x x x Special studies x x M (10–15 weekse) x x Special studies x x M (10–15 weekse) M (10–15 weekse) M (10–15 weekse) x x Special studies x x x x x Special studies x x Postpartum (18–24 months) x x x 2 weeks – 2 months postpartum Prepuberty (5–6 years) x x x Midpuberty Postpuberty x x x x x x x x x x x x x x x x x x Special studies Special studies Special studies Special studies —— At 6, 12, 24 and 36 months, then annually until puberty —— x x x x x x x x x x x x 2 weeks – 2 months postpartum x x x x x x x x x x x x x x Special studies Special studies Special studies Special studies —— At 6, 12, 24 and 36 months, then annually until puberty —— x x x x GIS, geographic information system; F, father; M, mother; x, period when samples are to be collected. aAt the time of enrollment. bMedia with extant laboratory methods for likely target chemical agent. Note that for nonpersistent chemicals, multiple samples of biologic specimens may be needed during this time because of possible variability in biologic exposure level due to the short half-life of the chemical. This is particularly important if a critical exposure period is being considered. Alternatively, pilot data may demonstrate the lack of need for frequent sampling if constant exposure occurred in a stable environment. cTimed specimen collection; morning void specimen with creatinine measurement. dPediatric urine bag or diaper sample for non-toilet-trained children. If not diaper, spot samples or multiple spots. eFor example, weeks 10–15 of gestation are a critical period for breast development. fWhen blood collection is at a young age, piggyback on lead screen at 12 and 24 months that is recommended by the Centers for Disease Control and Prevention. gEnvironmental sampling, dietary assessment, questionnaires, and ecologic analysis are necessary for persistent chemicals to deter- mine either route or pathway of exposure; otherwise, exposure can be established through the analysis of biologic specimens. Information should be obtained proximate to the sampling period for biologic specimens. 1104 VOLUME 113 | NUMBER 8 | August 2005 • Environmental Health Perspectives