Ehrlich et al. After stratifying by protocol type, we identified a potentially more sensitive subgroup of women. There was a stronger association between urinary BPA concentrations and implantation failure among women undergoing the flare or antagonist protocol (alternative/ low responder protocol) as compared with women undergoing the luteal protocol, though numbers were small within strata and there was considerable overlap in 95% CI for corresponding estimates between the two groups. Women on the flare/antagonist protocol were older with diminished ovarian reserve and response than women on the luteal protocol. Although speculative, diminished ovarian reserve may impart increased sensitivity to implantation failure in relation to BPA exposure. Implantation in animals and humans is hormonally modulated by E2 and P via regu­ lation of expression of endometrial proteins that play an important role in endometrial receptivity at the time of implantation (Cooke et al. 1997). Increased secretion of E2 in the preovulatory phase is necessary to stimulate the proliferation and differentiation of the uterine epithelial cells. In the luteal phase, continued production of P stimulates the proliferation and stimulation of the stromal cells, thus preparing the endometrium for suc­ cessful embryo implantation (Norwitz et al. 2001). E2 and P also play a role in embryo development and embryo migration through the oviduct, though the latter is less relevant in women undergoing IVF because eggs are retrieved from the ovary and cultured embryos are transferred directly into the uterine cav­ ity, bypassing the oviduct. At the level of the embryo, E2 plays an important role in the activation of the dormant blastocyst, which is critical for successful implantation (Paria et al. 1993). Previous findings from the study cohort indicated that higher urinary BPA con­ centrations were associated with decreased peak serum E2 levels and decreased number of eggs retrieved in women undergoing IVF (MokL­in et al. 2010). Evidence from animal studies sug­ gests that BPA’s estrogenic properties may disrupt implantation via two mechanisms (Berger et al. 2007, 2008, 2010; Varayoud et al. 2011): a) accelerating the rate of blasto­ cyst development and thus leading to mis­ match in timing with the appropriate uterine receptivity window (Takai et al. 2001), and b) directly decreasing uterine receptivity to blastocyst implantation (Berger et al. 2010). Environmentally relevant doses or low concen­ trations (1–3 nM) of BPA have been shown to significantly increase the developmental rates of two­cell mouse embryos to blastocysts. At a high concentration (100 mM), BPA sig­ nificantly decreased the in vitro developmental rates of the embryos (Takai et al. 2000). This 982 could result in an embryo uterine mismatch and impair successful implantation. In a recent study, high doses of BPA administered subcutaneously (3.375, 6.75, and 10.125 mg/day) to mice during the peri­ implantation phase resulted in a dose­ dependent increase in luminal area (i.e., altered endometrial morphology) and decreased expression of estrogen receptor α (ERα) and progesterone receptor (PR) via BPA action on the ER and decreased PR expression leading to implantation failure (Berger et al. 2010). Timing of exposure was critical, and a single 6.75m­g dose of subcutaneously administered BPA on day 0 or 10.125 mg on day 1 of preg­ nancy was sufficient to significantly reduce the number of implantation sites in mice. However, when the same dose was adminis­ tered on day 2 of pregnancy, it had no effect on the number of implantation sites observed (Berger et al. 2008). In contrast to humans, in mice, implantation can occur as late as day 4 of pregnancy because blastocysts can remain in a state of quiescence until uterine conditions are optimal for embryo implantation (Carson et al. 2000). A previous study by Berger and colleagues found that an average of 68.84 mg of BPA ingested per day (substantially higher than typical daily doses to which humans are exposed) resulted in a complete block of implantation, whereas comparable results were achieved with a dose of only 6.75 mg administered via subcutaneous administration (Berger et al. 2007). The most common route of human exposure to BPA is through inges­ tion (Vandenberg et al. 2007). A recent study in rats showed that BPA at doses as low as 0.05 mg/kg/day [the U.S. Environmental Protection Agency (EPA) oral reference dose (U.S. EPA 1993)] resulted in decreased Hoxa 10 expression, an essential transcription factor required for endometrial receptivity and implantation (Varayoud et al. 2011). Downr­egulation of the ER α and PR was also observed, accompanied by a signifi­ cantly decreased number of implantation sites. One potential limitation of the present study is that there is within­individual vari ­ ability in urinary BPA concentrations and col­ lecting single spot urine samples may result in exposure misclassification (Braun et al. 2011; Ye et al. 2011). However, a previous study in our cohort showed that a single urine sam­ ple had moderate sensitivity for predicting an individual’s longert­erm exposure over several weeks or months (Mahalingaiah et al. 2008). The sensitivity of classifying a subject in the highest tertile using a single urine sample was 0.64. In the present study, during each cycle, urine samples were collected twice over a period of 2 weeks. The Spearman correlation of within­cycle urinary BPA concentrations for this short time interval was 0.18. Although two measures of urinary BPA concentration are better than a single spot sample at clas­ sifying BPA exposure, we cannot be certain that we collected samples within the relevant window of exposure given the short halfl­ife of BPA (Volkel et al. 2002) and its high variabil­ ity over time. The exact timing of urine collec­ tion was dependent on the date and time of medical appointments and eggr­etrieval proce ­ dures and were not necessarily first morning or fasting urines. Another potential limitation is uncertainty regarding the generalizability of the results to women who conceive naturally. It is possible that women undergoing IVF may be more sensitive to BPA exposure for a variety of rea­ sons, including their underlying infertility, the in vitro conditions of early embryonic development, or the ovarian hyperstimulation protocols. Despite uncertainty regarding gen­ eralizability to women conceiving naturally, using IVF as a model to study human repro­ duction has several strengths. This includes the assessment of early developmental end points that are not observable in women conceiving naturally but have been shown to be susceptible to environmental chemi­ cal exposure in experimental studies (Berger et al. 2007, 2008, 2010; Hunt et al. 2003; Takai et al. 2000). Furthermore, because approximately 10–15% of the population in the United States and developed countries are infertile (Fritz and Speroff 2011), the results of the present study may be generalizable to a large portion of the general population. Additional strengths include the preconcep­ tion prospective study design, allowing us to collect exposure information before the outcome of interest and performing accurate clinical ascertainment of implantation fail­ ure. One of the limitations of conventional epidemiologic designs in naturally conceiving women is that it is logistically challenging and expensive to collect and analyze daily urines for multiple months to assess implantation failure (Wilcox et al. 1988). Finally, by applying a GEE approach to account for correlated outcomes across cycles within a given woman, we were able to opti­ mize all the data available to us, which is par­ ticularly important given the relatively small size of our sample population. Alternative modeling approaches such as nonlinear mixed effect models may be preferable, particularly given the potential for ORs to overestimate relative risks for common outcomes (e.g., the 42% implantation failure in our study popu­ lation), but they could not be applied given the limited sample size. However, results based on only first cycle and on log binomial models were consistent with those presented. As recruitment continues and our study population increases, we plan to extend these analyses to explore the contribution of the male gametes and male exposures to BPA to ovlume 120 | number 7 | July 2012 • Environmental Health Perspectives