This study investigated the effects of preimplantation BPA exposure on the embryo and the uterus, two factors critical for embryo implantation in mice. A study by Berger et al demonstrated that BPA given s.c. (10.125 mg/mouse/day, ~400 mg/kg/day) to CF-1 mice from gestation day 1.5 to day 4.5 significantly reduced the percentage of females with visible implantation sites and the number of implantation sites detected on day 6.5 [44
]. In our study, C57BL6 mice were treated with 100 mg/kg/day BPA s.c. from gestation day 0.5 to day 3.5 and no embryo implantation was detected on gestation day 4.5 (). These results indicate that the regimen used in our study was more sensitive than the one used by Berger et al [44
]. Two main factors could be involved: different mouse strain sensitivities and/or the treatment regimens. C57BL6 is one of the most sensitive strains to endocrine disruption [50
], although the relative sensitivity of CF-1 to endocrine disruptors is unknown. Treatment regimen (timing and duration) could be another important factor. Exposure to endocrine disruptors on gestation day 0.5 could be more sensitive than on gestation days 2.5 and 3.5 in disrupting embryo implantation based on the data from methoxychlor (MXC), another environmental estrogen [51
]. In addition, all four injections (day 0.5~day 3.5) in our study were in the preimplantation period, whereas only three injections (day 1.5~day 4.5) in Berger et al [44
] fell in the preimplantation period (implantation normally occurs ~gestation day 4.0 in mice).
An abrupt drop of implantation rates as the dose was raised from 40 to 100 mg/kg/day BPA in the treated groups suggests a threshold for toxicity of BPA on the events critical for successful embryo implantation, for example preimplantation embryo development, embryo transport, and uterine receptivity. These events were all adversely affected in the 100 mg/kg/day BPA-treated group (section 3.1~3.4), and each of these adverse effects could lead to failed embryo implantation.
BPA treatment affects preimplantation embryos both in vitro
and in vivo
. In vitro
studies have demonstrated that exposure of two-cell embryos to 100 µM BPA in culture for 48 hours significantly increased the degeneration of preimplantation embryos, whereas more of those exposed to 1 nM BPA reached the blastocyst stage [52
]. Our results indicate that preimplantation BPA exposure at 100 mg/kg/day adversely affected preimplantation embryo development and embryo transport (). The tube-locking effect of BPA on embryo transport may reflect the estrogenic effect of BPA [18
Although BPA at high doses can affect embryo implantation () [44
] and BPA can have various effects on the uterus [38
], the effects of BPA on uterine receptivity, which is also indispensable for successful embryo implantation, was unknown. Preimplantation treatment of 100 mg/kg/day BPA is detrimental not only to the embryos (Sections 3.2 & 3.3) but also to the establishment of uterine receptivity, which was demonstrated by the embryo transfer study. Since the blastocysts were transferred to the BPA-treated pseudopregnant mice, which received the last dose only three hours prior to embryo transfer, the transferred blastocysts were exposed to residual BPA. It is possible that failed implantation in the embryo transfer study might be the result of blastocyst exposure to BPA. However, two lines of evidence would argue against this possibility. First, hatched blastocysts (data not shown) were flushed from the BPA-treated uteri; second, when BPA was tested for its estrogenicity in a delayed implantation rat model, up to 200 mg/kg BPA single s.c. injection on the day before induction of implantation (equivalent to gestation day 3.5 in this study) could induce embryo implantation [17
], indicating that exposure to BPA (up to 200 mg/kg) right before implantation did not harm the ability of the blastocysts to implant in rats.
The progesterone receptor (PR) contributes to embryo implantation [53
], and it has dynamic spatiotemporal expression patterns in the uterus during peri-implantation [47
] (). Loss of PR in uterine epithelium is associated with the establishment of uterine receptivity in all mammals examined [54
], while sustained PR expression in the uterine epithelium during the expected “implantation window” has been associated with defective uterine receptivity in both the human and the mouse [56
]. Our hourly time-course study demonstrates that PR disappears from uterine luminal epithelium several hours after implantation sites become detectable by blue dye reaction and right before decidualization occurs [49
]. Therefore, PR expression in the uterine luminal epithelium can be used as a sensitive temporal marker in the early implantation process. However, if embryo implantation has proceeded to the decidualization stage, PR is no longer a good molecular marker for detecting delayed implantation. Based on immunohistochemistry, the sustained expression of PR in the luminal epithelium of gestation day 4.5 uteri exposed to 40 and 100 mg/kg/day BPA confirmed delayed implantation () and failed implantation (), respectively.
Preimplantation BPA exposure affects not only embryo implantation processes but also post-implantation processes, such as increased post-implantational death, which was suggested by reduced litter size (), and increased postnatal death () from 40 mg/kg/day BPA-treated group. The female offspring had normal embryo implantation, indicating that the effect of preimplantation exposure to 40 mg/kg/day BPA on embryo implantation was not manifested in the next generation.
The focus of this study was to differentiate the effects of preimplantation BPA exposure on embryo and on uterine receptivity. Both of these two aspects are critical for successful embryo implantation, for which the molecular mechanism is still not fully understood. BPA at high doses was used as a pharmacologic agent to study its effects on embryo implantation. Based on the data from this study, many aspects, which are not the focus of the current study but future directions, can be potentially addressed. For example, what specific genes are differentially regulated in the uterus by BPA that could be associated with the adverse effect of BPA on uterine receptivity, thus embryo implantation, and how these genes are differentially regulated by BPA in the uterus. Such information will provide more insight into the molecular mechanism of the establishment of uterine receptivity. In addition, such information can also be used to assess any potential effects of long-term exposure to environmentally relevant BPA levels on genes critical for the establishment of uterine receptivity, thereby helping with risk assessment.