Effects of preimplantation 100 mg/kg/day BPA treatment on embryo transport and embryo development detected on gestation day 3.5. A. Percentage of pregnant females with embryo localization in the uterus and oviduct. * P=0.00031 when the numbers of mice with oviduct retention were compared. B. Percentage of pregnant females with embryos in different developmental stages. * P=0.0023 when the numbers of mice with delayed embryo development (morula and/or earlier stages) were compared. A & B. N=8 in control and N=5 in 100 mg/kg/day BPA-treated group. C. Percentage of embryos in earlier stages than morula, and blastocyst stages. A representative image of embryos at each of these three stages from 100 mg/kg/day BPA-treated group is shown on the right. All the embryos from the control group were in the blastocyst stage. * P=4.01E-09. N= 34 in control and N=22 in 100 mg/kg/day BPA-treated group.

Immunohistochemical detection of progesterone receptor (PR) in gestation day 3.5 and day 4.5 uteri upon preimplantation BPA treatment. Uterine cross sections (10 µm) were processed for detecting PR localization (brown staining). A representative section was from each of the following groups: A. Gestation day 3.5, 0 mg/kg/day BPA (control). B. Gestation day 4.5, 0 mg/kg/day BPA (control). C. Gestation day 3.5, 40 mg/kg/day BPA. D. Gestation day 4.5, 40 mg/kg/day BPA. E. Gestation day 3.5, 100 mg/kg/day BPA. F. Gestation day 4.5, 100 mg/kg/day BPA. No specific staining was detected in the negative control (data not shown). Red star, embryo; LE, luminal epithelium; S, stroma; GE, glandular epithelium; D, decidual z one; Myo, myometrium. Scale bar: 100 µm.

Effects of preimplantation bisphenol A (BPA) exposure on embryo implantation detected on gestation day 4.5. A. Implantation rates. They were determined by calculating the ratio of the total number of mice with implantation sites over the total number of plugged mice in each group × 100. N=5–14; * P<0.001 compared to control. E2, 17β-estradiol, was included as a positive control for estrogenicity. B. The average number of implantation sites. The implantation sites were detected as blue bands shown in Figure 3. The number of implantation sites was counted for each mouse and the average was determined for each dose group. Only the mice with implantation sites were counted. N=5–12. Error bars: standard error.

Effects of preimplantation 40 mg/k g/day BPA treatment on embryo implantation detected on gestation day 4.5 (A~F) and day 5.5 (G~J). A & B. Two representative uteri from the control group showing defined implantation sites as distinctive blue bands (red arrows). C & D. Two representative uteri with implantation sites from 40 mg/kg/day BPA-treated group. Red arrows in C indicate defined implantation sites. Red arrow heads in C and D indicate the locations of faint blue bands (implantation sites) suggesting delayed implantation. E. A uterus from 40 mg/kg/day BPA-treated group showing no detectable implantation sites (E) but with hatched embryos flushed from the uterus. F. A representative embryo from the uterus in E. G. A representative uterus from gestation day 5.5 control uterus showing implantation sites (red arrows). H~J. Images of three gestation day 5.5 uteri from 40 mg/kg/day BPA-treated mice showing implantation sites (red arrow heads) with variable sizes but at relatively earlier stages (especially those in H and I) compared to those in the control (G).

Effects of preimplantation BPA (40 mg/kg/day) exposure on pregnancy outcome. A. Gestation period. *, P=0.00096. All the animals in the control group (0 mg/kg/day BPA) happened to have the same gestation period; the standard error for this group was zero. B. Litter size. *, P=0.046. A & B, N=4–7. Error bars: standard error. C. Postnatal survival rate. *, P=0.0036. N=31 in control and 36 in BPA-treated group.