58 Transcervical transfer of blastocysts reveals detrimental effect on implantation rate in di (2-ethylhexyl) phthalate-exposed mice

Abstract

Transcervical transfer is widely used in assisted reproduction techniques, such as cloning and the production of transgenic animals. Using this technique, the number of transferred blastocysts and implanted embryos may be quantified, representing a useful method for assessing the effect of xenobiotics on quality and functionality of the blastocyst. Di (2-ethylhexyl) phthalate (DEHP) is one of the most widely used plasticizers worldwide that causes several detrimental effects, such as decreased embryo quality and a low implantation rate when laboratory animals are treated with very high doses, ranging from 3000 to 3500 µg·kg⁻¹d⁻¹. The aim of this research was to evaluate the effect of environmentally relevant doses of DEHP on blastocyst functionality and ability to implant in an ex vivo study. The CD-1 mice were exposed subchronically (1 month) to 0, 20, 200, and 2000 µg·kg⁻¹d⁻¹ of DEHP via deposition in the mouth, and then they were subjected to a transcervical transfer procedure. The donor female mice were superovulated with pregnant mare serum gonadotropin (5 IU) IP, followed by hCG (5 UI) IP 48 h later. They were then mated with males of proven fertility. The recipient females (unexposed mice) had the same superovulation protocol as donors, but were mated with vasectomized males. Blastocysts were obtained 96 h post-hCG by flushing the oviduct with Gardner-blastocyst medium and were classified according to morphological characteristics. Only expanded blastocysts were transferred transcervically under general anaesthesia protocol using isoflurane (induction: 3-5% and maintenance 2-2.5%). Four days after transcervical transfer, recipient females were injected with 200 µL of Chicago Blue 1% IV, killed by cervical dislocation 2 h later, following which implantation sites were counted and the implantation rate was calculated with the following formula (implanted blastocysts × 100/transferred blastocysts). A decreased implantation rate was evident in 2000 µg·kg⁻¹d⁻¹ DEHP-treated females compared with controls (DEHP 2000: 11.67% ± 9.64 v. control: 71.67% ± 6.0; P < 0.05 according to 1-way ANOVA analysis followed by the Bonferroni post-hoc test). Taken together, these data suggest that the dose of 2000 µg·kg⁻¹d⁻¹ DEPH impairs embryo implantation. We confirm that transcervical transfer is a useful technique in toxicological studies to assess detrimental effects on implantation.