247 Delayed implantation reduces fetal growth alterations observed in assisted reproductive technology pregnancies

Superovulation, in vitro exposure, and embryo transfer are examples of assisted reproductive technologies (ART) that can negatively affect embryo development and lead to fetal growth alterations, pregnancy failure, and health problems in offspring. Enhanced signalling by exosomes and other biological systems that can effectively repair potential blastocyst damage increase during delayed implantation (DI), which may all contribute to better pregnancy outcomes. In this study, we tested whether DI improves the development of ART fetuses by focusing on their morphology, which is due to the previous state of the blastocyst, including ART-related damage. To this aim, superovulated mice females were mated with fertile males and then ovariectomised to prevent implantation. At Day 8 post-coitum, embryos were collected (DI group). Control embryos (non-delayed implantation [NDI group]) were collected at Day 4 post-coitum. Collected embryos (31 NDI and 48 DI) were shortly kept in vitro (up to 1 h) and then transferred to pseudopregnant recipients at 4 dpc for implantation and further development. At 15.5 dpc, recipients were sacrificed, and fetuses were collected to evaluate their development stage by histological analysis using hematoxylin and eosin staining, and then analysed in reference to Kaufman Atlas of Mouse Development (CTR). Our study showed that crown-rump length of NDI fetuses is 8.65 mm, which corresponds to 13.5 dpc (Carnegie Stages 18), whereas DI fetuses are 10.62 mm, corresponding to 14.5 dpc (Carnegie Stages 20). Notably, the NDI group was heterogenous (standard deviation: 1.429, coefficient of variation: 15.70%) and the fetuses varied in size in comparison to CTR (one-sample Wilcoxon test, P = 0.0010, discrepancy = −3.35 mm). NDI was characterised by several morphological aberrancies indicative of underdevelopment in comparison to both DI and CTR. These included heart underdevelopment, mainly atria, septum, and pericardial deformity; low heterogeneity in the marginal layer, intermediate, and ventricular zone (brain-part underdevelopment); delayed pituitary formation, decreased liver vascularisation; and a lower degree of lung capillarisation. DI group was homogeneous (standard deviation: 0.5602, coefficient of variation: 5.174%). The size variation in the DI group fetuses was lower (one-sample Wilcoxon test, P = 0.0313, discrepancy = −1.385 mm) and shows no signs of underdevelopment. Our study showed that DI can correct alterations in prenatal development seen in NDI fetuses. DI fetus morphology resembles that of those naturally conceived (CTR). The larger size of DI fetuses suggests more-prompt implantation after transfer to prereceptive recipients, likely due to their increased size and more intensive signalling through exosomes. Our study suggests that DI allows embryos to repair ART-related morphological damage to the heart, brain, liver, and lungs. This might be a solution to ensure development of healthy offspring when using ART.

This work was supported by the National Science Centre, Poland (GA no. 2019/35/B/NZ4/03547 and 2021/41/B/NZ3/03507).