Post-warming culture of human vitrified blastocysts with prolactin improves trophoblast outgrowth.

BACKGROUND: Human embryos express the prolactin (PRL) receptor at the morula and blastocyst stages. Treatment with PRL from cleavage to the blastocyst stage improves blastocyst outgrowth on fibronectin-coated dishes. However, whether post-warming PRL treatment of blastocysts cultured without PRL could improve outgrowth competence remains unknown. Furthermore, the optimal time for post-warming PRL treatment remains to be ascertained. This study investigated the effects of PRL treatment during recovery culture on human blastocyst outgrowth and its related genes. METHODS: In total, 374 discarded vitrified blastocysts were randomly allocated to two groups, to be cultured with (n = 208) or without PRL (control; n = 166) for 120 min for recovery, and then plated on fibronectin-coated dishes. The expression level of PRL-interacting genes, blastocyst adhesion rate, outgrowth area, distance of trophoblast migration, and outgrowth degeneration were examined. RESULTS: The mRNA expression of ezrin, radixin, and moesin, which regulate cell adhesion and invasion by controlling actin reorganization during epithelial-to-mesenchymal transition (EMT), was stimulated by PRL treatment for 120 min. The expression of EMT-related genes, transforming growth factor ß1, snail1, and twist1 was also promoted following treatment with PRL for 120 min. PRL-treated blastocysts also exhibited augmented expression of cadherin 2 and transcriptional repression of cadherin 1. Higher mRNA expression of integrin-based focal adhesion-related genes, ITGA5 and ITGB1, was observed after treatment with PRL for 120 min than in the non- and shorter-treatment groups. PRL treatment for 120 min did not alter the rate of blastocyst adhesion to fibronectin-coated dishes 96 h after the outgrowth culture assay. However, multiple linear regression analysis revealed that the outgrowth area was significantly increased in PRL-treated blastocysts. The migration distance of trophoblast cells was significantly increased and degeneration rate was significantly decreased after PRL treatment. Furthermore, a more beneficial effect of PRL treatment on blastocyst outgrowth was observed when the blastocysts were vitrified on day 5 than when they were vitrified on day 6. CONCLUSIONS: Post-warming culture of human vitrified blastocysts with PRL for 120 min promoted trophoblast outgrowth in vitrified human blastocysts. Furthermore, PRL treatment may reduce outgrowth degeneration by increasing resistance to apoptosis during trophoblast migration.

Effects of fatty acid supplementation during vitrification and warming on the developmental competence of mouse, bovine and human oocytes and embryos.

RESEARCH QUESTION: Does fatty acid supplementation in vitrification and warming media influence developmental competence in oocytes after vitrification and warming? DESIGN: Mouse oocytes and four-cell embryos were vitrified and warmed with solutions supplemented with fatty acid and cultured to the blastocyst stage. To study lipid metabolism after vitrification, quantitative real-time polymerase chain reaction was used to analyse the expression of genes related to beta oxidation in mouse embryos vitrified and warmed with or without fatty acids. The effects of fatty acid supplementation in the warming solutions on the developmental competence of bovine and human embryos were analysed. Blastocyst outgrowth assay was used to evaluate the potential of human blastocysts for adhesion to fibronectin. RESULTS: The neutral lipid content of mouse oocytes in the fatty acid 1% supplementation group was significantly higher than in the fatty acid 0% group (P = 0.0032). The developmental rate to the blastocyst stage was significantly higher in the fatty acid 1% group than in the fatty acid 0% group in mice (P = 0.0345). Fatty acid supplementation in warming solution upregulated Acaa2 and Hadha in mouse embryos. Fatty acids significantly improved the developmental ability of bovine embryos to the blastocyst stage (P = 0.0048). Warming with 1% fatty acid supplementation significantly increased the proportion of human blastocysts with morphological grade A inner cell mass (P = 0.0074) and trophectoderm (P = 0.0323). CONCLUSIONS: Fatty acid supplementation in the warming solutions improved the developmental competence of vitrified-warmed mouse oocytes by activating the beta-oxidation pathway. Fatty acid supplementation enhanced the developmental rate of bovine embryos to the blastocyst stage and improved morphological characteristics of human embryos vitrified at the cleavage stage.

Prolactin receptor expression and its role in trophoblast outgrowth in human embryos.

RESEARCH QUESTION: What is the gene expression pattern of prolactin receptor (PRLR) in human pre-implantation embryos and what are its functions during the embryonic development and adhesion process? DESIGN: A total of 405 discarded human vitrified oocytes and embryos donated for research by consenting couples were used in this study. The oocytes and embryos were used to analyse PRLR expression and to evaluate the influence of prolactin (PRL) supplementation in the embryo culture medium on embryo developmental competence and viability. The rates of blastocyst development and adhesion, outgrowth area, cytoskeletal reorganization and nascent adhesion formation were compared between groups. RESULTS: PRLR expression increased significantly after embryo compaction (P < 0.0001) and blastulation (P < 0.0001). Supplementation of the embryo culture medium with PRL did not improve the developmental rate and morphological grade. In contrast, blastocyst outgrowth was significantly increased in embryos cultured with PRL (P = 0.0004). Phosphorylation of JAK2, downstream of the prolactin receptor family, was markedly higher in the PRL-treated embryos than in embryos cultured without PRL. Furthermore, the expression of mRNAs encoding ezrin-radixin-moesin proteins and epithelial-mesenchymal transition-related genes was stimulated by the activation of PRL-JAK2 signalling. The PRL-treated embryos had higher mRNA expression of integrins than non-treated embryos, and transcriptional repression of cadherin 1 was observed after PRL treatment. More nascent adherent cells expressed focal adhesion kinase and paxillin in PRL-treated embryos than in non-treated embryos. CONCLUSIONS: Human embryos express PRLR at the morula and blastocyst stages, and PRLR signalling stimulates blastocyst adhesion by promoting integrin-based focal adhesions and cytoskeletal organization during trophoblast outgrowth.