O-064 Recovery culture of human cryopreserved blastocysts with prolactin after warming improves trophoblast outgrowth
Authors: Ezoe K , Fujiwara N , Miki T , Kato K 
Published: June 2022

Abstract

Study question
Does prolactin (PRL) treatment during recovery culture affect human blastocyst outgrowth?

Summary answer
PRL treatment for 120 min promoted trophoblast outgrowth in cryopreserved human blastocysts by upregulating the expressions of epithelial-to-mesenchymal transition (EMT) and focal adhesion-related genes.

What is known already
Human embryos express the PRL receptor at the morula and the blastocyst stages. This expression correlates with the blastocyst diameter and the time required for the embryos to reach the blastocyst stage. Treatment with PRL from cleavage to the blastocyst stage improves blastocyst outgrowth to fibronectin. However, whether PRL treatment after warming cryopreserved blastocysts cultured to the blastocyst stage without PRL influences outgrowth competence remains unknown. Furthermore, the optimal time for post-warming PRL treatment remains to be ascertained.

Study design, size, duration
A total of 374 discarded human vitrified blastocysts donated for research by consenting couples were used. The study was approved by the Institutional Review Board. The blastocysts were randomly allocated to two groups, to be cultured in medium either with PRL (n = 208) or without PRL (control; n = 166). The gene expression level, blastocyst adhesion, outgrowth area, and distance of trophoblast migration were compared between the groups.

Participants/materials, setting, methods
Vitrified human blastocysts were cultured for 120 min after warming. Some blastocysts were treated with PRL for 15–120 min during the recovery period. The blastocysts were plated on fibronectin-coated dishes and cultured to assess blastocyst adhesion and outgrowth. The expressions of PRL-interacting genes were assessed by quantitative RT-PCR 12 h after outgrowth culture. The migration distance at the outer edge of the trophoblast cells was examined using time-lapse systems.

Main results and the role of chance
The mRNA expressions of ezrin, radixin, and moesin, which regulate cell adhesion and invasion by controlling actin reorganisation during EMT, was stimulated by PRL treatment for 120 min. The expressions of EMT-related genes, transforming growth factor β1, snail1, and twist1 were also promoted by treatment with PRL for 120 min. The blastocysts treated with PRL also exhibited augmented expression of cadherin2 and transcriptional repression of cadherin1. Higher mRNA expressions of integrin-based focal adhesion-related genes, ITGA5 and ITGB1, were observed after treatment with PRL for 120 min compared to that in the other 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 blastocysts treated with PRL. The migration distance of trophoblast cells was significantly increased after PRL treatment. Furthermore, a more beneficial effect of prolactin treatment on blastocyst outgrowth was observed when the blastocysts were vitrified on day 5 compared to that when the blastocysts were vitrified on day 6. Moreover, the outgrowth area was increased by PRL treatment when the blastocyst diameter was larger than 180 µm.

Limitations, reasons for caution
The results may vary between in vivo and in vitro conditions. Further clinical studies are required to explore the clinical efficacy of PRL treatment.

Wider implications of the findings
This study showed that PRL treatment for 120 min improved trophoblast migration in cryopreserved human blastocysts. Therefore, recovery culture with PRL treatment post-warming followed by blastocyst transfer could improve pregnancy outcomes.

Trial registration number
not applicable