P-170 Comparison of the effect of human polarized and non-polarized Granulosa cells co-culture on mouse embryo development
Authors: Sedaghat Shoar M , Dalman A , Rezazadeh Valojerdi M 
Published: June 2022

Abstract

Study question
Do human polarized Granulosa cells (GCs), improve the development of mouse embryos after co-culture, compared to human non-polarized GCs and standard culture?

Summary answer
Yes, human polarized GCs improve blastulation and hatching rate and also embryos quality after co-culture, compared to human non-polarized GCs and standard embryo culture.

What is known already
Co-culture embryo with the cells of the female reproductive tract is one of the methods that, in addition to meeting the metabolic needs of the embryo, compensate for the lack of interactions between the embryo and the female reproductive tract during in vitro culture. Various studies have shown that co-culturing of embryos with GCs improves embryo quality and clinical outcomes. However, GCs morphology is essential for proper function changes during in vitro culture to fibroblast-like appearance. Matrigel can effectively maintain epithelial cells' physiology, morphology, and polarity like GCs in vitro and provide more efficient feeder cells.

Study design, size, duration
GCs were cultured on Matrigel to maintain epithelial nature. The effect of Matrigel on GCs morphology, ultrastructure, and the transcription level of specific and apoptotic genes was studied following one and six days of culture and compared to culturing without Matrigel. Then Polarized and non-polarized GCs were applied for co-culturing with embryos. Blastulation and hatching rate, cell count in the blastocyst stage, and expansion were compared between two co-culture groups and standard embryo culture.

Participants/materials, setting, methods
GCs were enzymatically isolated from oocyte surroundings of fertile women who had undergone IVF treatment due to male factors and cultured with or without Matrigel overnight. Pronucleus embryos were extracted from adult female NMRI mice and cultured randomly for up to five days in three groups: standard culture, co-culture with non-polarized GCs, and co-culture with polarized GCs.

Main results and the role of chance
Histological results showed that Matrigel maintains the round shape of GCs, but in the group without Matrigel, GCs were slender and spindle-shaped. Ultrastructure sections showed in culture on Matrigel, Multiple cell junctions connected GCs, lipid droplets were homogeneously spread on the cytoplasm, and Cylinder- shape mitochondria were also detected. GCs culture without Matrigel showed thick cell extensions and gaps between cells and also clustered lipid droplets on one side of the cytoplasm. Gene expression studies indicated culturing GCs on Matrigel is associated with maintaining function and greater cell viability, which had a positive effect on co-culture with embryos; In the co-culture group with polarized GCs, the blastulation rate, as well as hatching or hatched blastocyst, was significantly higher compared to the control group (P-value&lt;0.05). Also, the number of blastomeres increased dramatically in both co-culture groups compared to the standard culture (P-value&lt;0.05). The approximate ratio of the area of inner cell mass to the total area of blastocytes was also significantly lower in the co-culture group with polarized GCs compared to the control group and culture with non-polarized GCs (P-value&lt;0.05), indicating more blastocyst expansion in the co-culture group with polarized GCs.

Limitations, reasons for caution
Because of ethical issues, mice embryos were used in this study instead of human embryos.

Wider implications of the findings
In the presence of polarized granulosa cells, the number of embryonic cells, especially trophectoderm cells, blastocyst expansion, blastulation, and hatching rate increase. It seems that granulosa cell culture on Matrigel can effectively simulate the in vivo environment for the embryo and lead to more successful assisted reproductive techniques.

Trial registration number
98000112