P–178 Mural granulosa cells of the human follicles indirectly show death molecular signals not depending on different ovarian stimulation protocols
Authors: Ruvolo G , Geraci F , Roccheri M C , Alessandro R , Bosco L 
Published: August 2021

Abstract

Study question
Could the expression of the anti-apoptotic molecules AKT, p-Akt and ERK1/2 in Mural Granulosa Cells (MGC) be considered as marker of oocyte quality?

Summary answer
MGCs activate cell death pathways in analyzed follicles and it is not influenced by different stimulation protocols and it is not correlated to oocyte competence.

What is known already
It has been previously demonstrated that apoptosis rate of mural granulosa or cumulus cells (CC) were correlated with follicular oocyte number, age, embryo numbers in IVF/ICSI and also clinical pregnancy. Moreover, our previous data demonstrated that in selected patients, who received recombinant LH associated with recombinant FSH (rFSH), the DNA fragmentation in cumulus cells was significantly lower and pregnancy rate was higher, compared to patients treated with rFSH alone. However, to date little is known about the differences between MGC and CC regarding death/survival pathways and whether the two cell types respond in the same way.

Study design, size, duration
Molecular study on MGCs to investigate the role of the surviving/apoptotic molecules AKT, p-Akt and ERK1/2 and their relationship with the administration of exogenous r-LH combined with r-FSH administration in ovarian stimulation comparing with r-FSH alone. We analyzed also the oocyte competence, for each follicle, according to the embryo development during in vitro culture and the pregnancy outcome. We included fifty-three normo-responder women undergoing ICSI in two years.

Participants/materials, setting, methods
Patients were divided into two groups: 1) 34 women were stimulated with r-FSH and used as control group, 2) 19 women were stimulated with r-FSH combined with r-LH. Mural granulosa cells isolated singularly from 255 MII oocytes of the 53 patients were used for the study. The study was conducted in public university laboratory. MGCs obtained from each single follicle were suspended in medium, without serum. For immunoreaction anti-AKT, p-AKT, ERK1/2 antibodies were used.

Main results and the role of chance
Out of 255 MII oocytes collected, 197 were fertilized and the derived embryos had the following evolution: 117 transferred, 57 vitrified and 23 arrested during in vitro culture. 58 oocytes were not analyzed because of failed fertilization or because of their immature condition (GV or MI).
In the MGCs isolated from the follicle of each oocyte generating an embryo, the expression AKT, pAKT and ERK1/2 was analyzed and associated with embryo quality and pregnancy outcomes.
Immunoblot analysis on granulose cells showed no statistically significant differences in protein level in MGCs isolated from oocytes that have generated transferred embryos (blastocyst at day 5 or 6) comparing with embryos who arrested during in vitro culture. No differences were found also in MGCs collected from the follicles derived from r-FSH ovarian stimulation compared to r-FSH+r-LH. Moreover, no difference was highlighted even between protein level and pregnancy outcomes. The results seem to demonstrate that the MGCs primarily have an endocrine function and support the growth of the follicle, and finally follow a specific death pathway. This condition is not influenced by different ovarian stimulation protocol, in contrast with CC, and is not correlated to oocyte competence, embryo quality and clinical outcomes.

Limitations, reasons for caution
Only a limited number of patients have been observed.
Wider implications of the findings: Our current and past results suggest that the evaluation of cumulus cells and mural granulosa cells in the same follicle show different expression of molecules involved in the apoptotic pathway and therefore they cannot be used, as molecular markers, in the same way, to assess the competence of oocytes.

Trial registration number
Not applicable