Markers of stem cells in human ovarian granulosa cells: is there a clinical significance in ART?

Abstract

BackgroundThe purpose of the study was to determine the incidence of gene expression of Oct-4 and DAZL, which are typical markers for stem cells, in human granulosa cells during ovarian stimulation in women with normal FSH levels undergoing IVF or ICSI and to discover any clinical significance of such expression in ART.MethodsTwenty one women underwent ovulation induction for IVF or ICSI and ET with standard GnRH analogue-recombinant FSH protocol. Infertility causes were male and tubal factor. Cumulus–mature oocyte complexes were denuded separately and granulosa cells were analyzed for each patient separately using quantitative reverse-transcription–polymerase chain reaction analysis for Oct-4 and DAZL gene expression with G6PD gene as internal standard.ResultsG6PD and Oct-4 mRNA was detected in the granulosa cells in 47.6% (10/21). The median of Oct-4 mRNA/G6PD mRNA was 1.75 with intra-quarteral range from 0.10 to 98.21. The OCT-4 mRNA expression was statistically significantly correlated with the number of oocytes retrieved; when the Oct-4 mRNA expression was higher, then more than six oocytes were retrieved (p=0.037, Wilcoxon rank-sum). No detection of DAZL mRNA was found in granulosa cells. There was no additional statistically significant correlation between the levels of Oct-4 expression and FSH basal levels or estradiol peak levels or dosage of FSH for ovulation induction. No association was found between the presence or absence of Oct-4 mRNA expression in granulosa cells and ovarian response to gonadotropin stimulation. Also, no influence on pregnancy was observed between the presence or absence of Oct-4 mRNA expression in granulosa cells or to its expression levels accordingly.ConclusionsExpression of OCT-4 mRNA, which is a typical stem cell marker and absence of expression of DAZL mRNA, which is a typical germ cell marker, suggest that a subpopulation of luteinized granulosa cells in healthy ovarian follicles (47.6%) consists of stem cells, which are not originated from primordial germ cells. Absence of Oct-4 gene expression in more than half of the cases means probably the end of the productive journey of these cells, towards the oocyte.

Plasticity of granulosa cells: on the crossroad of stemness and transdifferentiation potential

“…Additionally, GCs were positive for various markers of mesenchymal stem cells: CD29, CD44, CD90, CD105, CD117, and CD166, but not for CD73 [41]. Another study by Varras et al [77] showed that OCT-4 gene was expressed in GCs of women included in the assisted reproduction program, namely in 48 % of the studied women; however, the expression of this gene was demonstrated only by real time-PCR methodology. They carefully analyzed if OCT-4 expression was related to female clinical parameters (i.e., age, BMI, duration and causes of infertility, previous assisted reproduction attempts, basal serum FSH and LH levels, serum levels of PRL, serum estradiol levels on the fifth day of rFSH administration and on the day of hCG administration, the total dose of rFSH, the duration of treatment, the type of assisted reproduction, the number of aspirated follicles etc.…”

“…), but they did not find any correlation. Unlike Kossowska-Tomaszczuk et al [41] study in which GCs were pooled from various infertile women, Varras et al [77] analyzed the gene expression of GCs in each single patient with tubal factor of infertility or with infertile partner. Although the OCT-4 transcription factor is a solid marker of cell stemness, we need to be careful when selecting the appropriate primers.…”

“…Kossowska-Tomaszczuk et al [41] isolated GCs from aspirated follicular fluid of infertile patients based on FSHR; Varras et al [77] collected GCs from cumulus-oocyte complex during oocyte retrieval from fertile patients; and Mattioli et al [47] scraped GCs from internal face of the follicle wall obtained from prepubertal pigs. First variable which can affect differences in stem cell markers is fertility since it was found that the cause of female infertility can drastically affect the GCs viability [34]; therefore this factor should always be taken into account when discussing the stem cell characteristics of GCs.…”

“…All four layers are also functionally heterogeneous, secrete different molecules and express various hormone receptors [55]. The GCs are subject to increased attention since it has recently been shown that the subpopulation of GCs within the growing follicle is not terminally differentiated but showing stem cell characteristics [39,41,47,77].…”

Selected molecular and physiological aspects of mammalian ovarian granulosa cells in primary culture

“…It has been shown in several publications that some GCs in the growing follicle are not fully differentiated and exhibit stem cell properties across mammals, i.e. : ovine, porcine and human GCs (28,29,38,49). Besides stem cell properties, GCs also present high telomerase activity and are capable of differentiation into cell types that are not present within ovaries.…”