Apoptosis is a genetically determined and biologically functional mode of cell death. It plays important roles in numerous physiological events in mammals, including ovarian follicular atresia, decidualisation and placentation during embryo implantation, as well as many pathological conditions [
44,
45]. The major features of apoptosis are internucleosomal DNA fragmentation, cell shrinkage, plasma membrane blebbing, formation of apoptotic bodies and phagocytosis of apoptotic bodies by macrophages [
46]. During follicular growth, more than 99% of follicles disappear, primarily due to apoptosis of granulosa cells. Follicular atresia is a hormonally regulated process, and different factors are affecting the decision to die at different stages of ovarian follicle development [
47]. Atretogenic factors include caspases, protein bax, members of the tumor necrosis factor family (TNF-α, Fas, FasL, TRAIL), tumor suppressor protein P53, members of transforming growth factor-beta family (factor NODAL and AMH), c-Myc, endothelins, androgens and GnRH [
44]. Successful follicle development depends on the presence of survival factors that promote follicle growth and also protect cells from apoptosis. These include factors produced within the ovary as well as the gonadotropins LH and FSH. Some of the paracrine factors that promote survival during the growth and differentiation of follicles include kinase Akt, members of bcl-2 family, KIT-ligand and c-KIT receptors, stem cell factor (SCF), members of TGF-beta family (activin, factors BMP-4 and BMP-7, growth differention factor-9), oestrogens, insulin and IGFs, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), TGF-α, interleukin 1b (IL-1b), growth hormone (GH) and the member of inhibitor of apoptosis, survivin [
44,
47,
48]. Most of the inhibitors of follicle atresia are regulated by FSH and LH. When the growing follicles reach the antral stage, they express receptors for FSH and become dependent on FSH stimulation. Sufficient FSH concentrations are critical for survival of follicles that have differentiated to the antral stage or beyond. During each reproductive cycle, increasing FSH concentrations rescue developing follicles. LH is important for follicles approaching ovulation and expressing LH receptor [
47]. FSH and LH influence oocyte growth and maturation through the sterol pathways in mice [
49]. Follicular fluid meiosis-activating sterol (FF-MAS) is found at high concentrations in the follicular fluid of mammals including humans in response to gonadotropins and is proved to be stimulatory to oocyte meiotic resumption, while lanosterol 14α-demethylase (LDM), a key enzyme that converts lanosterol to FF-MAS seems to have a positive effect on the oocyte plasma maturation for fertilization and early embryo development in mice [
50]. In addition, epidermal growth factor receptor (EGFR) activation, by protein kinase C (PKC) signal pathway, participates in FSH-induced oocyte maturation in pigs [
51]. It is well known that the expression of the LH receptor (LHR) in cumulus cells is related with FSH-induced meiotic resumption of cumulus enclosed oocytes (CEOs). An important new step towards understanding the physiological actions of gonadotropins during oocyte maturation is the finding that in mice the LHR expression in cumulus cells has a functional role during FSH-induced oocyte maturation, which process is possibly regulated by MAPK cascade [
52]. In addition to all that it has been found that in mice FSH increases cAMP-dependent protein kinase (PKA) levels and induces cAMP response element-binding protein (CREB) phosphorylation and cytochrome P
450 lanosterol 14α-demethylase (CYP51) expression in cumulus cells before the oocyte meiotic resumption [
53]. In the absence of survival factors, endogenous apoptosis pathways within the follicle become activated and lead to follicular atresia [
47]. The present study showed the expression of survivin in luteinized granulosa cells from a sample of Greek patients that underwent IVF or ICSI. To the best of our knowledge, this is the first report which determined the frequency of survivin expression in these cells from a sample of Greek patients. Therefore, the present study gives an additional estimation on the frequency of expression of the survivin in luteinized granulosa cells, which is detected in almost all of the studied cases from Greek patients. However, more studies are needed to analyze larger numbers from Greek patients and investigate therefore whether geographic or ethnic prevalence of survivin gene expression could be caused by environmental or genetic factors. The expression of survivin, in granulosa cells reported herein, is in agreement with the previous findings from Fujino et al. [
34], who studied the expression of survivin gene expression in granulosa cells from infertile Japanese patients and found such expression in all granulosa cells. In addition, the present study found a statistically significant increased expression of survivin in granulosa cells of women who had tubal factor infertility compared with normal women (male factor infertility). This finding suggests a protective role of survivin in the ovarian microenvironment from situations such as inflammation (salpingitis), hydrosalpinx, tubal ligation and salpingectomy. Ovrieto et al., (2011) studied the effects of salpingectomy due to hydrosalpinx on the outcomes of assisted reproduction and embryo transfer and found a significant reduction in ipsilateral ovarian response to ovulation induction as regards the development of follicles [
54]. It is therefore likely that survivin might try to protect the ovaries, with possible influenced perfusion due to the ipsilateral salpingectomy. In cases with tubal inflammation or hydrosalpinges survivin might try to protect the ovaries from follicular apoptosis in a paracrine environment. However, more studies are needed to support our hypothesis probably using animal models.
In the present study the capacity for fertilization (fertilizability) was 100%, regardless IVF or ICSI method used. An increased survivin expression in granulosa cells was found in women undergoing IVF compared with ICSI. Nakahara et al., (1997) suggested that when the quality of eggs is small, measured by apoptosis in granulosa cells, then the eggs are more likely to be fertilized by ICSI compared with IVF method [
11]. However, Clavero et al., (2003) found that the rate of apoptosis in granulosa cells was not associated with the maturity of the oocytes and the ability for fertilization in ICSI or the quality of follicles during the ovulation induction [
55]. Despite the controversy that exists in the literature about the effects of apoptosis in luteinized granulosa cells as predictor of oocytes quality during ovulation induction in cycles of IVF or ICSI, the estimation of survival factors in these cells might have prognostic role. An example supporting the validity of using survival factors for prognosis provides the IGFs family. It has been found that the IGF1, IGF2 and their receptors are down regulated in ovarian granulosa cells of women with diminished ovarian reserve (DOR) compared to those with normal ovarian reserve (NOR) undergoing in vitro fertilization (IVF) [
56]. However, more research is needed about the clinical significance of the expression of some other survival factors in the luteinized granulosa cells. Data generated using mice genetic models perturbing ovulation and fertility indicates that the EGF-like growth factors amphiregulin, epiregulin, and betacellulin are induced by LH and activate the EGF receptor pathway in granulosa cells of preovulatory follicles to impact ovulation [
57,
58]. Whether this network also plays a critical role in humans and whether it can be used as a biological marker of follicle development or for the improvement of fertility remains to be determined [
57]. We enounce our hypothesis about the levels of survivin mRNA expression in ovarian granulosa cells in tubal factor infertility. Some patients in the subpopulation of women with tubal factor undergoing assisted reproduction and embryo transfer probably could benefit in assessing oocyte quality by measuring the levels of survivin expression in their granulosa cells. Therefore, if the survivin levels in granulosa cells are low, then ICSI should be concerned, as ICSI is an invasive method and good oocyte quality is not required. On the other hand, if survivin levels are highly expressed in granulosa cells then IVF should be preferred, as IVF is a non-invasive method and therefore normal sperm-egg interaction and good oocyte quality is essential. However, the cut-off survivin mRNA expressed levels have to be determined in such cases. Fujino et al., (2008) found that there was a statistically significant correlation in the levels of survivin expression in granulosa cells among women with endometriosis and normal women. In women with endometriosis lower survivin levels were found [
34]. However, one factor to be considered is that in the present study only normal women (male factor infertility) and women with tubal factor infertility were studied. Women with endometriosis were not included since endometriosis promotes apoptosis [
11]. Also, in the present study women with polycystic ovarian syndrome were not included because androgens promote apoptosis. Fujino at el, (2008) found no statistically significant difference in levels of survivin expression between normal women (male factor infertility) and women with tubal factor infertility [
34], as we found in this study. Fujino et al., (2008) found that the survivin expression in granulosa cells was higher in all pregnant women than in not pregnant women [
34]. However, this did not result from the findings of this study. As there is some controversy in this field more studies should be taken. In addition, it would be interest further studies to investigate expression with any clinical significance of survivin gene in granulosa cells of patients with diminished ovarian reserve (DOR) as such population was not included in our study.