Apoptosis and endometrial receptivity: Relationship with in vitro fertilization treatment outcome

Abstract

Apoptosis is an important process in the reconstruction of endometrium within the menstrual cycle. The balance between cell proliferation and apoptosis regulates the periodic repair and shedding of endometrial cells and leads to the menstruation or prepare the mucosal layer of endometrium for the implantation of the embryo. Many factors with pro- and antiapoptotic action, such as B cell lymphoma/leukemia-2 and inhibitors apoptosis proteins families, caspases, tumor necrosis factor receptors, phosphatase and tensin homolog, proliferator-activated receptor gamma, microRNAs and others are differently expressed in the endometrial tissue at phases of menstrual cycle. Receptivity of the endometrium at the period of “window of implantation” is associated with the significant increase of apoptosis in endometrium to allow the embryo to be successfully implanted. The impairment of apoptosis regulation in the endometrium at this period often is observed in infertile women with endometriosis, tubal factor, polycystic ovary syndrome, etc.. In many cases the impairment of apoptosis regulation in the endometrium is the main cause of in vitro fertilization (IVF) treatment failure in these patients. As of today, the exact mechanisms and factors mediating the apoptotic process in normal endometrium and in infertile women are not fully understood. Herein, the literature data concerning the endometrial apoptosis regulation in general, and in light of the influence of apoptosis upon IVF treatment outcome are reviewed. The possibility to use some parameters of endometrial apoptosis for prediction of the successful pregnancy achievement in women participating in IVF protocols also is discussed.

Key Words: Apoptosis; Endometrium; Receptivity; In vitro fertilization

Core tip: Endometrial receptivity depends on many factors and apoptosis regulation as well. Compromised fertility and in vitro fertilization (IVF) failure is often associated with the impairment of endometrial apoptosis during “window of implantation”. Understanding of the molecule mechanisms involved in apoptosis regulation in the infertile women might have a great value and let us to use them as predictors of endometrial dysfunctions to improve implantation rate in IVF program.

INTRODUCTION

In the last decades in vitro fertilization (IVF) is widely used for the treatment of infertility in couples with unexplained subfertility, male subfertility, endometriosis or tubal pathology[1]. The impact of different clinical parameters (infertility etiology, female age, time of infertility, thickness of the endometrial tissue, the quantity of follicles and amount of the progesterone on the day of hCG injection, etc.) on the outcome of the IVF treatment was proved[1-4]. Analysis of the data received from different clinical centers shows that maximum achievable pregnancy rate in IVF doesn’t exceed 50% per embryo transfer cycle and the average clinical pregnancy rate is between 27% and 40%[5-8]. Despite some evident technical improvement of IVF protocol, it is clear now that the percentage of the pregnancy onset cannot be made higher due to the manipulation with the embryo transfer procedure and conditions of embryo cultivation or by optimal choice of blastocysts[9]. Many studies have demonstrated that the function and receptivity endometrium must be considered as the main limiting factors in the pregnancy onset after IVF, because for accomplishing an implantation, pregnancy and subsequent live child birth endometrium should be ready to accept the embryo[9-11]. One can see now that overcoming the difficulties in improvements to ART will require a deeper insight in the contact between the endometrium and embryo[12].

It is well known that the success of the embryo implantation depends on some morphologic and biochemical modifications of the human endometrium during menstrual cycle[9]. These processes are controlled by a plethora of different factors, including ovarian steroids and its receptors, cytokines, growth factors, adhesion molecules, transcriptional factors and many others[13]. Recent investigations have demonstrated an importance of apoptosis in the processes of endometrial tissue reconstruction during menstrual cycle[10]. The balance between cell proliferation and apoptosis regulates the periodic repair and shedding of endometrial cells and leads to the menstruation or prepare the mucosal layer of endometrium for the implantation of the embryo[14].

According to classical definition, given by Kerr et al[15] in 1972, “apoptosis, or programmed cell death, is characterized by the fragmentation and enfolding of cell compartments into membrane-covered apoptotic bodies that are removed without any immune response or damage of the surrounding cells”. Caspases, or intracellular cysteine proteases, are the main enzymes in the process of apoptosis cascades execution[16]. At the beginning of the apoptotic pathway the initiator caspases, like capsase-8 and -9 are activated, and the later morphological changes of the apoptotic cell are mediated by the action of effector caspases, like caspase-3[16]. Now two apoptotic signaling pathways, the extrinsic and intrinsic, have been established. The extrinsic apoptotic cascade is activated by ligation of specific death receptors, expressed on the cell-surface, i.e., Fas, tumor necrosis factor (TNF) receptor or TNF-related apoptosis-inducing ligand (TRAIL) receptor[17]. After binding with specific ligands these death receptors are oligomerized, involving Fas-associated death domain (FADD) and procaspase-8 with subsequent appearance of the death-inducing signaling complex, activation of caspase-8 and development of intracellular apoptotic signaling cascades. This results in morphological and biochemical changes in the cell, characteristic for apoptosis[18]. The intrinsic mechanism of apoptosis is activated by a large spectrum of intracellular death signals, such as damage of DNA, stress, and diminishing of the growth factors value[19]. In the result of these apoptotic signals, the mitochondrial membrane became permeabilized and a lot of pro-apoptotic proteins that are typically located in the intermembrane space, i.e., cytochrome c, release into the cytoplasm to trigger the caspase-9 activation[20]. Subsequently both extrinsic and intrinsic apoptosis pathways result in the activation of the caspase-3. They are regulated by numerous molecules with pro- and anti-apoptotic action, among them the best known factors which are belonged to the B cell lymphoma/leukemia-2 (Bcl-2) and inhibitors apoptosis proteins (IAP) families.

The Bcl-2 family comprises 25 members with pro- and anti-apoptotic effects. Bcl-2-family members that have four Bcl-2 homology (BH) domains (Bcl-2, MCL-1, A1/Bfl-1, Bcl-B/Bcl2L10, and BCL-xL) possess the anti-apoptotic action due to inhibition of their pro-apoptotic counterparts via protein-protein interactions[21,22]. The pro-apoptotic family members are divided into two subgroups: those having multiple BH domains (effector proteins), such as Bcl-2-associated X protein (BAX), BAK, and BOK (Bcl-2 related ovarian killer), and containing only the BH3 domain (BID, BIM, PUMA, NOXA, BIK, BAD, HRK, and BMF)[21,22]. The members of IAPs family prevent the activation of caspases-3, -7 and -9 and thereby inhibit apoptosis[23]. Today 8 human IAP family members are identified and divided into 3 classes (1, 2 and 3) according to the presence or absence of a RING or caspase activation recruitment domain and the homology of their baculovirus IAP repeat domains[23]. The best known member of class 1 IAP family is X-linked IAP (XIAP), which is also known as hILP, MIHA and BIC4. It inhibits caspases-3, -7 and -9, but it does not influence on caspase-8[23]. The class 2 IAP family member NAIP is expressed in adult liver, placenta and central nervous system and inhibits caspases-3 and -7, but not caspases-1, -4, -5, or -8. Class 3 IAP members such as survivin is mostly expressed in the fetal tissues, but not in most normal adult tissues[23]. Recently it was demonstrated that survivin is also expressed in the endometrium of healthy women[24]. In addition to these factors there are many others proteins and enzymes which are directly or indirectly involved in the apoptosis of different types of endometrial cells during menstrual cycle. Study of the distribution and expression of these apoptosis regulators can give us new data concerning endometrium functioning in normal and pathological conditions. And understanding of the molecule mechanisms involved in apoptosis regulation in the infertile women might have a great value and let us to use them as predictors of endometrial dysfunctions to improve implantation rate in IVF program.

LITERATURE RESEARCH

In this study we review the data of the most important factors regulating apoptosis in normal endometrium at proliferative and secretory phase of cycle and in endometrium of women with compromised infertility, who participate in IVF protocols. The literature data concerning the role of apoptosis in the establishment of endometrial receptivity during the “window of implantation” and association of the parameters of endometrial apoptosis with the IVF treatment outcome also has been described. To this purpose we conducted a search at http://www.ncbi.nlm.nih.gov/pubmed/electronic database using the following keywords: (1) “endometrium and apoptosis”; (2) “endometrial receptivity”; (3) “endometrium and IVF”; (4) “implantation window”; (5) “endometrium and IVF success”; and (6) “endometrium, infertility, apoptosis”. A literature review of English-language papers published by May 2015 has been performed. The selection of articles was based on their titles and abstracts with subsequent analysis the texts of the related articles.

APOPTOSIS IN THE NORMAL ENDOMETRIUM

Endometrium is a mucosal inner layer of the myometrium[25]. Steroid hormones (estrogens, progesterone, androgens and glucocorticoids) tightly regulate endometrium function and morphological changes during menstrual cycle[25,26]. Noyes, Hertig and Rock had described these morphological changes in 1950[26]. These changes are characterized by the alteration of the cellular morphology and expression of some molecules on the cell membrane as well as a synthesis and production of different biologically active factors[26]. The dominant hormone within proliferative phase of the menstrual cycle is estrogen, the secretory phase is determined by high production of the progesterone[9]. There are several types of cells in the endometrial tissue, including luminal and glandular epithelial cells, stromal fibroblastic cells, immune cells and blood vessels[25]. The number, activity, structure and function of the endometrial tissue cells are significantly changed during the proliferative and secretory phases of menstrual cycle. It was established that endometrial cell proliferation and cell death are directly regulated by numerous factors, including cytokines, growth factors, proteolytic enzymes, transcriptional factors and different apoptosis-related factors as well[9,27,28].

The proliferative phase is associated with follicular growth and increased estrogen secretion, leading to endometrial reconstruction[29]. This phase of menstrual cycle is accompanied by the active cells proliferation and angiogenesis to provide the nutrition of the developing new endometrium[9]. All tissue components (glands, stromal and endothelial cells) show proliferation, with DNA nuclear and RNA cytoplasmic syntheses, which peak on days 8-10 of the cycle, reflecting maximum concentration of estrogen receptors in the endometrium[29]. It was demonstrated that insulin-like growth factors (IGF), vascular endothelial growth factor, epidermal growth factor (EGF) system, transforming growth factor (TGF)-α and amphiregulin are highly expressed in endometrium during proliferative phase[9,30,31]. The action of all these factors is directed towards the tissue proliferation, and very little apoptosis was detected in the endometrium during this period[32]. It was supposed that the high level of Bcl-2 expression by the glandular epithelial cells leads to the inhibition of apoptosis in the endometrial tissue during proliferative phase of cycle[32-34]. Bcl-2 as apoptosis inhibitor reduces oxygen free radicals, blocks the intracellular Ca²⁺ influx into cell organelles, decreases p53-dependent apoptosis, and antagonizes c-Myc[35-37]. High level of Bcl-2 expression likely mediates the reduction of mitochondrial-induced apoptosis of the endometrial epithelial cells and leads to proliferation of endometrial cells, thereby making the endometrial tissue thicker[33,34]. Estrogen increases the cellular proliferation during the proliferative phase of menstrual cycle due to inhibition of the tumor suppressor gene phosphatase and tensin homolog (PTEN)[9]. This gene increases the apoptosis of endometrial cells by down-regulating the Akt-depending signal pathway[38].

Progesterone plays the main role in the secretory endometrial transformation during the second half of menstrual cycle, suppressing proliferation and inducing cell differentiation[39]. After ovulation, the granulosa cells undergo luteinization and form part of the corpus luteum, which secretes progesterone[40]. The influence of the progesterone on epithelial cell proliferation is proved by the observation that progesterone completely inhibits estrogen-induced DNA synthesis and suppresses cell proliferation[41]. Progesterone also antagonizes the stimulatory action of many oncogenes that are likely to mediate estrogen-induced growth[29]. Progesterone inhibits the estrogen regulation of cyclin D1 nuclear translocation resulting in the hypophosphorylation of Rb and p107 proteins and a block in G1-S phase development[42]. The inhibitory action of progesterone on Akt-depended signal pathway with subsequent suppression of cells proliferation was also demonstrated[43].

The apoptosis in the normal secretory endometrium is proposed to promote elimination of the senescent or dysfunctional cells and to provide the tissue repair at each menstrual cycle[44]. The abundance of the apoptotic cells is maximal in the glandular epithelium and much less in the stroma[44]. It was demonstrated that different apoptosis-related factors are involved in the regulation of secretory endometrial cell transformation. It was found that after entering the secretory phase, Bcl-2 expression in endometrium is declined[32,33]. At the same time the increase of the proapoptotic factor BAX expression was noted in the endometrium at this phase of cycle[33]. The function of BAX is opposite to that of Bcl-2. It can accelerate the apoptosis and the ratio of Bcl-2: BAX defines if a cell lives or dies[33]. The BAX proteins form BAX/BAX homodimers, which lead to cytochrome C release and caspases activation with subsequent induction of the endometrial epithelial cells apoptosis[45,46]. It was also shown that endometrial stromal cells apoptosis in the secretory phase of cycle was increased due to caspase activity, based on the up-regulation of specific receptors Fas and TRAIL-R2[47]. Different expression of DNA fragmentation factor of 45 kDa (DFF45) was found in endometrium during the menstrual cycle. In normal endometrium, a lowest DFF45 expression was detected in the late proliferative phase secretory endometrium and maximal endometrial tissue staining for DFF45 was observed in an early secretory phase of the menstrual cycle[48-50]. Much more DFF45-positive cells were found in the endometrial glands in comparison to stroma, irrespective of menstrual cycle phase[48,49]. It is known that the DNA damage during apoptosis is a consequence of activation of the DFF40/DFF45 complex. DFF40 (DNA fragmentation factor of 40 kDa) causes the direct DNA fragmentation while DFF45 acts as a DFF40 inhibitor and as its chaperone[49]. Therefore, the DFF45 is required for adequate DFF40 synthesis and the high level of DFF45 expression in the secretory endometrium can reflect the high intensity of endometrial cells apoptosis. Recently it was shown that the immunoreactivity of the peroxisome proliferator-activated receptor gamma (PPARγ) protein was more pronounced in secretory-phase endometrium than in that the proliferative endometrium[51]. PPARγ agonists can trigger terminal differentiation, suppress cell proliferation, increase apoptosis, and inhibit inflammation in many cancer models, so the high level of PPARγ expression can mediate the induction of apoptosis in the secretory endometrial tissue. Immunoelectron microscopic study of Fas and FasL molecules expression demonstrated their different expression in human endometrial glandular cells during the menstrual cycle[52]. Fas and FasL were found mostly on the Golgi apparatus during the late proliferative phase and on apical membranes and Golgi-transporting vesicles during the late secretory phase. Thus, it was shown that apoptosis of human endometrial glandular cells is suppressed by Bcl-2 expression on the proliferative phase and induced by the Fas/FasL system in an autocrine or paracrine way on the secretory phase[34].

Thus, literature data clearly demonstrate the tight association of endometrial apoptosis intensity with the hormonally-dependent changes of endometrial tissue morphology. The summarized data, concerning the expression of pro- and anti-apoptotic factors during different phases of the menstrual cycle, is presented in the Table 1.

Table 1 Regulation of apoptosis in normal endometrium during menstrual cycle.

Proliferative phase		Secretory phase
Increasedexpression	Inhibited expression	Increased expression	Inhibited expression
Bcl-2	p53	BAX	Bcl-2
Fas, FasL	PTEN	Fas, FasL
	PPARγ	TRAIL-R
	DFF45	PTEN
		Caspase-3
		PPARγ
		DFF 45

Bcl-2: B cell lymphoma/leukemia-2; PTEN: Phosphatase and tensin homolog; PPARγ: Peroxisome proliferator-activated receptor gamma; TRAIL: TNF-related apoptosis-inducing ligand; DFF45: DNA fragmentation factor of 45 kDa.

APOPTOSIS AND ENDOMETRIAL RECEPTIVITY

It is well known, that a short period of time during the mid- to late-secretory phase exhibits a highest readiness of endometrium for embryo implantation and this period is called as the “window of implantation”[9]. This period is extremely important for the preparation of endometrium to the acceptance of the implanted embryo. In humans the endometrium becomes susceptible to blastocyst implantation at 6-8 d after ovulation and remains susceptible for approximately 4 d (cycle days 20-24)[25]. Last years there have appeared a number of studies aimed at the identification of potential markers of receptivity[53]. These studied were devoted to the investigation of the molecular changes and apoptosis regulation that takes place in the endometrium during the “window of implantation”. It was shown that apoptosis in the human endometrium plays an essential role for endometrial receptivity and early implantation. An imbalance of pro- and anti-apoptotic events in the secretory endometrium seems to be involved in implantation disorders and consecutive pregnancy complications[47].

It was established that at the beginning of the implantation window the changes of apoptosis in endometrium become most intensive. Increase of apoptosis activity may have significance for the decidualization processes establishment in endometrium during the late secretory phase. It was shown that decidualization includes differentiation and apoptosis of epithelial as well as stromal cell compartments[53]. The morphological changes that characterize decidualization take place in the endometrium without respect to conception[9]. All the cells in the endometrium are influenced by these changes. Epithelial cells are undergoing glandular secretory transformation. Stromal cells decidualization is associated with production and secretion of numerous decidual proteins such as prolactin, IGF binding protein-1 (IGBP-1) and tissue factor[54,55]. Endometrium is infiltrated by a large amount of different types of immune cells, including NK, T-lymphocytes, and macrophages[9]. On the secretory phase, vascular remodeling takes place, with the main angiogenic mechanisms leading to coiling and intussusceptions of the spiral arteries[56]. All these changes prepare endometrium for the successful implantation of the blastocyst.

Successful implantation supposedly is associated with the apposition and attachment of the embryo to the endometrial epithelial cells and adequate invasion in the endometrial stroma[9]. It was established that several mechanisms facilitate the embryo adhesion. Pinopodes change the concentration of endometrial fluids at the implantation site[9]. Mucins, in particular MUC1, take part in the selection of implantation site, because MUC1 barrier on the luminal epithelium prevents the interactions between the embryo and the endometrium, but the blastocyst itself cleaves MUC1 and defines the most appropriate site of implantation[57]. Integrins, especially α_νβ₃, and their receptor osteoponinare are highly expressed in the endometrium at the period of the window of implantation[56]. The important role of leukaemia-inhibitory factor (LIF), matrix metalloproteinase’s family, TGF-β, colony stimulating factor-1, transcription factor home box gene HOXA10, cytokines IL-1, IL-11, IL-15 in implantation also has been clearly demonstrated[9,58,59]. But during this step of embryo attachment endometrial stroma is not susceptible to the apoptotic events[9].

Apoptosis evidently takes part in the regulation of the process of blastocyst invasion. It was shown that on the initial steps of implantation the uterine epithelium of the implantation chamber undergoes apoptosis by the influence of the interacting blastocyst in mouse model[60]. With progressing of implantation, the regression of decidual cells results in a restricted and coordinated invasion of trophoblast cells into the maternal compartment due to the balanced expression of BAX, Bcl-2 and caspase-9 proteins in the decidual compartment and the high level of caspase-3 synthesis in the apoptotic uterine epithelium[60]. It was also shown that apoptosis-inducing factor might play an important role during mouse embryo implantation[61]. Tumor suppressor p53 is important for embryonic implantation because of transcriptional up-regulation of uterine LIF. It was reported, that simultaneous activation of p53 and estrogen receptor α took place in the endometrial tissues during implantation to coordinately regulate LIF production[62].

So, apoptosis is crucial for embryo implantation, rescuing the endometrium from apoptosis in the apposition phase and then providing the successful invasion of blastocyst by locally induced cells death in the site of the embryo and endometrial surface contact. But it is obvious that many apoptosis-regulating factors directly involved in the interaction between the embryo and endometrium have not yet been studied both in healthy women and in the infertile patients.

APOPTOSIS IN THE ENDOMETRIUM OF INFERTILE WOMEN

It is well established that in women with certain gynecologic diseases, including endometriosis, tubal disease, and polycystic ovary syndrome (PCOS), endometrial receptivity is compromised, leading to infertility and pregnancy loss[58]. Supposedly, regulation of apoptosis in the endometrium of infertile women also is impaired. It was reported that the endometrial Wilms tumor suppressor gene (WT1) in fertile women is highly expressed during the period of the window of implantation, but the endometrial WT1 expression was decreased in patients with PCOS during the secretory phase of the cycle[63]. The group of women with PCOS also was characterized by the higher stromal expression of Bcl-2 and p27, lower expression of EGF receptor in comparison with the fertile women. It was found, that apoptosis level was significantly reduced in the endometrial samples of women with PCOS[63]. These changes can hinder the success of decidualization and endometrial receptivity in infertile patients with PCOS[63]. In another work the changes in the apoptosis-related genes expression in the endometrium during the window of implantation were demonstrated in PCOS patients[64]. Five apoptosis-associated genes (including Bcl-2) were up regulated and four (including FADD) were downregulated. Authors concluded that the diminishing of the cell apoptosis at the period of the window of implantation in PCOS patients can result in the reduced endometrial receptivity[64].

Another gynecological pathology - chronic endometritis also can compromise human fertility and lead to abnormal uterine bleeding, pain, and reproductive failures. In women with chronic endometritis the expressions of Bcl-2 and BAX in endometrium were up-regulated, while caspase-8 was down-regulated[65]. Possibly the altered expression of these genes in the endometrium can be responsible for the impaired endometrial receptivity and the presence of endometrial hyperplastic lesions in women affected by chronic endometritis[65].

The spontaneous apoptosis, which is regulated by Bcl-2, BAX, p53, during the window of implantation in women with unexplained infertility was studied In another work[66]. It was found, that reduced amount of apoptotic cells, weak immunoreactivity of p53 and strong immunoreactivity of Bcl-2 took place in the endometrium of infertile women compared with the fertile women. Authors suggest that this finding might be an important factor of defective implantation compromising fertility in this group of patients[66].

The changed endometrial microRNAs (miRNAs) expression profile was shown during the window of implantation for women with repeated implantation failure (RIF)[67]. In recent years miRNAs are intensively studied. Now miRNAs are considered as key posttranscriptional regulators. As members of small non-coding RNA family, miRNAs are transcribed from specific genes spread over multiple locations in all human chromosomes except the Y chromosome. The mature miRNAs incorporate into the RNA induced silencing complex and, through complementary binding to the 3′ UTR of specific target genes, post-transcriptionally regulate their expression[68]. It was identified 13 miRNAs, differentially expressed in endometrial samples of patients with RIF. Among these genes 10 miRNAs were more expressed (including miR 145, 23b and 99a) and 3 were less expressed[12]. These miRNAs participate in the regulation of p53 signaling and cell cycle pathways. It was also shown that during window of implantation in the natural menstrual cycle, endometrial miRNA 22 was considerably higher in patients with RIF in comparison with control group[12]. Simultaneously, experiments using animal models demonstrated that miRNA 22 up-regulation contributed to the inhibition of the embryo implantation in mice[69]. Thus, these promising results allow to conclude, that the RIF-associated miRNAs could be used as new candidates for diagnosis and treatment of embryo implantation failures[12].

Significant changes of endometrial apoptosis were noted in women with endometriosis. Now it is widely accepted that pathogenesis of endometriosis is directly connected with the resistance of eutopic endometrial cells to apoptosis-induced signals which enable endometrial cells to escape immunosurveillance in the peritoneal cavity and to be implanted and growing in ectopic location[28,32,70]. Numerous literature data support this hypothesis. About 20 years ago the high level of Bcl-2 expression was for the first time demonstrated in the endometrial stroma of women with endometriosis and later these data was confirmed by different groups[28,32,70,71]. It was also shown that increased expression of anti-apoptotic factors and decreased expression of pro-apoptotic factors took place in the eutopic endometrium from women with endometriosis compared with endometrium from healthy women[72]. Both mRNA and protein amounts of several members of IAP family with anti-apoptotic action (c-IAP1, c-IAP2, XIAP and Survivin) were more expressed in women with endometriosis then in healthy donors[73]. In the endometrium of women with ovarian endometriosis the lower level of DFF45 expression was observed then that in both normal eutopic proliferatory and secretory endometrium. These results also directly evidence in favor of reduced apoptosis in the endometrium of patients with endometriosis[48]. Our research group also studied the expression of some factors with pro- and anti-apoptotic action in the endometrium of infertile women with endometriosis. We have found that in the endometrium of women with endometriosis during “window of implantation” the level of XIAP mRNA expression was significantly higher than that in the endometrium of healthy women[74]. Evidently taking together all these data about the impairment of endometrial apoptosis can explain the severe deficiency of endometrial receptivity which was noted for patients with endometriosis[28,32].

We have studied the character of apoptosis in the endometrium of women with tubal factor infertility, because these patients represent the most numerous clinical group of women participating in IVF protocols, so the estimation of their endometrial receptivity is of grate clinical value for improvement of IVF success. We have studied 73 women with tubal factor infertility, which planned to participate in IVF program. In women with tubal factor of infertility the increased level of the expression of pro-apoptotic factor PTEN mRNA with simultaneously elevated expression of mRNAs of anti-apoptotic factors XIAP and HSP27 were noted comparing to that in healthy women[74]. It is well known that XIAP is one of the important inhibitors of apoptosis, which is able to bind with caspase-9, -2 and -7 and inactivate their activity[75]. The XIAP expression is strongly elevated in patients with different types of tumors and the high level of XIAP production is associated with the poor prognosis[75]. The elevated expression of HSP27 also effectively protects cells from apoptosis[76]. It was found that HSP27 interacts and inhibits components of both stress- and receptor-induced apoptotic pathways[76]. It was shown that HSP27 could prevent activation of caspases by direct sequestering cytochrome c released from mitochondria into cytosol[76]. As we have mentioned above, normally during the window of implantation the increase of apoptosis in endometrial tissue is noted, so the high level of the production of anti-apoptotic factors might be estimated as negative factor which can reduce the receptivity of endometrium of women with tubal factor infertility. But it must be noted that the synthesis of PTEN was considerably increased in endometrium of women with tubal factor infertility. The tumor suppressor PTEN is also known as mutated gene in multiple advanced cancer 1, was discovered independently by two groups in 1997[38]. Somatic mutations of PTEN were identified as a prevalent event in different type of tumors, including tumors of the endometrium, brain, skin and prostate[38]. PTEN is a non-redundant, evolutionarily conserved dual-specific phosphatase. PTEN is capable to remove phosphates from protein and lipid substrates[77]. The primary target of PTEN is the lipid second messenger intermediate PIP3 (phosphatidylinositol 3, 4, 5-trisphosphate). PTEN removes the phosphate from the tree-position of the inositol ring to generate PIP2 (phosphatidylinositol 4,5-bisphosphate) thereby preventing intracellular signaling through the PI3K/Akt pathway[77]. It is known that Akt is the main downstream effector of PI3K (phosphoinositide 3-kinase) signaling that can phosphorylate a wide range of substrates and, thus, activate cell growth, proliferation and survival[38,77]. Today it is well known that PTEN function affects different cellular processes such as cell-cycle progression, cell proliferation, apoptosis, aging, DNA damage response, angiogenesis, muscle contractility, and other[77]. Recently the participation of PTEN in trophoblast invasion and decidual regression during human pregnancy was demonstrated[78]. Taking into account these properties of PTEN we suggested that the high level of PTEN mRNA expression in the endometrium of patients with tubal factor infertility might be estimated as positive mechanism, which compensates the over expression of anti-apoptotic factors and facilitates the preparing of endometrium to the implantation[74].

Summarized data about the character of apoptosis regulation in the endometrium of women with infertility are present in the Table 2.

Table 2 Expression of apoptosis-related genes in the endometrium of women with fertility problems during window of implantation.

Clinical group	Studied gene	Character of expression	Ref.
Chronic endometritis	Bcl-2	Up-regulated	Yan et al[64]
	BAX	Up-regulated
	Caspase-8	Down-regulated
Unexplained infertility	Bcl-2	Strong immunoreactivity	Vatansever et al[66]
	p53	Weak immunoreactivity
RIF	miR 145, 23b,99a (involved in p53 signaling)	Over expressed	Revel[12]
PCOS	Bcl-2 and p27	Increased expression	Gonzalez et al[63]
	FADD	Down-regulated	Yan et al[64]
Tubal factor	PTEN, XIAP, HSP27	Increased expression	Antsiferova et al[74]
Endometriosis	Bcl-2	Up-regulated	Jones et al[71]; Harada[32]; Nasu et al[28]
	IAP-proteins (c-IAP1, c-IAP2, XIAP, Survivin)	Up-regulated	Antsiferova et al[74]; Uegaki et al[73]
	DFF45	Low level	Banas et al[48]

Bcl-2: B cell lymphoma/leukemia-2; RIF: Repeated implantation failure; PCOS: Polycystic ovary syndrome; FADD: Fas-associated death domain; PTEN: Phosphatase and tensin homolog; XIAP: X-linked inhibitors apoptosis proteins; DFF45: DNA fragmentation factor of 45 kDa.

ENDOMETRIAL APOPTOSIS AND IVF TREATMENT OUTCOME

Now it is evident that endometrial gene expression during receptive phase is associated with the IVF treatment outcome in infertile women. Recently the comparison of apoptosis-related genes expression in the endometrium of patients who achieved a successful pregnancy and those of patients who was not successful after at least two failed ICSI cycles was performed. And this comparative analysis showed a significant different expression of genes, involving in apoptosis, such as caspase-8, -10, FADD, APAF1, and ANXA4 in the endometrium of women with different ICSI outcome[79].

We also studied the relationship between the character of apoptosis-related genes expression in endometrium during window of implantation and successes of IVF treatment in women with tubal factor of infertility and with endometriosis. We found that in women with tubal factor of infertility implantation failures were associated with the lowest amount of PTEN mRNA expression in the endometrium[74]. These results evidence that this factor is essential for implantation and estimation of PTEN synthesis in the endometrium and can be used as the predictor of endometrial receptivity at least in infertile women with tubal factor. In group of women with endometriosis the pregnancy ongoing was achieved in those patients who initially had the minimal level of anti-apoptotic factors XIAP and HSP27 expression in the endometrium. So, it can be concluded that the high level of the activity of anti-apoptotic factors negatively influence the endometrial receptivity in infertile women with endometriosis. Probably the future investigations will allow us to identify the best receptive endometrial gene expression profile which can be used as an effective prognostic tool for IVF patients[79].

CONCLUSION

Normally, in secretory phase of menstrual cycle, namely during the window of implantation, the increase of tissue apoptosis is noted. Probably, this phenomenon is important for the establishment of endometrial receptivity and provides the adequate invasion of the implanted blastocyst in the endometrial stroma. Regulation of apoptosis is impaired in infertile women. In most cases the impairment of endometrial receptivity is associated with the decrease of apoptosis in endometrium during period of “window implantation”. The estimation of the activity of apoptosis in the endometrium during the window of implantation surely gives us the important information about the endometrial receptivity. The further investigations of this problem would likely let us to better understand the mechanisms of endometrial receptivity and to develop new predictors of IVF outcome.