**2.1 Endometrial gene expression during the time of embryo implantation**

In a restricted period, called implantation window (IW), endometrium is most receptive for the embryo attachment. In humans IW is temporally confined to days 20-24 of menstrual

The Tissue Specific Role

of Estrogen and Progesterone in Human Endometrium and Mammary Gland 41

Nowadays the number of couples seeking for aid to achieve pregnancy is constantly

Ovarian stimulation and ovulation induction with gonadotrophin administration has been a success from the 1960s (Fowler and Edwards, 1957). Ovulation induction leads to multifollicular growth instead of a single follicle in natural cycles escalating possible successful fertilisation. Still, the general success rates for clinical pregnancies have stayed around 30- 40% for more than three decades (Department of Health and Human Services Centres for Disease Control and Prevention Report 2001). The focus to develop more effective ovarian stimulation protocols to increase the number of oocytes and embryos obtained from one cycle has by some means overlooked the relevance of supraphysiological levels of ovarian steroid hormones and their collateral effect on the endometrium (Simon et al., 2008). In modern IVF, drugs used to stimulate ovaries during the follicular phase include clomiphene citrate, urinary and recombinant gonadotrophins and gonadotrophin releasing hormone (GnRH) agonists and antagonists (Edwards et al., 2005). The usage of ovarian stimulating drugs often results in shorter luteal phase of the endometrium, which is therefore no longer synchronized with embryo development. The use of GnRH agonists may have a negative effect on implantation. Several studies observing endometrial biopsies from patients undergoing IVF treatment show 1-3 day advancement in endometrial development (Lass et al., 1998; Nikas et al 1999). The formation of pinopodes, considered as morphological markers for receptive endometrium, has also been shifted to day 17 or 18 compared to day 20 in normal cycle (Stavreus-Evers et al., 2001). Elevated concentrations of E2 and subtle P4 increases in the late follicular phase lead to modulated steroid hormone receptor profile (Papanikolau et al., 2005). Histological study has shown down-regulation of the ERs and PRs and pinopode expression in stimulated cycles compared to natural cycles (Develioglu et al., 1999). There is some evidence of a negative impact of supraphysiological steroid levels on endometrium because increased pregnancy rates have been observed in the presence of reduced production of serum E2. This explains the fact that there are higher pregnancy and implantation rates recorded for oocyte recipients versus donors who have only P4 support prior to embryo transfer (Check et al., 1995). A premature reduction in PRs in the early luteal phase has been found after ovarian stimulation. Horcajadas and colleagues have demonstrated that gene expression profiling of the endometrium is different between natural and controlled

increasing as at least every tenth couple requires infertility treatments.

ovarian stimulation cycles in the receptive phase (Horcajadas et al., 2008).

stimulation and response might have a beneficial effect on implantation potential.

**2.3 E2 and P4 endometriosis** 

There are ways to restore the length of luteal phase by stimulating corpus luteum with hCG or by supplementing the luteal phase with steroids, such as E2 and P4 (Smitz et al., 1992). Also, to overcome the side effects caused by high doses of drugs milder stimulation protocols have been developed (Olivennes et al., 2002; Nargund and Frydman, 2007; Pennings and Ombelet, 2007; Ubaldi et al., 2007). The evidence regarding a potentially negative effect of supraphysiological steroid levels on endometrial receptivity (Simon et al., 1995; Devroey et al., 2004), corpus luteum function (Fauser and Devroey, 2003; Beckers et al., 2006), oocyte and embryo quality (Valbuena et al., 2001; Baart et al., 2007) indicate that limited ovarian

The ovarian steroid hormones play also a central role in pathogenesis of several uterine disorders, including endometriosis, which is characterized by the presence of endometrial tissue outside the uterine cavity like the peritoneum and ovary. It has been shown that both

cycle (8-10 days after ovulation). During this time period corpus luteum induces high level of P4 and stable level of E2 expression. For successful pregnancy the apposition, adhesion and invasion of developing embryo is needed which can only happen if the endometrium is at the right developmental stage posessing a receptive atmosphere.

There is a certain group of women who repeatedly fail to achieve pregnancy in spite of good quality embryos transferred during IVF (*in vitro* fertilisation) treatments. This has led to the search for better solutions to improve implantation rates. In a molecular level embryo implantation is a dialog between blastocyst and receptive endometrium which is mediated by various growth factors, cytokines, lipid mediators, transcription factors and other putative molecules often regulated by steroid hormones. In recent years, numerous studies applying global gene expression analysis have found a wide range of genes up- or down regulated in human endometrium during the IW (Carson et al., 2002; Kao et al., 2002; Riesewijk et al., 2003; Horcajadas et al., 2004; Krikun et al., 2005; Mirkin et al., 2005; Simon et al., 2005; Punyadeera et al., 2005; Talbi et al., 2006; Horcajadas et al., 2008; Haouzi et al., 2009a,b; Altmäe et al., 2010). Each study has brought out candidate genes believed to be crucial in embryo implantation process but the overlap of potential marker genes between different publications has still remained relatively low. However, today there are already some biomarkers confirmed in separate studies which are pivotial during implantation process. For example, the most potential endometrial marker identified is leukemia inhibitory factor (LIF) and its importance has been proven in animal and human studies (*Stewart et al., 1994;* Arici et al., 1995; Steck et al., 2004). Unfortunately, the development of recombinant human LIF (r-fLIF) has not met the expectations of increasing implantation rates in infertile women (Brinsden et al., 2009). The localization of immune system related molecules like cytokines, IL-6 and IL-11, has been identified in endometrial cells and they have shown coincidental expression changes at the time of high levels of E2 and P4 (Tabibzadeh et al., 1995; Robertson et al., 2000; Vandermolen and Gu, 1996; Cork et al., 2001; Dimitriadis et al., 2000; von Rango et al., 2004). The two integrins, α4βl and αvβ3, appear to be good markers of the receptive endometrium in normal fertile women (Lessey et al., 1994). Recognized growth factors related to endometrial receptivity and implantation are transforming growth factor β (TGF-β), epidermal growth factor (EGF), heparin bindingepidermal growth factor (HB-EGF) and inlsulin like growth factor (IGF) (Jones et al., 2006b; Hofmann et al., 1991; Dadi et al., 2007; Lessey et al., 2002; Stavreus-Evers et al., 2002). Growth factors and their respective receptors have shown to enhance embryo development and improve implantation rates in IVF cycles (Kabir-Salmani et al., 2004).

It is more likely that there is no single molecule, which could solve the implantation issue and help patients with recurrent implantation failures. As a complex process implantation seems to depend on many factors, which influence the development of the embryo and endometrial dating in synchronized manner. Moreover, the individual differences and monthly cyclic changes of the regenerative tissue make the search for universal markers relevant to implantation complex.

### **2.2 The influence of the IVF treatment on endometrial receptivity**

Since the first announcement of successful IVF treatment in 1978 (Steptoe and Edwards, 1978) assessed fertilization procedures have been increasingly used world-wide. Based on the report by European Society of Human Reproduction and Embryology (ESHRE) in 2008, more than three million babies have been born with the help of IVF (ESHRE 2008).

cycle (8-10 days after ovulation). During this time period corpus luteum induces high level of P4 and stable level of E2 expression. For successful pregnancy the apposition, adhesion and invasion of developing embryo is needed which can only happen if the endometrium is

There is a certain group of women who repeatedly fail to achieve pregnancy in spite of good quality embryos transferred during IVF (*in vitro* fertilisation) treatments. This has led to the search for better solutions to improve implantation rates. In a molecular level embryo implantation is a dialog between blastocyst and receptive endometrium which is mediated by various growth factors, cytokines, lipid mediators, transcription factors and other putative molecules often regulated by steroid hormones. In recent years, numerous studies applying global gene expression analysis have found a wide range of genes up- or down regulated in human endometrium during the IW (Carson et al., 2002; Kao et al., 2002; Riesewijk et al., 2003; Horcajadas et al., 2004; Krikun et al., 2005; Mirkin et al., 2005; Simon et al., 2005; Punyadeera et al., 2005; Talbi et al., 2006; Horcajadas et al., 2008; Haouzi et al., 2009a,b; Altmäe et al., 2010). Each study has brought out candidate genes believed to be crucial in embryo implantation process but the overlap of potential marker genes between different publications has still remained relatively low. However, today there are already some biomarkers confirmed in separate studies which are pivotial during implantation process. For example, the most potential endometrial marker identified is leukemia inhibitory factor (LIF) and its importance has been proven in animal and human studies (*Stewart et al., 1994;* Arici et al., 1995; Steck et al., 2004). Unfortunately, the development of recombinant human LIF (r-fLIF) has not met the expectations of increasing implantation rates in infertile women (Brinsden et al., 2009). The localization of immune system related molecules like cytokines, IL-6 and IL-11, has been identified in endometrial cells and they have shown coincidental expression changes at the time of high levels of E2 and P4 (Tabibzadeh et al., 1995; Robertson et al., 2000; Vandermolen and Gu, 1996; Cork et al., 2001; Dimitriadis et al., 2000; von Rango et al., 2004). The two integrins, α4βl and αvβ3, appear to be good markers of the receptive endometrium in normal fertile women (Lessey et al., 1994). Recognized growth factors related to endometrial receptivity and implantation are transforming growth factor β (TGF-β), epidermal growth factor (EGF), heparin bindingepidermal growth factor (HB-EGF) and inlsulin like growth factor (IGF) (Jones et al., 2006b; Hofmann et al., 1991; Dadi et al., 2007; Lessey et al., 2002; Stavreus-Evers et al., 2002). Growth factors and their respective receptors have shown to enhance embryo development

at the right developmental stage posessing a receptive atmosphere.

and improve implantation rates in IVF cycles (Kabir-Salmani et al., 2004).

**2.2 The influence of the IVF treatment on endometrial receptivity** 

relevant to implantation complex.

It is more likely that there is no single molecule, which could solve the implantation issue and help patients with recurrent implantation failures. As a complex process implantation seems to depend on many factors, which influence the development of the embryo and endometrial dating in synchronized manner. Moreover, the individual differences and monthly cyclic changes of the regenerative tissue make the search for universal markers

Since the first announcement of successful IVF treatment in 1978 (Steptoe and Edwards, 1978) assessed fertilization procedures have been increasingly used world-wide. Based on the report by European Society of Human Reproduction and Embryology (ESHRE) in 2008, more than three million babies have been born with the help of IVF (ESHRE 2008). Nowadays the number of couples seeking for aid to achieve pregnancy is constantly increasing as at least every tenth couple requires infertility treatments.

Ovarian stimulation and ovulation induction with gonadotrophin administration has been a success from the 1960s (Fowler and Edwards, 1957). Ovulation induction leads to multifollicular growth instead of a single follicle in natural cycles escalating possible successful fertilisation. Still, the general success rates for clinical pregnancies have stayed around 30- 40% for more than three decades (Department of Health and Human Services Centres for Disease Control and Prevention Report 2001). The focus to develop more effective ovarian stimulation protocols to increase the number of oocytes and embryos obtained from one cycle has by some means overlooked the relevance of supraphysiological levels of ovarian steroid hormones and their collateral effect on the endometrium (Simon et al., 2008). In modern IVF, drugs used to stimulate ovaries during the follicular phase include clomiphene citrate, urinary and recombinant gonadotrophins and gonadotrophin releasing hormone (GnRH) agonists and antagonists (Edwards et al., 2005). The usage of ovarian stimulating drugs often results in shorter luteal phase of the endometrium, which is therefore no longer synchronized with embryo development. The use of GnRH agonists may have a negative effect on implantation. Several studies observing endometrial biopsies from patients undergoing IVF treatment show 1-3 day advancement in endometrial development (Lass et al., 1998; Nikas et al 1999). The formation of pinopodes, considered as morphological markers for receptive endometrium, has also been shifted to day 17 or 18 compared to day 20 in normal cycle (Stavreus-Evers et al., 2001). Elevated concentrations of E2 and subtle P4 increases in the late follicular phase lead to modulated steroid hormone receptor profile (Papanikolau et al., 2005). Histological study has shown down-regulation of the ERs and PRs and pinopode expression in stimulated cycles compared to natural cycles (Develioglu et al., 1999). There is some evidence of a negative impact of supraphysiological steroid levels on endometrium because increased pregnancy rates have been observed in the presence of reduced production of serum E2. This explains the fact that there are higher pregnancy and implantation rates recorded for oocyte recipients versus donors who have only P4 support prior to embryo transfer (Check et al., 1995). A premature reduction in PRs in the early luteal phase has been found after ovarian stimulation. Horcajadas and colleagues have demonstrated that gene expression profiling of the endometrium is different between natural and controlled ovarian stimulation cycles in the receptive phase (Horcajadas et al., 2008).

There are ways to restore the length of luteal phase by stimulating corpus luteum with hCG or by supplementing the luteal phase with steroids, such as E2 and P4 (Smitz et al., 1992). Also, to overcome the side effects caused by high doses of drugs milder stimulation protocols have been developed (Olivennes et al., 2002; Nargund and Frydman, 2007; Pennings and Ombelet, 2007; Ubaldi et al., 2007). The evidence regarding a potentially negative effect of supraphysiological steroid levels on endometrial receptivity (Simon et al., 1995; Devroey et al., 2004), corpus luteum function (Fauser and Devroey, 2003; Beckers et al., 2006), oocyte and embryo quality (Valbuena et al., 2001; Baart et al., 2007) indicate that limited ovarian stimulation and response might have a beneficial effect on implantation potential.
