**Biomarkers Related to Endometrial Receptivity and Implantation**

Mark P. Trolice1 and George Amyradakis2 *1Associate Professor, Department of Obstetrics & Gynecology, University of Central Florida School of Medicine, Orlando, FL Director, Fertility Center of Assisted Reproduction & Endocrinology, Winter Park, FL 2Resident Physician, Winnie Palmer Hospital for Women & Babies, Orlando, FL USA*

## **1. Introduction**

206 Advances in Embryo Transfer

Stephens L.E., Sutherland A.E., Klimanskaya I.V., Andreiux A., Meneses J., Pedersen R.A.,

failure and peri-implantation lethality. *Genes Dev,* Vol. 9, pp. 1883-1895 Surveyor G.A., Gendler S., Pemberton L., Das S., Chakraborty I., Pimental R. (1995).

Veeck L.L. (1999). *An atlas of human gametes and conceptuses*. New York, Parthenon, pp. 46-51 Vlahos N.F., Lipari C.W., Bankowski B. (2006). Effect of luteal-phase support on endometrial

Yeh, J.C., Hiraoka N. (2001). Novel Sulfated Lymphocyte Homing Receptors and Their

Zhou Y., Damsky C., Fisher S. (1997). Preeclampsia is associated with failure of human

endovascular invasion in this syndrome? *J Clin Invest*. Vol.99, pp. 2152-2164

*Endocrinology*, Vol. 136, pp. 3639-3647

4049

Vol.105, pp. 957-969

Damsky C.H. (1995). Deletion of beta 1 integrins in mice results in inner cell mass

Expression and steroid hormonal control of muc-1 in the mouse uterus.

L-selectin ligand expression after recombinant follicle-stimulating hormone and ganirelix acetate for in Vitro Fertilization. *Clin Endocrine Met*, Vol. 91(10), pp. 4043-

Control by a Corel Extension Beta 1,3-N,acetylglucosaminyltransferase. *Cell*

cytotrophoblasts to mimic a vascular adhesion phenotype: one cause of defective

Implantation is a process requiring the delicate interaction between the embryo and a receptive endometrium. This intricate interaction requires a harmonized dialogue between embryonic and maternal tissues. [Aghajanova et al., 2008; Simon et al., 2000] The three stages of implantation are: apposition, adhesion, and invasion. Apposition describes trophoblast cells adhering to the receptive endometrial wall. Adhesion to the basal lamina and stromal extracellular matrix occurs in the presence of specific hormones, cytokines, and adhesion molecules. Once the blastocyst is anchored to the endometrial wall, it will become enclosed by an outer layer of syncytiotrophoblast, and an inner layer of cytotrophoblast. As the syncytiotrophoblast erodes the endometrium, the blastocyst will burrow into it and implantation will occur. [Ganong, 2005] During the last few years, research pursues enhancing both the quality of the embryo as well as understanding the highly dynamic tissue of the endometrial wall. Despite morphological and chromosomal criteria to improve the quality of transferred embryos, implantation rates remain at 25-35%. [Boomsma &Macklon, 2006]

The priming of the endometrium to optimize the window of implantation phase has been a subject of interest for decades, and much work has gone into understanding the preparation and capability of the endometrial wall to create a hospitable environment for the interaction with the blastocyst. While an embryo factor accounts for one third one implantation failure, lack of uterine receptivity explains approximately two thirds of implantation failures. [Achache, 2006; Ledee-Bataille et al., 2002] The actions of numerous cytokines, hormones, immunoglobulins, and other factors, are all orchestrated into preparing the endometrium for implantation. The morphological changes towards a receptive endometrium have been described as early as 1950 by Noyes, Hertig, and Rock [Strowitzki, 2006] and occur under the control of the sexual steroid hormones estrogen, and progesterone; with estrogen being the determinant hormone in the proliferative phase and progesterone being the determinant hormone in the secretory phase

During the luteal implantation phase; corresponding to cycle days 20-24, or seven to nine days after ovulation, the endometrium is receptive to the oncoming blastocyst. [Goiran &

Biomarkers Related to Endometrial Receptivity and Implantation 209

adhesions between cells and extracellular components. [Ceydell, 2006] In addition, integrins participate in many physiologically important processes including embryological development, haemostasis, thrombosis, wound healing, immune and non-immune defense mechanisms and oncogenic transformation. Specifically, the αvβ3 integrin as well as its ligand osteoponin was positively detected by immunohistochemistry on the endometrial luminal epithelial surface, which first interacts with the trophoblast. [Achache & Revel, 2006; Apparao ET AL., 2001] The expression of the endometrial stromal integrins may be modulated by several factors and the expression of the αvβ3 integrin in the endometrial stroma was demonstrated to be stimulated by IL- α, IL- β and TNF-α. [Ceydell, 2006] Integrins have been proposed as markers for endometrial receptivity, and the αvβ3

Selectins are lectin like proteins and include E-, L-, and P-selectins, all of which were originally thought o be expressed solely by hemangioblast descendants. P- selectins are expressed on the surface of platelets, E- selectins are expressed on activated endothelial cells, and L-selectins are expressed on lymphocytes. Glycoproteins carrying oligosaccharide formations including CD34, GlyCAM-1, PSGL-1, podocalyxin, and endoglycan, are recognized by the selectin molecules. [Foulk et al., 2007] Selectins are responsible for the tether and roll mechanism on endothelial surfaces. Once leucocytes slow down and subsequently arrest, integrin activation triggers adhesion and transmigration through the vascular endothelium. [Torry et al., 2007] Recently, Genbacec et al. [Genbacev et al., 2003] have shown that hatched blastocysts expressed L-selectin and used this molecule to mediate its attachment to the luminal epithelial surface via MECA-79, its carbohydrate ligands, and related epitopes. [Foulk et al., 2007] Also, Foulk and Zdravkovic have shown that lack of expression of the L-selectin ligand MECA-79 in mid-luteal endometrial biopsies were

Heparin binding-epidermal growth factor (HB-EGF) interacts with the EGF receptor and belongs to the epidermal growth factor family. It has been shown that HB-EGF expression is low during the proliferative endometrial phase, attaining its highest measure immediately prior to the implantation window, suggesting that it may have a role during the blastocyst implantation process. [Cavagna & Mantese, 2003; Lessey et al., 2002] It has been suggested that HB-EGF promotes implantation and trophoblast invasion through paracrine/autocrine signaling as cells penetrate the stroma. HB-EGF has also been shown to inhibit apoptosis and induces an invasive trophoblast phenotype. The co-existence of HB-EGF and pinopodes has been investigated with electron microscopy and immunochemistry, and shows that the expression of HB-EGF is highest when fully developed pinopodes are present, supporting the role of HB-EGF in the implantation

Chorionic gonadotropin is one of the early embryonic secretions from the trophoblast cells of the pre-implantation embryo. This helps maintain the corpus luteum of pregnancy, and leads to the modifications in morphology and endometrial gene expression preparing for

glycoprotein particularly has been directly associated with implantation.

indicative or low or no chance of pregnancy. **Heparin binding-epidermal growth factor**

process. [Cavagna & Mantese, 2003, Stavreus et al., 2001]

**Chorionic gonadotropin and Notch 1**

implantation.

**L-selectin**

Mignot, 1999] Essential expression of proteins, cytokines, and peptides can be detected at this time and serve as biomarkers for maximal endometrial receptivity. [Singh & Aplin, 2009; Lessey et al., 2002] The detection and investigation of biochemical markers during the implantation phase is an area of research receiving much interest and may serve to establish future treatments to help maximize the effectiveness of assisted reproductive techniques (ART) in the near future. According to Zhu, biomarkers are those that are present in the endometrium during the implantation phase, close to the implantation site, and disappear thereafter. [Cavagna & Mantese, 2003] This chapter will discuss biomarkers and their role in the attachment and invasion process during the implantation phase.

## **2. Biomarkers**

## **HLA-G**

Human leukocyte antigen G (HLA-G) is a major histocompatability complex (MHC) class Ib gene thought to play an essential role in implantation by modulating cytokine secretion to maintain local immunotolerance and modulate cytokine secretion to control trophopblastic cell invasion. [Roussev & Coulam, 2007] At first, HLA-G was proposed as a protector against natural killer (NK)-cell-mediated cytolysis of target cells and to prevent allorecognition by maternal cytotoxic lymphocytes. Recently, it has been shown tha these proteins regulate immune cells including T cells, NK cells, and antigen-presenting cells. [Fournel, 2000] Due to its essential role in the implantation process, recent attention has been focused on HLA-G and its diagnostic and therapeutic clinical applications. This has included the evaluation of couples with recurrent miscarriages and the mutation of the HLA-G gene. Serum sHLA-G levels during pregnancy may in the future become a diagnostic tool for evaluation of successful implantation but has yet to be established. [Roussev & Coulam, 2007]

#### **Pinopodes**

Pinopodes are organelles shown to be present on the endometrial wall during the implantation phase. They have been detected by electron microscopy and are specific markers for uterine receptivity. Progesterone dependent, pinopodes are present 20-21 days into the luteal cycle. [Cavagna & Mantese, 2003] Their function has not fully been established, but pinopodes are thought to play a role in protecting the blastocyst from being swept by the cilia on the endometrial wallpromoting withdrawal of uterine fluid and facilitating molecular adhesion of the pinopodes with the blastocyst. The life span of fully developed pinopodes lasts no more than 48 hours suggesting a transient cell state. Following ovarian stimulation with clomiphene citrate and human chorionic gonadotropin (hCG), pinopodes formed a little earlier, on days 17 or 18 than in the natural state.. [Cavagna & Mantese, 2003] It is thus possible that ovarian stimulation and early pinopode formation may have a role in shifting the window of receptivity resulting in asynchrony between the endometrium and blastocyst thereby negatively influencing implantation rates with IVF.

#### **Integrins**

Integrins are a family of transmembrane glycoproteins, formed by the interaction of two different, non-covalently linked α and β subunits. [Achache & Revel, 2006] They are adhesion molecules which participate in cell-adhesions and have also shown to play part in adhesions between cells and extracellular components. [Ceydell, 2006] In addition, integrins participate in many physiologically important processes including embryological development, haemostasis, thrombosis, wound healing, immune and non-immune defense mechanisms and oncogenic transformation. Specifically, the αvβ3 integrin as well as its ligand osteoponin was positively detected by immunohistochemistry on the endometrial luminal epithelial surface, which first interacts with the trophoblast. [Achache & Revel, 2006; Apparao ET AL., 2001] The expression of the endometrial stromal integrins may be modulated by several factors and the expression of the αvβ3 integrin in the endometrial stroma was demonstrated to be stimulated by IL- α, IL- β and TNF-α. [Ceydell, 2006] Integrins have been proposed as markers for endometrial receptivity, and the αvβ3 glycoprotein particularly has been directly associated with implantation.

## **L-selectin**

208 Advances in Embryo Transfer

Mignot, 1999] Essential expression of proteins, cytokines, and peptides can be detected at this time and serve as biomarkers for maximal endometrial receptivity. [Singh & Aplin, 2009; Lessey et al., 2002] The detection and investigation of biochemical markers during the implantation phase is an area of research receiving much interest and may serve to establish future treatments to help maximize the effectiveness of assisted reproductive techniques (ART) in the near future. According to Zhu, biomarkers are those that are present in the endometrium during the implantation phase, close to the implantation site, and disappear thereafter. [Cavagna & Mantese, 2003] This chapter will discuss biomarkers and their role in

Human leukocyte antigen G (HLA-G) is a major histocompatability complex (MHC) class Ib gene thought to play an essential role in implantation by modulating cytokine secretion to maintain local immunotolerance and modulate cytokine secretion to control trophopblastic cell invasion. [Roussev & Coulam, 2007] At first, HLA-G was proposed as a protector against natural killer (NK)-cell-mediated cytolysis of target cells and to prevent allorecognition by maternal cytotoxic lymphocytes. Recently, it has been shown tha these proteins regulate immune cells including T cells, NK cells, and antigen-presenting cells. [Fournel, 2000] Due to its essential role in the implantation process, recent attention has been focused on HLA-G and its diagnostic and therapeutic clinical applications. This has included the evaluation of couples with recurrent miscarriages and the mutation of the HLA-G gene. Serum sHLA-G levels during pregnancy may in the future become a diagnostic tool for evaluation of successful implantation but has yet to be established.

Pinopodes are organelles shown to be present on the endometrial wall during the implantation phase. They have been detected by electron microscopy and are specific markers for uterine receptivity. Progesterone dependent, pinopodes are present 20-21 days into the luteal cycle. [Cavagna & Mantese, 2003] Their function has not fully been established, but pinopodes are thought to play a role in protecting the blastocyst from being swept by the cilia on the endometrial wallpromoting withdrawal of uterine fluid and facilitating molecular adhesion of the pinopodes with the blastocyst. The life span of fully developed pinopodes lasts no more than 48 hours suggesting a transient cell state. Following ovarian stimulation with clomiphene citrate and human chorionic gonadotropin (hCG), pinopodes formed a little earlier, on days 17 or 18 than in the natural state.. [Cavagna & Mantese, 2003] It is thus possible that ovarian stimulation and early pinopode formation may have a role in shifting the window of receptivity resulting in asynchrony between the endometrium and blastocyst thereby negatively influencing implantation rates with IVF.

Integrins are a family of transmembrane glycoproteins, formed by the interaction of two different, non-covalently linked α and β subunits. [Achache & Revel, 2006] They are adhesion molecules which participate in cell-adhesions and have also shown to play part in

the attachment and invasion process during the implantation phase.

**2. Biomarkers**

[Roussev & Coulam, 2007]

**Pinopodes**

**Integrins**

**HLA-G**

Selectins are lectin like proteins and include E-, L-, and P-selectins, all of which were originally thought o be expressed solely by hemangioblast descendants. P- selectins are expressed on the surface of platelets, E- selectins are expressed on activated endothelial cells, and L-selectins are expressed on lymphocytes. Glycoproteins carrying oligosaccharide formations including CD34, GlyCAM-1, PSGL-1, podocalyxin, and endoglycan, are recognized by the selectin molecules. [Foulk et al., 2007] Selectins are responsible for the tether and roll mechanism on endothelial surfaces. Once leucocytes slow down and subsequently arrest, integrin activation triggers adhesion and transmigration through the vascular endothelium. [Torry et al., 2007] Recently, Genbacec et al. [Genbacev et al., 2003] have shown that hatched blastocysts expressed L-selectin and used this molecule to mediate its attachment to the luminal epithelial surface via MECA-79, its carbohydrate ligands, and related epitopes. [Foulk et al., 2007] Also, Foulk and Zdravkovic have shown that lack of expression of the L-selectin ligand MECA-79 in mid-luteal endometrial biopsies were indicative or low or no chance of pregnancy.

#### **Heparin binding-epidermal growth factor**

Heparin binding-epidermal growth factor (HB-EGF) interacts with the EGF receptor and belongs to the epidermal growth factor family. It has been shown that HB-EGF expression is low during the proliferative endometrial phase, attaining its highest measure immediately prior to the implantation window, suggesting that it may have a role during the blastocyst implantation process. [Cavagna & Mantese, 2003; Lessey et al., 2002] It has been suggested that HB-EGF promotes implantation and trophoblast invasion through paracrine/autocrine signaling as cells penetrate the stroma. HB-EGF has also been shown to inhibit apoptosis and induces an invasive trophoblast phenotype. The co-existence of HB-EGF and pinopodes has been investigated with electron microscopy and immunochemistry, and shows that the expression of HB-EGF is highest when fully developed pinopodes are present, supporting the role of HB-EGF in the implantation process. [Cavagna & Mantese, 2003, Stavreus et al., 2001]

#### **Chorionic gonadotropin and Notch 1**

Chorionic gonadotropin is one of the early embryonic secretions from the trophoblast cells of the pre-implantation embryo. This helps maintain the corpus luteum of pregnancy, and leads to the modifications in morphology and endometrial gene expression preparing for implantation.

Biomarkers Related to Endometrial Receptivity and Implantation 211

progesterone induces calcitonin gene expression in the endometrium. [Cavagna & Mantese, 2003; Kumar, 1998] By immunoreactivity for calcitonin mRNA,calcitonin seems to be absent during the proliferative and ovulatory phase. This finding may be another reason to suspect that calcitonin may be a marker for uterine receptivity. Calcitonin controls calcium homeostasis by binding to specific receptors identifeied as CR1a and CR1b. [Sexton et al., 1993; Wang, 1998] CR1a receptors have been found to be present in murine oocytes and zygotes in low concentrations, but significant increase of this receptor was found in embryos between the 8 cell and blastocyst stage. Wang et al. have also shown blastocysts differentiate in vitro at an accelerated rate when treated with 10 nM calcitonin for 30 minutes. [Cavagna & Mantese, 2003; Wang, 1998] Though this seems to demonstrate the role of calcitonin in embryonic development, further studies will need to be conducted to show the definitive

As implantation takes place, the blastocyst needs access and connection to the maternal vascular system. For this to occur there needs to be an increase of vascular permeability at the site of implantation. [Chakraborty et al., 1996] Prostaglandins (PGs) are known to possess vasoactive factors,play a definitive role in ovulation, fertilization, and labor, and recently have shown to be crucial during the implantation process. [Achache & Revel, 2006,

Prostaglandins are eicosanoids consisting of four members, PGD2, PGE2, PGF2α, and prostacyclin (PGI2). These are generated by the action of two enzymes, cytosolic phospholipase A2 (cPLA2), and cyclooxygenase (COX). Song et al. have demonstrated female mice lacking the cPLA2 and COX enzymes are not able to produce PG, leading to significant implantation defects. cPLA2 knockout mice also exhibited pregnancy failures and small litter size, secondary to delayed implantation. Exogenous administration of PG was able to restore embryo implantation at the correct time. It is not clear whether diminished expression of PG prevents human fertility because mice lacking PG will be fertile but present with fine tuning details. Thus it is postulated a similar process in humans leading to delayed implantation could lead to early pregnancy loss. Further investigation on the role of PGs at the time of human implantation and its possible role in late-pregnancy

abnormalities needs to be further explored. [Achache & Revel, 2006, Song et al., 2006]

reproductive tract and in adult endometrium for adequate implantation events.

Homeobox genes HOXA-10 and HOXA-11 have been linked with endometrial receptivity. Mutations in these genes have lead to failure to achieve normal implantation in mice. [Cavagna & Mantese, 2003; Daftary & Taylor, 2001] Growth and development of the human endometrium have been linked with these genes, and shown to have significant upregulation in the mid-secretory phase correlating with the implantation window. Female mice with homozygous mutations in the HOXA-10 or HOXA-11 have been shown to be infertile due to endometrial factors. [Satokata et al., 1995] According to Benson et al., the HOXA-10 gene may be important during morphogenesis for proper patterning of the

In women with endometriosis, Taylor et al. observed HOX gene expression is altered resulting in endometrial molecular alterations resulting in decreased endometrial

role of calcitonin during implantation and development of the embryo.

**Prostaglandins**

Song et al., 2006]

**HOX genes**

The notch family of receptors mediates a highly conserved pathway that regulated differentiation and pro-survival signals from humans to varied species of invertebrates. [Afshar et al., 2007; Paria et al., 2002] Notch proteins are ligand-dependant transmembrane receptors that transduce extracellular signals responsible for cell-fate and differentiation throughout development. Notch signaling often restricts the differentiation fates of a cell, directing it to a specific cell fate in cooperation with other signals, while at the same time inhibiting differentiation toward an alternate fate and promoting survival. Evidence indicates that Notch signaling regulates all three branches of the fate cell decision tree; differentiation, cell cycle progression and apoptotic cell death. Recently Afshar et al. have shown the coexpression of αSMA and Notch 1, both arising from CG signaling, inhibits apoptosis of stromal cells during the establishment of pregnancy. Shedding of the uterine lining and the inability of the uterus to accept an embryo can be correlated with low expression of Notch 1. Survival of the uterine lining can be mediated by (h)CG supplementation or progesterone as they will induce the expression of Notch 1. [Afshar et al., 2007]

#### **Mucins**

Mucins are glycoproteins high in molecular weight, which contain at least 50% of carbohydrate O-linked to a theonine/serine rich peptide core. [Gendler et al., 1990] MUC-1 is a large glycoprotein with a molecular weight >250 kDa. [Achache & Revel, 2006] When highly expressed on a cell surface, MUC-1 produces a steric hindrance phenomenon interfering with cellular adhesion. Cell-cell and cell-matrix adhesions are inhibited in direct correlation to the length of the MUC-1 ectodomain. [Hilkens et al, 1992] The apical surface of most epithelial cells is protected by a thick glycocalyx composed mostly of mucins that are believed to protect the cell surface from pathological processes. In the endometrium, MUC-1 is probably the first molecule the blastocyst encounters on the endometrial wall before implantation. This interaction would seem to indicate the blastocyst might be deterred from the endometrial wall until a proper location is encountered for implantation. In mice, rats, and pigs it has been shown that MUC-1 is down-regulated during the window of receptivity and thus optimizing the interaction between blastocyst and uterine wall. Paradoxically in humans, it has been shown that MUC-1 is up-regulated during the pre-implantation period. Therefore, it was suggested that humans must have a mechanism to induce inhibitory factors to down-regulate the MUC-1 barrier. High progesterone levels apparently reduce MUC-1 levels, thus unmasking intracellular adhesion molecules (CAM) on the surface of the endometrium and increasing uterine receptivity. [Bowen et al., 1996] Immunohistochemistry and scanning electron microscopy have shown that the MUC1 epitope corresponds only to ciliated cells. But the surface of non-ciliated cells such as pinopods has not been correlated to MUC-1. It has been suggested that pinopodes are important in providing a MUC-1 free area for blastocyst implantation. It seems that even though MUC-1 appears to have negative effects on implantation, its upregulation and extension beyond the glycocalyx covering the endometrium suggest it may have a temporary role in directing the embryo to effective implantation. [Achache & Revel, 2006]

#### **Calcitonin**

Parafollicular cells of the thyroid release calcitonin in response to hypercalcemia to reduce calcium levels. Though its role remains to be determined, calcitonin is expressed in the human endometrium during the secretory phase with highest concentrations on luteal cycle days 19-21, coinciding with the implantation period. It has also been demonstrated progesterone induces calcitonin gene expression in the endometrium. [Cavagna & Mantese, 2003; Kumar, 1998] By immunoreactivity for calcitonin mRNA,calcitonin seems to be absent during the proliferative and ovulatory phase. This finding may be another reason to suspect that calcitonin may be a marker for uterine receptivity. Calcitonin controls calcium homeostasis by binding to specific receptors identifeied as CR1a and CR1b. [Sexton et al., 1993; Wang, 1998] CR1a receptors have been found to be present in murine oocytes and zygotes in low concentrations, but significant increase of this receptor was found in embryos between the 8 cell and blastocyst stage. Wang et al. have also shown blastocysts differentiate in vitro at an accelerated rate when treated with 10 nM calcitonin for 30 minutes. [Cavagna & Mantese, 2003; Wang, 1998] Though this seems to demonstrate the role of calcitonin in embryonic development, further studies will need to be conducted to show the definitive role of calcitonin during implantation and development of the embryo.

#### **Prostaglandins**

210 Advances in Embryo Transfer

The notch family of receptors mediates a highly conserved pathway that regulated differentiation and pro-survival signals from humans to varied species of invertebrates. [Afshar et al., 2007; Paria et al., 2002] Notch proteins are ligand-dependant transmembrane receptors that transduce extracellular signals responsible for cell-fate and differentiation throughout development. Notch signaling often restricts the differentiation fates of a cell, directing it to a specific cell fate in cooperation with other signals, while at the same time inhibiting differentiation toward an alternate fate and promoting survival. Evidence indicates that Notch signaling regulates all three branches of the fate cell decision tree; differentiation, cell cycle progression and apoptotic cell death. Recently Afshar et al. have shown the coexpression of αSMA and Notch 1, both arising from CG signaling, inhibits apoptosis of stromal cells during the establishment of pregnancy. Shedding of the uterine lining and the inability of the uterus to accept an embryo can be correlated with low expression of Notch 1. Survival of the uterine lining can be mediated by (h)CG supplementation or progesterone as

Mucins are glycoproteins high in molecular weight, which contain at least 50% of carbohydrate O-linked to a theonine/serine rich peptide core. [Gendler et al., 1990] MUC-1 is a large glycoprotein with a molecular weight >250 kDa. [Achache & Revel, 2006] When highly expressed on a cell surface, MUC-1 produces a steric hindrance phenomenon interfering with cellular adhesion. Cell-cell and cell-matrix adhesions are inhibited in direct correlation to the length of the MUC-1 ectodomain. [Hilkens et al, 1992] The apical surface of most epithelial cells is protected by a thick glycocalyx composed mostly of mucins that are believed to protect the cell surface from pathological processes. In the endometrium, MUC-1 is probably the first molecule the blastocyst encounters on the endometrial wall before implantation. This interaction would seem to indicate the blastocyst might be deterred from the endometrial wall until a proper location is encountered for implantation. In mice, rats, and pigs it has been shown that MUC-1 is down-regulated during the window of receptivity and thus optimizing the interaction between blastocyst and uterine wall. Paradoxically in humans, it has been shown that MUC-1 is up-regulated during the pre-implantation period. Therefore, it was suggested that humans must have a mechanism to induce inhibitory factors to down-regulate the MUC-1 barrier. High progesterone levels apparently reduce MUC-1 levels, thus unmasking intracellular adhesion molecules (CAM) on the surface of the endometrium and increasing uterine receptivity. [Bowen et al., 1996] Immunohistochemistry and scanning electron microscopy have shown that the MUC1 epitope corresponds only to ciliated cells. But the surface of non-ciliated cells such as pinopods has not been correlated to MUC-1. It has been suggested that pinopodes are important in providing a MUC-1 free area for blastocyst implantation. It seems that even though MUC-1 appears to have negative effects on implantation, its upregulation and extension beyond the glycocalyx covering the endometrium suggest it may have a temporary role in directing the embryo to effective

Parafollicular cells of the thyroid release calcitonin in response to hypercalcemia to reduce calcium levels. Though its role remains to be determined, calcitonin is expressed in the human endometrium during the secretory phase with highest concentrations on luteal cycle days 19-21, coinciding with the implantation period. It has also been demonstrated

they will induce the expression of Notch 1. [Afshar et al., 2007]

implantation. [Achache & Revel, 2006]

**Calcitonin**

**Mucins**

As implantation takes place, the blastocyst needs access and connection to the maternal vascular system. For this to occur there needs to be an increase of vascular permeability at the site of implantation. [Chakraborty et al., 1996] Prostaglandins (PGs) are known to possess vasoactive factors,play a definitive role in ovulation, fertilization, and labor, and recently have shown to be crucial during the implantation process. [Achache & Revel, 2006, Song et al., 2006]

Prostaglandins are eicosanoids consisting of four members, PGD2, PGE2, PGF2α, and prostacyclin (PGI2). These are generated by the action of two enzymes, cytosolic phospholipase A2 (cPLA2), and cyclooxygenase (COX). Song et al. have demonstrated female mice lacking the cPLA2 and COX enzymes are not able to produce PG, leading to significant implantation defects. cPLA2 knockout mice also exhibited pregnancy failures and small litter size, secondary to delayed implantation. Exogenous administration of PG was able to restore embryo implantation at the correct time. It is not clear whether diminished expression of PG prevents human fertility because mice lacking PG will be fertile but present with fine tuning details. Thus it is postulated a similar process in humans leading to delayed implantation could lead to early pregnancy loss. Further investigation on the role of PGs at the time of human implantation and its possible role in late-pregnancy abnormalities needs to be further explored. [Achache & Revel, 2006, Song et al., 2006]

#### **HOX genes**

Homeobox genes HOXA-10 and HOXA-11 have been linked with endometrial receptivity. Mutations in these genes have lead to failure to achieve normal implantation in mice. [Cavagna & Mantese, 2003; Daftary & Taylor, 2001] Growth and development of the human endometrium have been linked with these genes, and shown to have significant upregulation in the mid-secretory phase correlating with the implantation window. Female mice with homozygous mutations in the HOXA-10 or HOXA-11 have been shown to be infertile due to endometrial factors. [Satokata et al., 1995] According to Benson et al., the HOXA-10 gene may be important during morphogenesis for proper patterning of the reproductive tract and in adult endometrium for adequate implantation events.

In women with endometriosis, Taylor et al. observed HOX gene expression is altered resulting in endometrial molecular alterations resulting in decreased endometrial

Biomarkers Related to Endometrial Receptivity and Implantation 213

2006; Hilton, 1992] Proliferation, cell survival, and differentiation, are some of the autocrine and paracrine effects of LIF, and have led researchers into investigating its function in blastocyst development and implantation. A study by Stewart [Stewart, 1994] showed that female mice expressing homozygous LIF gene deficiency displayed failed embryo implantation. Further evidence of the importance of LIF was observed as LIF supplementation rescued embryo implantation in the previously affected mice. LIF expression was observed to reach maximum concentrations in the mid- to late-secretory phase. Endometrial biopsies have shown LIF mRNA expression on days 18 to 28 of the menstrual cycle with maximum expression on day 20. [Charnock-Jones et al., 1994] Infertile patients and those with repeated implantation failureshave been shown to have abnormal levels of LIF supporting the role of LIF as a fundamental element in the implantation process. [Achache & Revel, 2006] Preclinical and clinical trials have investigated the effects of recombinant human LIF (r-hLIF) in improving endometrial receptivity. [Brinsden et al., 2003] In light of the importance of LIF in the implantation process, r-hLIF could be an

Recently, Feroze-Zaidi el al. demonstrated that women with unexplained fertility or recurrent implantation failure after IVF showed an abnormal expression of the SGK1 gene in the luminal epithelial cells during the midsecretory receptive phase corresponding with the implantation window. [Fakhera et al., 2007] Regulation of epithelial Na+ channels (ENaCs) is known to be controlled by SGK1. Uterine fluid homeostasis could thus be directly influenced by SGK1 leading to decreased uterine receptivity and disruption of successful implantation. Differentiating human endometrial stromal cells (HESCs) also activate SGK1, which stimulates the expression of prolactin (PRL), a most important decidual marker gene.

FOXO proteins are known to be able to regulate genes involved in proaptotic properties, and also genes involved in differentiation, cell cycle arrest, DNA repair, and oxidative defenses. [Fakhera et al., 2007; Sunters et al., 2003] Phosphorylation of transcription factors regulating expression of FOXO proteins, are targeted by kinases including SGK1 which serve to inactivate such proteins. [Brunet et al., 2001; Rena et al., 2003] Increased activity of SKG1 in the midsecretory phase may disrupt implantation by disrupting normal activity of

IL-6 is a cytokine classically known to induce immunoglobulin production in activated B cells, but also found to display a wide variety of functions outside the B-lymphocyte system. IL-6 expression in the human endometrium has been detected with the highest levels corresponding to the luteal phase. [Achache & Revel, 2006] mRNA expression of IL-6 steadily increases during the mid- to late-secretory phase and then decreases again in the late-secretory phase. During the crucial window of implantation, immunoreactivity for IL-6 becomes markedly detectable. The epithelial and glandular cells are the areas where the protein is mostly pronounced, compared to the stroma. During the window of implantation, receptors for IL-6 can be found not only in the endometrium, but are also expressed in the blastocyst, suggesting the paracrine/autocrine role of IL-6 during the peri-implantation period. Experiments performed using mice with disrupted IL-6 genes have shown despite

ENAC-mediated Na+ and water transport or by interrupting focal apoptosis.

important tool in the near future to optimize endometrial receptivity.

**Serum-and Glucocorticoid-Regulated Kinase 1 (SGK1)**

[Brosens & Gellersen, 2003, 2006]

**Interleukin-6 (IL-6)**

receptivity. These observations further support the importance HOX gene expression may have during implantation process.

#### **Angiogenesis**

Vascular development at the maternal fetal interface is an essential component for successful implantation and development. Trophoblasts, natural killer cells, and other cell types are responsible for this development. Trophoblasts are well known to produce angiogenic growth factors. [Cross et al., 2002; Torry et al., 2007] Ungranulated uterine natural killer cells (uNK) precursors are recruited to the endometrium during the transition of the endometrium to the secretory phase. Progesterone allows the development of the preuNK into large granulated uNK cells. These appear to be present during the implantation phase and have a role in releasing cytokines responsible for angiogenesis in early pregnancy, and development of spiral arteriole formation as the pregnancy progresses. [Leonard et al., 2006] In vitro models of mice have suggested that progesterone serves to upregulate decidua IL-15, in turn serving as a main activator of uNK population. [Leede-Bataille et al., 2005]

Other cells such as B and T lymphocytes have also been implicated in angiogenesis during the early phases of pregnancy. B lymphocytes have been shown to express the c-Myc oncogene, which can induce angiogenesis by producing VEGF. [Ruddell et al., 2003] Vascular endothelial growth factor (VEGF) is a known angiogenic substance involved in the process of vascular proliferation. [Tammela et al., 2005] During the peri-implantation phases, certain VEGF receptors appear to be expressed and function to optimize blastocyst implantation by mediating vascular permeability. These are VEGFR-1, VEGFR-2, and NRP-1. [Halder et al., 2000; Torry et al., 2007] The function and expression of VEGF have shown to be pivotal for angiogenesis during the implantation process and early placental development. Disturbance of this process could lead to implantation failure and early pregnancy loss.

#### **Insulin like growth factor-II (IGF-II)**

Insulin-like growth factors along with their binding proteins are thought to be responsible for differentiation, endometrial growth, angiogenesis, and apoptosis. [Cavagna & Mantese, 2003] IGF-II in particular is a known mediator of trophoblast function and is required for suitable placental growth and transport function. [Herr et al., 2003] Trophoblasts havebeen shown to express IGF-II while vessels near the implantation site have similarly been shown to expresses IGF-II receptors indicating IGF-II may directly act as an angiogenic growth factor. [Torry et al., 2007] In mice, IGF-II has demonstrated its vessel proliferation potential by inducing angiogenic growth factors such as VEGF and proliferin. Insulin like growth factors are regulated by insulin-like growth factor binding proteins (IGFBP). [Cavagna & Mantese, 2003] Litcht et al. have shown that secretion of IGFBP-1 by the endometrium occurs approximately 10 days after the LH surge, correlating with the implantation window. [Licht et al., 2002] With IGFBP-1 being the predominant regulatory factor for IGF-II, it may play an important role in endometrial receptivity and implantation.

#### **Leukemia Inhibitory Factor (LIF)**

In 1992, Hilton demonstrated LIF to be a haemapoietic factor by its capability to stimulate macrophage differentiation of the mouse myeloid leukemia cell line. [Achache & Revel,

receptivity. These observations further support the importance HOX gene expression may

Vascular development at the maternal fetal interface is an essential component for successful implantation and development. Trophoblasts, natural killer cells, and other cell types are responsible for this development. Trophoblasts are well known to produce angiogenic growth factors. [Cross et al., 2002; Torry et al., 2007] Ungranulated uterine natural killer cells (uNK) precursors are recruited to the endometrium during the transition of the endometrium to the secretory phase. Progesterone allows the development of the preuNK into large granulated uNK cells. These appear to be present during the implantation phase and have a role in releasing cytokines responsible for angiogenesis in early pregnancy, and development of spiral arteriole formation as the pregnancy progresses. [Leonard et al., 2006] In vitro models of mice have suggested that progesterone serves to upregulate decidua IL-15, in turn serving as a main activator of uNK population. [Leede-

Other cells such as B and T lymphocytes have also been implicated in angiogenesis during the early phases of pregnancy. B lymphocytes have been shown to express the c-Myc oncogene, which can induce angiogenesis by producing VEGF. [Ruddell et al., 2003] Vascular endothelial growth factor (VEGF) is a known angiogenic substance involved in the process of vascular proliferation. [Tammela et al., 2005] During the peri-implantation phases, certain VEGF receptors appear to be expressed and function to optimize blastocyst implantation by mediating vascular permeability. These are VEGFR-1, VEGFR-2, and NRP-1. [Halder et al., 2000; Torry et al., 2007] The function and expression of VEGF have shown to be pivotal for angiogenesis during the implantation process and early placental development. Disturbance of this process could lead to implantation failure and early

Insulin-like growth factors along with their binding proteins are thought to be responsible for differentiation, endometrial growth, angiogenesis, and apoptosis. [Cavagna & Mantese, 2003] IGF-II in particular is a known mediator of trophoblast function and is required for suitable placental growth and transport function. [Herr et al., 2003] Trophoblasts havebeen shown to express IGF-II while vessels near the implantation site have similarly been shown to expresses IGF-II receptors indicating IGF-II may directly act as an angiogenic growth factor. [Torry et al., 2007] In mice, IGF-II has demonstrated its vessel proliferation potential by inducing angiogenic growth factors such as VEGF and proliferin. Insulin like growth factors are regulated by insulin-like growth factor binding proteins (IGFBP). [Cavagna & Mantese, 2003] Litcht et al. have shown that secretion of IGFBP-1 by the endometrium occurs approximately 10 days after the LH surge, correlating with the implantation window. [Licht et al., 2002] With IGFBP-1 being the predominant regulatory factor for IGF-II, it may

In 1992, Hilton demonstrated LIF to be a haemapoietic factor by its capability to stimulate macrophage differentiation of the mouse myeloid leukemia cell line. [Achache & Revel,

play an important role in endometrial receptivity and implantation.

have during implantation process.

**Angiogenesis**

Bataille et al., 2005]

pregnancy loss.

**Insulin like growth factor-II (IGF-II)**

**Leukemia Inhibitory Factor (LIF)**

2006; Hilton, 1992] Proliferation, cell survival, and differentiation, are some of the autocrine and paracrine effects of LIF, and have led researchers into investigating its function in blastocyst development and implantation. A study by Stewart [Stewart, 1994] showed that female mice expressing homozygous LIF gene deficiency displayed failed embryo implantation. Further evidence of the importance of LIF was observed as LIF supplementation rescued embryo implantation in the previously affected mice. LIF expression was observed to reach maximum concentrations in the mid- to late-secretory phase. Endometrial biopsies have shown LIF mRNA expression on days 18 to 28 of the menstrual cycle with maximum expression on day 20. [Charnock-Jones et al., 1994] Infertile patients and those with repeated implantation failureshave been shown to have abnormal levels of LIF supporting the role of LIF as a fundamental element in the implantation process. [Achache & Revel, 2006] Preclinical and clinical trials have investigated the effects of recombinant human LIF (r-hLIF) in improving endometrial receptivity. [Brinsden et al., 2003] In light of the importance of LIF in the implantation process, r-hLIF could be an important tool in the near future to optimize endometrial receptivity.

## **Serum-and Glucocorticoid-Regulated Kinase 1 (SGK1)**

Recently, Feroze-Zaidi el al. demonstrated that women with unexplained fertility or recurrent implantation failure after IVF showed an abnormal expression of the SGK1 gene in the luminal epithelial cells during the midsecretory receptive phase corresponding with the implantation window. [Fakhera et al., 2007] Regulation of epithelial Na+ channels (ENaCs) is known to be controlled by SGK1. Uterine fluid homeostasis could thus be directly influenced by SGK1 leading to decreased uterine receptivity and disruption of successful implantation. Differentiating human endometrial stromal cells (HESCs) also activate SGK1, which stimulates the expression of prolactin (PRL), a most important decidual marker gene. [Brosens & Gellersen, 2003, 2006]

FOXO proteins are known to be able to regulate genes involved in proaptotic properties, and also genes involved in differentiation, cell cycle arrest, DNA repair, and oxidative defenses. [Fakhera et al., 2007; Sunters et al., 2003] Phosphorylation of transcription factors regulating expression of FOXO proteins, are targeted by kinases including SGK1 which serve to inactivate such proteins. [Brunet et al., 2001; Rena et al., 2003] Increased activity of SKG1 in the midsecretory phase may disrupt implantation by disrupting normal activity of ENAC-mediated Na+ and water transport or by interrupting focal apoptosis.

#### **Interleukin-6 (IL-6)**

IL-6 is a cytokine classically known to induce immunoglobulin production in activated B cells, but also found to display a wide variety of functions outside the B-lymphocyte system. IL-6 expression in the human endometrium has been detected with the highest levels corresponding to the luteal phase. [Achache & Revel, 2006] mRNA expression of IL-6 steadily increases during the mid- to late-secretory phase and then decreases again in the late-secretory phase. During the crucial window of implantation, immunoreactivity for IL-6 becomes markedly detectable. The epithelial and glandular cells are the areas where the protein is mostly pronounced, compared to the stroma. During the window of implantation, receptors for IL-6 can be found not only in the endometrium, but are also expressed in the blastocyst, suggesting the paracrine/autocrine role of IL-6 during the peri-implantation period. Experiments performed using mice with disrupted IL-6 genes have shown despite

Biomarkers Related to Endometrial Receptivity and Implantation 215

maintainance of adherens junctions in epithelial cells. [Singh & Aplin, 2009] E-cadherin suppression is responsible for cell-cell adhesion dysfunction. Riethmacher et al. demonstrated that targeted mutation of the E-cadherin gene resulted in defective pre-

During the luteal phase, E-cadherin mRNA levels are significantly elevated and regulation seems to be mainly controlled by intracellular calcium levels. E-cadherin cytoskeletal organization and disassembly at the adherens junction are mediated by rising levels of calcium which work by acting on signaling pathways. In vitro studies have shown that calcitonin produces a transient rise in intracellular calcium levels, suppressing E-cadherin at cellular contact sites. These experiments were performed by Li et al. on

Calcitonin appears to be an important regulator of implantation. Progesterone acts to increase calcitonin levels, which in-turn acts to increase intracellular calcium thus regulating E-cadherin expression. E-cadherin then seems to serve two main functions of uterine receptivity: adhesiveness in the preliminary phases; and inactivation by the actions of progesterone and calcitonin in the secretory phase to allow epithelial cell disassociation and

Cyclins are known to control mitotic phase progression in cells. The G1 to S phase transition is controlled by the rate limiting step of Cyclin E, whereas prevention of the cell cycle progression is controlled by the p27 cyclin-dependent kinase inhibitor. [Kliman et al., 2006] While the plausible role of cyclin E involves proliferation, p27 is mostly responsible for differentiation. [Dubowy et al., 2003] Conbsistent with these actions, estrogen has positive regulatory effects on Cyclin E andprogesterone seems to induce a dominant p27 state. Cyclin E activity is present in the cytoplasm of epithelial cells whereas p27 activity is exclusively active in the nucleus. While present in the early phases of the mestrual cycle, Cyclin E reactivity seems to rapidly decrease after cycle day 19; this could be explained by its subsequent movement towards the nucleus where it binds to p27 thereby becoming

The Endometrial Function Test (EFT) is a means to assess Cyclin E by immunohistochemically staining endometrial biopsies using antibodies against Cyclin E; and also as a means to identify an abnormally developing endometrium. [Dubowy et al., 2003] EFT showing a persistence of Cyclin E was associated with glandular developmental arrest (GDA), and observed in women with infertility. The overexpression of Cyclin E seemed to indicate that cells were arrested at an earlier phase of the menstrual cycle, possibly due to a premature expression of p27. [Kliman et al., 2006] The development of the EFT associated with cyclin markers and their correlation to estrogen and progesterone could serve as an important tool in the near future to assess endometrial receptivity and the effects

CSF-1 is a haemopoietic growth factor inducing proliferation and differentiation of cells belonging to the mononuclear phagocytic lineage. Pollard et al. have demonstrated that

of exogenous hormone administration in infertile patients. [Kliman et al., 2006]

implantation development in mice.

implantation. [Achache & Revel, 2006]

**Colony Stimulating Factor-1 (CSF-1)**

cultured Ishikawa cells.

**Cyclin E and p27**

inactivated.

implantation , the growth and development of the blastocyst becomes compromised. [Achache & Revel, 2006; Salamonsen et al., 2000] This suggests that even though IL-6 may not be an essential element for implantation, the lack of its presence could still explain infertility in some cases. Recent findings of patients with recurrent abortions have shown that IL-6 endometrial m-RNA is suppressed in the mid-secretory phase, thus supporting the role and importance of IL-6 in infertility.

#### **Interleukin-1 (IL-1)**

IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra) are all included in the family of IL-1, and serve as pivotal mediators of the immunologic and inflammatory response. In past experiments with mice, knockout mice for IL-1 were still able to proceed with implantation, but of interest, mice who received intraperitoneal injections of the IL-1ra displayed blastocysts unable to implant on the endometrial wall. Simon attributed this to the down regulation of crucial integrins at the luminal epithelial surfaces by the IL-1ra. In humans, it has been observed that administration of IL-1 causes an increase of β3 expression in the culture media of EECs thereby optimizing blastocyst implantation. [Achache & Revel, 2006] Leptin has also been shown to increase integrin β3 expression. Interestingly enough, IL-1β acts in stimulating leptin secretion and up-regulating its Ob-R receptor in EECs. IL-1 RtI mRNA and protein have shown to be present in maximal levels during the luteal phase in the human epithelial endometrium, and expression of the IL-1 antagonist has been shown to be reduced during the period of the implantation window. This finding suggests that suppression of the IL-1 antagonist during this crucial period of implantation maximizes successful implantation. [Boucher et al., 2001]

In women with endometriosis, the levels of IL-1ra and IL-1α were found to be markedly increased when compared to control groups in the PF and serum, and may serve as an explanation of the pathogenesis and infertility in such patients. [Kondera-Anasz et al. 2005]

#### **Leptin**

Acting both at the endocrine and paracrine level, leptin has been associated with regulation of body weight and reproductive function. [Cervero et al., 2004] Leptin is the product of the OB gene. Studies with rodents have determined this ligand-receptor system to be necessary for implantation. Receptors associated with leptin include total leptin receptor (OB-RT), the long form (OB-RL), and HuB219.1 and HuB219.3 short isoforms found in the endometrium. Studies with mice expressing ob/ob mutations resulted in phenotypically obese and sterile mice. Exogenous leptin treatment was able to restore sterility in these mice, but food restriction was not, implicating leptin as a requirement for normal reproductive functioning. [Cervero et al., 2004] Additionally, leptin has also been shown to increase integrin β3 expression, an important ligand protein essential for endometrial receptivity and implantation. The leptin receptors OB-RT, OB-RL, HuB219.1, and HuB219.3 have all demonstrated maximal expression in the late luteal phase. [Achache & Revel, 2006]

#### Cadherins

Cadherins are responsible for calcium-dependent cell-to-cell adhesion mechanisms and belong to a group of glycoproteins divided into N-, P-, and E-cadherins, all displaying specific functions and tissue distributions. Of all the cadherins, E-cadherin is the most studied pertaining to implantation, is ubiquitous, and is believed to be responsible for maintainance of adherens junctions in epithelial cells. [Singh & Aplin, 2009] E-cadherin suppression is responsible for cell-cell adhesion dysfunction. Riethmacher et al. demonstrated that targeted mutation of the E-cadherin gene resulted in defective preimplantation development in mice.

During the luteal phase, E-cadherin mRNA levels are significantly elevated and regulation seems to be mainly controlled by intracellular calcium levels. E-cadherin cytoskeletal organization and disassembly at the adherens junction are mediated by rising levels of calcium which work by acting on signaling pathways. In vitro studies have shown that calcitonin produces a transient rise in intracellular calcium levels, suppressing E-cadherin at cellular contact sites. These experiments were performed by Li et al. on cultured Ishikawa cells.

Calcitonin appears to be an important regulator of implantation. Progesterone acts to increase calcitonin levels, which in-turn acts to increase intracellular calcium thus regulating E-cadherin expression. E-cadherin then seems to serve two main functions of uterine receptivity: adhesiveness in the preliminary phases; and inactivation by the actions of progesterone and calcitonin in the secretory phase to allow epithelial cell disassociation and implantation. [Achache & Revel, 2006]

## **Cyclin E and p27**

214 Advances in Embryo Transfer

implantation , the growth and development of the blastocyst becomes compromised. [Achache & Revel, 2006; Salamonsen et al., 2000] This suggests that even though IL-6 may not be an essential element for implantation, the lack of its presence could still explain infertility in some cases. Recent findings of patients with recurrent abortions have shown that IL-6 endometrial m-RNA is suppressed in the mid-secretory phase, thus supporting the

IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra) are all included in the family of IL-1, and serve as pivotal mediators of the immunologic and inflammatory response. In past experiments with mice, knockout mice for IL-1 were still able to proceed with implantation, but of interest, mice who received intraperitoneal injections of the IL-1ra displayed blastocysts unable to implant on the endometrial wall. Simon attributed this to the down regulation of crucial integrins at the luminal epithelial surfaces by the IL-1ra. In humans, it has been observed that administration of IL-1 causes an increase of β3 expression in the culture media of EECs thereby optimizing blastocyst implantation. [Achache & Revel, 2006] Leptin has also been shown to increase integrin β3 expression. Interestingly enough, IL-1β acts in stimulating leptin secretion and up-regulating its Ob-R receptor in EECs. IL-1 RtI mRNA and protein have shown to be present in maximal levels during the luteal phase in the human epithelial endometrium, and expression of the IL-1 antagonist has been shown to be reduced during the period of the implantation window. This finding suggests that suppression of the IL-1 antagonist during this crucial period of implantation maximizes

In women with endometriosis, the levels of IL-1ra and IL-1α were found to be markedly increased when compared to control groups in the PF and serum, and may serve as an explanation of the pathogenesis and infertility in such patients. [Kondera-Anasz et al. 2005]

Acting both at the endocrine and paracrine level, leptin has been associated with regulation of body weight and reproductive function. [Cervero et al., 2004] Leptin is the product of the OB gene. Studies with rodents have determined this ligand-receptor system to be necessary for implantation. Receptors associated with leptin include total leptin receptor (OB-RT), the long form (OB-RL), and HuB219.1 and HuB219.3 short isoforms found in the endometrium. Studies with mice expressing ob/ob mutations resulted in phenotypically obese and sterile mice. Exogenous leptin treatment was able to restore sterility in these mice, but food restriction was not, implicating leptin as a requirement for normal reproductive functioning. [Cervero et al., 2004] Additionally, leptin has also been shown to increase integrin β3 expression, an important ligand protein essential for endometrial receptivity and implantation. The leptin receptors OB-RT, OB-RL, HuB219.1, and HuB219.3 have all

demonstrated maximal expression in the late luteal phase. [Achache & Revel, 2006]

Cadherins are responsible for calcium-dependent cell-to-cell adhesion mechanisms and belong to a group of glycoproteins divided into N-, P-, and E-cadherins, all displaying specific functions and tissue distributions. Of all the cadherins, E-cadherin is the most studied pertaining to implantation, is ubiquitous, and is believed to be responsible for

role and importance of IL-6 in infertility.

successful implantation. [Boucher et al., 2001]

**Interleukin-1 (IL-1)**

**Leptin**

Cadherins

Cyclins are known to control mitotic phase progression in cells. The G1 to S phase transition is controlled by the rate limiting step of Cyclin E, whereas prevention of the cell cycle progression is controlled by the p27 cyclin-dependent kinase inhibitor. [Kliman et al., 2006] While the plausible role of cyclin E involves proliferation, p27 is mostly responsible for differentiation. [Dubowy et al., 2003] Conbsistent with these actions, estrogen has positive regulatory effects on Cyclin E andprogesterone seems to induce a dominant p27 state. Cyclin E activity is present in the cytoplasm of epithelial cells whereas p27 activity is exclusively active in the nucleus. While present in the early phases of the mestrual cycle, Cyclin E reactivity seems to rapidly decrease after cycle day 19; this could be explained by its subsequent movement towards the nucleus where it binds to p27 thereby becoming inactivated.

The Endometrial Function Test (EFT) is a means to assess Cyclin E by immunohistochemically staining endometrial biopsies using antibodies against Cyclin E; and also as a means to identify an abnormally developing endometrium. [Dubowy et al., 2003] EFT showing a persistence of Cyclin E was associated with glandular developmental arrest (GDA), and observed in women with infertility. The overexpression of Cyclin E seemed to indicate that cells were arrested at an earlier phase of the menstrual cycle, possibly due to a premature expression of p27. [Kliman et al., 2006] The development of the EFT associated with cyclin markers and their correlation to estrogen and progesterone could serve as an important tool in the near future to assess endometrial receptivity and the effects of exogenous hormone administration in infertile patients. [Kliman et al., 2006]

#### **Colony Stimulating Factor-1 (CSF-1)**

CSF-1 is a haemopoietic growth factor inducing proliferation and differentiation of cells belonging to the mononuclear phagocytic lineage. Pollard et al. have demonstrated that

Biomarkers Related to Endometrial Receptivity and Implantation 217

cadherins serve as adhesion molecules for nidation. This fine orchestration of biomarkers and timed events has lead scientists toward improved understanding of the endometrium and its role during implantation. During the last decades, many advances have been made to improve ovulation and the quality of embryos. While remarkable advances such as IVF and other ART have been achieved, scientists are starting to realize the importance of a "fertile ground" at the embryo-uterus interface. Current research leading to the better understanding of biomarkers and endometrial receptivity may lead to optimization of embryo implantation in the future. Screening for receptivity markers and treating patients accordingly may allow for increasing use of a single embryo transfer with IVF leading to fewer complications encountered from multiple gestations. Patients will in tandem benefit by avoiding high costs of recurrent ART treatments and emotional despair from failed procedures. Some physicians are already taking proactive approaches in assessing endometrial receptivity by assessing biomarkes such as integrins, cyclin E, p27, and recently, even genes from endometrial biopsies. Such screenings may be standard in the near future and may lead to favorable treatments with subsequent higher rates of

[1] Foulk RA, Zdravkovic T, Genbavec O, Prokobphol A. Expression of L-selectin ligand

[2] Torry DS, Leavenworth J, Chang M, Maheshwari V, Groesch K, Ball ER, Torry RJ. Angiogenesis in implantation. J Assist Reprod Genet 2007;24:303-315. [3] Roussev RG, Coulam CB. HLA-G and its role in implantation (review). J Assist Reprod

[4] Meyer WR, Castlebaum AJ, Somkutti S, Sagoskin AW, Doyle M, Harris JE, Lessey BA.

[5] Cheung W, Ng EH, Chung P. A randomized double-blind comparison of perifollicular

[7] Achache H, Revel A. Endometrial receptivity markers, the journey to successful embryo

[8] Diedrich K, Devroey P, Griesinger G. The role of the endometrium and embryo in human implantation. Human Reproduction Update 2007;13:365-377. [9] Afshar Y, Stanculescu A, Miele L, Fazleabas AT. The role of chorionic gonadotropin and

[10] Nikas G, Toner J, Jones H. Endometrial pinopodes indicate a shift in the window of

citrate at two different times. Human Reproduction 2002;17:2881-2884. [6] Fakhera F, Fusi L, Takano M, Higham J, Salker M, Goto T. Role and regulation of the

endometrium. Endocrinology 2007;148:5020-5029.

implantation. Human Reproduction Update 2006;6:731-746.

Notch 1 in implantation. J Assist Reprod Genet 2007;24:296-302.

receptivity in IVF cycles. Human Reproduction 1999;14:787-792.

MECA-79 as a predictive marker of human uterine receptivity. J Assist Reprod

Hydrosalpinges adversely affect markers of endometrial receptivity. Human

vascularity and endometrial receptivity in ovulatory women taking clomiphene

serum – and glucocorticoid-regulated kinase 1 in fertile and infertile human

successful implantation.

Genet 2007;24:316-321.

Genet 2007;24:288-295.

Reproduction 1997;12:1393-1398.

**5. References**

op/op mice with mutations in CSF gene displayed multiple skeletal defects and decreased implantation rates. Other studies have shown CSF-1 to also be an important factor when it comes to ovulation. Op/op mice compared to wild type mice showed significant lower follicular development and ovulation rates. It has been shown that women with lower preconceptional CSF-1 levels are more prone to recurrent abortions compared to women with higher preconceptional CSF-1 levels. [Cavagna & Mantese, 2003]

## **2. Clinical implications**

Continuing investigation into understanding and exploring new markers of endometrial receptivity remain a high priority in reproductive endocrinology. Recent studies performed by Haouzi and associates have found new genes expressed during the implantation window by the human endometrium. [Haouzi et al., 2009] This information along with knowledge of previously discussed biomarkers can lead investigators to a more thorough approach when performing endometrial biopsies during a natural cycle especially in patients who have had unsuccessful IVF cycles. The goal of such investigation is to better understand the requirements of a hospitable environment for blastocyst implantation. Such knowledge may decrease unsuccessful implantation and facilitate a single embryo transfer in a well known receptive environment during an IVF cycle.

## **3. Future applications**

With recent significant attention given to endometrial receptivity, it is with no surprise that new methods of investigating the endometrial factor are under investigation and may soon become routine when exploring causes for infertility. Recently performed studies have now started to analyze endometrial secretions prior to embryo transfers in IVF and IUI patients. [Boomsma et al., 2009] Recent research conducted by Boomsma and associates evaluated secretions of different cytokines including interleukins, tumor necrosis factor-α, macrophage migration inhibitory factor, eotaxin, monocyte chemotactic protein-1, and heparin-binding epidermal growth factor. [Boomsma et al., 2009] Such novel modalities may soon elucidate new therapies and treatments of defective endometrial receptivity.

## **4. Conclusion**

With the precisely timed roles of different cytokines, hormones, and immune regulatory mechanisms, implantation is an intricate process requiring the collaboration of synchronized timed events and chemical interactions. As previously discussed, the "window of implantation" corresponds to a short period of time between days 20 and 24 of the menstrual cycle when the endometrium becomes receptive to the oncoming blastocyst. During the first part of the menstrual cycle, estrogen is present as the predominant hormone causing endometrial cell proliferation. Progesterone secreted by luteinized follicles after ovulation in the latter phase of the menstrual cycle serves to induce cell differentiation.

Approximately five-six days after ovulation, the blastocyst will enter the uterine cavity in search of a well prepared endometrium for implantation. Biomarkers such as the ones previously discussed are vital to ensure this process is successful. Selectins and mucins play a role in leading the blastocyst to a receptive endometrium , while integrins and cadherins serve as adhesion molecules for nidation. This fine orchestration of biomarkers and timed events has lead scientists toward improved understanding of the endometrium and its role during implantation. During the last decades, many advances have been made to improve ovulation and the quality of embryos. While remarkable advances such as IVF and other ART have been achieved, scientists are starting to realize the importance of a "fertile ground" at the embryo-uterus interface. Current research leading to the better understanding of biomarkers and endometrial receptivity may lead to optimization of embryo implantation in the future. Screening for receptivity markers and treating patients accordingly may allow for increasing use of a single embryo transfer with IVF leading to fewer complications encountered from multiple gestations. Patients will in tandem benefit by avoiding high costs of recurrent ART treatments and emotional despair from failed procedures. Some physicians are already taking proactive approaches in assessing endometrial receptivity by assessing biomarkes such as integrins, cyclin E, p27, and recently, even genes from endometrial biopsies. Such screenings may be standard in the near future and may lead to favorable treatments with subsequent higher rates of successful implantation.

#### **5. References**

216 Advances in Embryo Transfer

op/op mice with mutations in CSF gene displayed multiple skeletal defects and decreased implantation rates. Other studies have shown CSF-1 to also be an important factor when it comes to ovulation. Op/op mice compared to wild type mice showed significant lower follicular development and ovulation rates. It has been shown that women with lower preconceptional CSF-1 levels are more prone to recurrent abortions compared to women

Continuing investigation into understanding and exploring new markers of endometrial receptivity remain a high priority in reproductive endocrinology. Recent studies performed by Haouzi and associates have found new genes expressed during the implantation window by the human endometrium. [Haouzi et al., 2009] This information along with knowledge of previously discussed biomarkers can lead investigators to a more thorough approach when performing endometrial biopsies during a natural cycle especially in patients who have had unsuccessful IVF cycles. The goal of such investigation is to better understand the requirements of a hospitable environment for blastocyst implantation. Such knowledge may decrease unsuccessful implantation and facilitate a single embryo transfer in a well known

With recent significant attention given to endometrial receptivity, it is with no surprise that new methods of investigating the endometrial factor are under investigation and may soon become routine when exploring causes for infertility. Recently performed studies have now started to analyze endometrial secretions prior to embryo transfers in IVF and IUI patients. [Boomsma et al., 2009] Recent research conducted by Boomsma and associates evaluated secretions of different cytokines including interleukins, tumor necrosis factor-α, macrophage migration inhibitory factor, eotaxin, monocyte chemotactic protein-1, and heparin-binding epidermal growth factor. [Boomsma et al., 2009] Such novel modalities may soon elucidate

With the precisely timed roles of different cytokines, hormones, and immune regulatory mechanisms, implantation is an intricate process requiring the collaboration of synchronized timed events and chemical interactions. As previously discussed, the "window of implantation" corresponds to a short period of time between days 20 and 24 of the menstrual cycle when the endometrium becomes receptive to the oncoming blastocyst. During the first part of the menstrual cycle, estrogen is present as the predominant hormone causing endometrial cell proliferation. Progesterone secreted by luteinized follicles after ovulation in the latter phase of the menstrual cycle serves to induce cell differentiation.

Approximately five-six days after ovulation, the blastocyst will enter the uterine cavity in search of a well prepared endometrium for implantation. Biomarkers such as the ones previously discussed are vital to ensure this process is successful. Selectins and mucins play a role in leading the blastocyst to a receptive endometrium , while integrins and

with higher preconceptional CSF-1 levels. [Cavagna & Mantese, 2003]

new therapies and treatments of defective endometrial receptivity.

**2. Clinical implications**

**3. Future applications**

**4. Conclusion**

receptive environment during an IVF cycle.

Biomarkers Related to Endometrial Receptivity and Implantation 219

[26] Yoo, Barlow, Mardon. Temporal ansd special regulation of expression of heparin-

[27] Stavreus E, Nikas G, Sahlin L, Eriksson H. Formation of pinopodes in human

[28] Paria BC, Reese J, Das SK, Dey SK. Deciphering the cross-talk of implantation: advances

[29] Artavanis-Tsakonas S, Matsuno K, Fortini ME. Notch signaling. Science 1995;268:225-

[30] Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal

[31] Gendler SJ, Lancaster CA, Taylor-Papadimitriou, Duhig J, Peat N, Lalani EN. Molecular

[32] Hilkens J, Ligtenberg MJ, Vos HL, Litvinov SV. Cell membrane associated mucins and their adhesion-modulating property. Trends Biochem Sci 1992;17:359-363. [33] Hey NA, Li TC, Devine PL, Graham RA. MUC1 in secretory phase endometrium:

[34] Bowen JA, Bazer FW, Burghardt RC. Spacial and temporal analyses of intergrin and

[35] Kumar S, Zhu LJ, Polihronis M, Cameron ST, Baird ST, Dua A. Progesterone induces

[36] Sexton PM, Houssami S, Hilton JM, Center RJ. Identification of brian isoforms of the rat

[37] Wang UK, Rout UK, Bagchi IC, Armant DR. Expression of calcitonin receptors in mouse

[38] Chakraborty I, Das SK, Wang J. Developmental expression of the cyclo-oxygenase-1 and

[39] Song H, Lim H, Paria BC, Matsumoto H, Swift LL, Morrow J, Bonventre JV, Dey SK.

[40] Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of

[41] Ma L, Benson GV, Lim H, Dey SK, Maas RL. Abdominal B (AbdB) Hoxa genes:

cloning and expression of human tumor-associated polymorphic epithelial mucin. J

expression in precisely dated biopsies in flushings from normal and recurrent

Muc-1 expression in porcine uterine epithelium and trophectoderm in vivo. Biol

calcitonin gene expression in human endometrium within the putative window of

preimplantation embryos and their function in regulationnof blastocyst

cyclo-oxygenase-2 genes in peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids. J Mol Endicronlol 1996;16:107-

Cytosolic phospholipase A2alpha is crucial [correction of A2alpha deficiency is crucial] for 'on-time' embryo implantation that directs subsequent development.

regulation in adult uterus by estrogen and progesterone and repression in

possible role in blastocyst implantation. Dev Genet 1997;21:102-108.

progesterone receptors. Fertil Steril 2001;76:782-791.

integration in development. Science 1999;284:770-776.

miscarriage patients. Hum Reprod 1995;10:2655-2662.

implantation. J Clin Endocrinol Metab 1998;83:4443-4450.

differentiation by calcitonin. Development 1998;125:4293-4302.

calcitonin receptor. Mol Endocrinol 1993;7:815-821.

and challenges. Science 2002;296:2185-2188.

Biol Chem 1990;265:15286-15293.

Reprod 1996;55:1098-1106.

Development 2002;129:2879–2889.

pregnancy. N Engl J Med 1999;340:1796–1799.

232.

122.

binding epidermal growth factor-like rowth factor in the human endometrium: a

endometrium is associated with the concentrations of progesterone and

[11] Damario MA, Lesnick TG, Lessey BA, Mandelin E, Rosenwalks Z. Endometrial markers

[12] Cavagna M, Mantese JC. Biomarkers of endometrial receptivity – a review. Placenta

[13] Strowitzki T, Germeyer A, Popovici R, Wolf M. The human endometrium as a fertility-

[14] Cittadini E. Human implantation: the new frontiers of human assisted reproductive

[15] Boomsma CM, Macklon MS. What can the clinician do to improve implantation?

[16] Horcajadas JA, Pellicer A, Simón C. Wide genomic analysis of human endometrial

[17] Ceydell N, Kaleli S, Calay Z, Akbas F. Difference in αvβ3 integrin expression in

[18] Goiran D, Mignot TM. Embryo-maternal interactions at the implantation site: a delicate

[19] Lessey BA, Gui Y, Aparao SL. Regulated-expression of heparin binding EGF-like

[20] Zhu LJ, Polihronis M, Bagchi MK. Calcitonin is a progesterone-regulated marker that

[21] Fournel S, Huc X, Alam A. Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand-

[22] Martel D, Frydman R, Glissant M. Scanning electron microscopy of postovulatory

[23] Apparao KB, Murray MJ, Fritz MA, Meyer WR. Osteoponin and its receptor

[24] Alon R, Feigelson S. From rolling to arrest on blood vessels: leukocyte tap dancing on

[25] Genbacev OD, Prakobphol A, Foulk RA, Krtolica A, Ilic D, Singer MS, et al. Trophoblast

equilibrium. Eur J Obstet Gynecol Reprod Biol 1999;83:85-100.

during implantation. Mol Reprod Dev 2002;62:446-455.

receptivity: new times, new opportunities. Human Reproduction Update

endometrial stromal cell in subgroups of women with unexplained infertility. European Journal of Obstetrics & Gynecology and Reproductive Biology

growth factor (HB-EGF) in the human endometrium: a potential paracrine role

forecasts the receptive state of endometrium during implantation. Endocrinology

mediated apoptosis in activated CD8+ cells interacting with CD8. J Immunol

human endometrium in spontaneous cycles and cycles stimulated by hormone

alphavbeta(3) integrin are coexpressed in the human endometrium during the mestrual cycle but regulated differentially. J lin Endocrinol Metab 2001;86:4991-

endothelial integrin ligands and chemokines at sub-second contacts. Semin

L-selectin mediated adhesion at the maternal-fetal interface. Science 2003;299:405-

determining factor. Human Reproduction Update 2006;5:617-630.

technologies. Reproductive BioMedicine Online 2007;1;1-3

Reproductive BioMedicine Online 2006;13:845-855.

2001;16:1893-1899.

2003;24:39-47.

2007;13:77-86.

2006;126:206-211.

1998;139:3923-3935.

2000;164:6100-6104.

Immunol 2002;14:93-104.

5000.

408.

treatment. J Endocronl 1987;114:319-324.

of uterine receptivity utilizing the donor oocyte model. Human Reproduction

Biomarkers Related to Endometrial Receptivity and Implantation 221

[57] Brinsden PR, Ndukwe G, Engrand P, Pinkstone S, Lancaster S, Macnamee MC and De

[58] Brosens JJ, Gellersen B. Death or survival: progesterone-dependent cell fate decisions in

[59] Gellersen B, Brosens J. Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair. J Endocrinol 2003;178:357–372. [60] Sunters A, Fernandez de Mattos S, Stahl M, Brosens JJ, Zoumpoulidou G, Saunders CA,

[61] Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW, Burgering BM,

[62] Brunet A, Park J, Tran H, Hu LS, Hemmings BA, Greenberg ME. Protein kinase SGK

[63] Rena G, Woods YL, Prescott AR, Peggie M, Unterman TG, Williams MR, Cohen P. Two

[64] Woods YL, Rena G, Morrice N, Barthel A, Becker W, Guo S, Unterman TG, Cohen P.

[66] Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol

[67] Vandermolen DT, Gu Y. Human endometrial interleukin-6 (IL-6): in vivo messenger

[68] von Wolff M, Thaler CJ, Zepf C, Becker V, Beier HM, Strowitzki T. Endometrial

[69] Tabibzadeh S, Kong QF, Babaknia A, May LT. Progressive rise in the expression of

[70] Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, Kishimoto T, Zinkernagel R,

[71] Salamonsen LA, Dimitriadis E, Robb L. Cytokines in implantation. Semin Reprod Med

implantation window. Hum Reprod 1995;10,2793–2799.

interleukin-6-deficient mice. Nature 1994;368,339–342.

vitro, a novel in vivo phosphorylation site. Biochem J 2001;355:597–607. [65] Revel A, Helman A, Koler M, Shushan A, Goldshmidt O, Zcharia E, Aingorn H,

the human endometrial stroma. J Mol Endocrinol 2006;36:389–398.

Annual Meeting, 2003; Madrid, Spain.

p27(KIP1). Mol Cell Biol 2000;20:9138–9148.

FKHRL1 (FOXO3a). Mol Cell Biol 2001;21:952–965.

2003;278:49795–49805.\

EMBO J 2002;21:2263–2271.

IL-1 beta. Fertil Steril 1996;66,741–747.

Endocrinol 2002;16,121–129.

2005;83,580–586.

2000;18,299–310.

1993;54,1–78.

Moustier B. Does recombinant leukemia inhibitory factor improve implantation in women with recurrent failure of assisted reproduction treatment? O-050 ESHRE

Coffer PJ, Medema RH, Coombes RC, Lam EW. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem

Raaijmakers JA, Lammers JW, Koenderman L, Coffer PJ. Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of

mediates survival signals by phosphorylating the forkhead transcription factor

novel phosphorylation sites on FKHR that are critical for its nuclear exclusion.

The kinase DYRK1A phosphorylates the transcription factor FKHR at Ser329 in

Vlodavsky I. Heparanase improves mouse embryo implantation. Fertil Steril

ribonucleic acid expression, in vitro protein production, and stimulation thereof by

expression and secretion of interleukin-6 throughout the menstrual cycle. Gynecol

interleukin-6 in human endometrium during menstrual cycle is initiated during the

Bluethmann H, Kohler G. Impaired immune and acute-phase responses in

mullerian duct by the synthetic estrogen diethylstilbestrol (DES). Dev Biol 1998;197:141–154.

[42] Daftary GS, Taylor HS. Molecular markers of implantation: clinical implications. Curr

[43] Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes in Hoxa 10

[44] Benson GV, Lim H, Paria BC, Satokata I, Dey SK, Maas RL. Mechanisms of reduced

[45] Cross JC, Hemberger L, Lu Y, Nozaki T, Masutami M. Trophoblast functions,

[46] Leonard S, Murrant C, Tayade C, Heuvel M, Watering R, Croy BA. Mechanisms

[47] Leede-Bataille N, Bonnet-Chea K, Hosny G, Dubanchet S, Frydman R, Chaouat G. Role

[48] Ruddell A, Mezquita P, Brandvold KA, Farr A, Iritani BM. B-lymphocyte-specific c-Myc

[50] Halder JB, Zhao X, Soker S, Paria BC, Klagsbrun M, Das SK. Differentail expression of

[51] Herr F, Liang OD, Herrero J, Lang U, Preissner KT, Han VK, et al. Possible angiogenic

[52] Volpert O, Jackson D, Bouck N, Linzer DI. The insulin-like growth factor II/mannose 6-

[53] Licht P, Russu V, Lehmeyer J, Moll J, Wildt L. Intrauterine microdyalisis reveals cycle-

secretion by human chorionic gonadotropin. Fertil Steril 2002;78:252-258.

[55] Stewart CL. Leukaemia inhibitory factor and the regulation of pre-implantation development of the mammalian embryo. Mol Reprod Dev 1994;39:233–238. [56] Charnock-Jones DS, Sharkey AM, Fenwick P and Smith SK. Leukaemia inhibitory factor

[54] Hilton DJ. LIF: lots of interesting functions. Trends Biochem Sci 1992;17:72–76.

lymphatics during lymphomagenesis. Am J Pathol 2003;163:2233-45. [49] Tammela T, Enholm B, Alitalo K, Paavonen K. The biology of vascular endothelial

1998;197:141–154.

Opin Obstet Gynecol 2001;13:269–274.

deficient mice. Nature 1995;374:460–463.

Endocrinol 2002;187:207-212.

Genesis 2000;26:213-24.

1996;137:3871–6.

1994;101:421–426.

expression. Development 1996;12:2687–2696.

hypotheses-a review. Placenta 2006;27:S40-6.

transfer failure. Fertil Steril 2005;83:598-605.

growth factors. Cardiovasc Res 2005;65:550-63.

to pregnancy. J Clin Endocrinol Metab 2003;88:4811–7.

mullerian duct by the synthetic estrogen diethylstilbestrol (DES). Dev Biol

fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10

angiogenesis and remodeling of the maternal vasculature in the placenta. Mol Cell

regulating immune cell contributions to spiral artery modification-Facts and

of the endometrial tripod interleukin-18, -15, and -12 in inadequate uterine receptivity in patients with a history of repeated in vitro fertilization- embryo

expression stimulates early and functional expansion of the vasculature and

VEGF (164)-specific receptor neuropilin-1 in the mouse uterus suggests a role for VEGF (164) in vascular permeability and angiogenesis during implantation.

roles of insulin-like growth factor II and its receptors in uterine vascular adaptation

phosphate receptor is required for proliferin-induced angiogenesis. Endocrinology

dependent regulation of endometrial insulin-like growth factor binding protein-1

mRNA concentration peaks in human endometrium at the time of implantation and the blastocyst contains mRNA for the receptor at this time. J Reprod Fertil

Biomarkers Related to Endometrial Receptivity and Implantation 223

[89] Fujimoto J, Ichigo S, Hori M, Tamaya T. Alteration of E-cadherin, alpha- and beta-

[90] Gumbiner BM. Cell adhesion: the molecular basis of tissue architecture and

[91] Li Q, Wang J, Armant DR, Bagchi MK, Bagchi IC. Calcitonin down-regulates E-cadherin

[92] Kliman HJ, Honig S, Walls D, Luna M, Mc Sweet JC, Copperman AB. Optimization of

[94] Kliman HJ. The soil test for your endometrium: the endometrial function test (EFT).

[95] Stanley ER, Guilbert LJ, Tushinski RJ, Bartelmez SH. CSF-1-a mononuclear phagocyte lineage specific haemopoietic growth factor. J Cell Biochem 1983;21:151–159. [96] Pollard JW, Hunt JS, Wiktor-Jedrzejczak WQ, Stanley ER. A pregnancy defect in the

[97] Cohen E, Zhu L, Pollard JW. Absence of colony stimulating factor-1 in osteopetrotic (cs

[98] Daiter E, Pampfer S, Yeung YG, Barad G, Stanley ER, Pollard JW. Expression of colony

[99] Katano K, Matsumoto Y, Ogasawara M, Aoyama T, Ozaki Y, Kajiura S, Aoki. Low

[100] Shinetugs B, Runesson E, Bonello NP, Brannstrom M, Norman RJ, Colony stimulating

[101] Simon C, Martin JC and Pellicer A. Paracrine regulators of implantation. Baillieres Best

[102] Enders A. A morphological analysis of the early implantation stages in the rat. Am J

[103] Ledee-Bataille N, Lapree-Delage G, Taupin JL, Dubanchet S, Frydman R and Chaouat

highly predictive of embryo implantation. Hum Reprod 2002;17:213–218.

[104] Ganong WF. Review of Medical Physiology. 22nd Edition 2005:411-467.

cycle. Gynecol Endocrinol 1996;10,187–191.

morphogenesis. Cell 1996;84,345–357.

Fertility today 2006;spring:108-111.

fertility. Dev Biol 1991:148:273–283.

Reprod Immunol 1997;38:1–5.

Pract Res Clin Obstet Gynaecol 2000;14:815–826.

1997;56:110–118.

1992:74:850–858.

1999;14:1302–1306.

Anat 1967;125:1–29.

2002;277,46447–46455.

56.

catenin mRNA expression in human uterine endometrium during the menstrual

expression in rodent uterine epithelium during implantation. J Biol Chem

endometrial preparation results in a normal endometrial test (EFT) and good reproductive outcome in donor ovum recipients. Physiology 2006;23:299-303. [93] Dubowy R, Feinberg R, Keefe D, Doncel G, Williams S, McSweet J, Kliman HJ.

Improved endometrial assessment using cyclin E and p27. Fertil Steril 2003;80:146–

osteopetrotic (op/op) mouse demonstrates the requirement for CSF-1 in female

fmop/cs fmop) mice disrupts estrous cycles and ovulation. Biol Reprod

stimulating factor-1 in the human uterus and placenta. J Clin Endocrinol Metab

serum M-CSF levels are associated with unexplained recurrent abortion. Am J

factor-1 concentrations in blood and follicular fluid during the human menstrual cycle and ovarian stimulation: possible role in the ovulatory process. Hum Reprod

G. Concentration of leukaemia inhibitory factor (LIF) in uterine flushing fluid is

[72] Lim KJ, Odukoya OA, Ajjan RA, Li TC, Weetman AP, Cooke ID. The role of T-helper

[74] Simon C, Frances A, Piquette GN, el Danasouri I, Zurawski G, Dang W, Polan ML.

[75] Simon C, Gimeno MJ, Mercader A, O'Connor JE, Remohi J, Polan ML, Pellicer A.

[77] Simon C, Piquette GN, Frances A, Polan ML. Localization of interleukin-1 type I

[78] Boucher A, Kharfi A, Al-Akoum M, Bossu P, Akoum A. Cycle-dependent expression of

[79] Kondera-Anasz Z, Sikora J, Mielczarek-Palacz A, Jonca M. Concentrations of interleukin

[80] Cervero A, Horcajadas JA, Martin J, Pellicer A, Simon C. The leptin system during

[81] Houseknecht KL, Baile CA, Matteri RL, Spurlock ME. The biology of leptin: a review. J

[82] Holness MJ, Munns MJ, Sugden MC. Current concepts concerning the role of leptin in

[83] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–432. [84] Chehab FF, Lim ME, Lu R. Correction of the sterility defect in homozygous obese

[85] Mounzih K, Lu R, Chehab FF. Leptin treatment rescues the sterility of genetically obese

[86] Gumbiner BM. Cell adhesion: the molecular basis of tissue architecture and

[87] Huber O, Bierkamp C, Kemler R. Cadherins and catenins in development. Curr Opin

[88] Riethmacher D, Brinkmann V and Birchmeier C. A targeted mutation in the mouse E-

cadherin gene results in defective preimplantation development. Proc Natl Acad

reproductive function. Mol Cell Endocrinol 1999;157:11–20.

ob/ob males. Endocrinology 1997;138:1190–1193.

morphogenesis. Cell 1996;84,345–357.

Embryonic implantation in mice is blocked by interleukin-1 receptor antagonist.

Embryonic regulation of integrins beta 3, alpha 4, and alpha 1 in human endometrial epithelial cells in vitro. J Clin Endocrinol Metab 1997;82,2607–2616. [76] Gonzalez RR, Leavis P. Leptin upregulates beta3-integrin expression and interleukin-

1beta, upregulates leptin and leptin receptor expression in human endometrial

receptor and interleukin-1 beta in human endometrium throughout the menstrual

interleukin-1 receptor type II in the human endometrium. Biol Reprod 2001;65,890–

(IL)-1alpha, IL-1 soluble receptor type II (IL-1 sRII) and IL-1 receptor antagonist (IL-1 Ra) in the peritoneal fluid and serum of infertile women with endometriosis. Eur J

human endometrial receptivity and preimplantation development. J of Clin

female mice by treatment with the human recombinant leptin. Nat Genet

cytokines in human reproduction. Fertil Steril 2000;73,136–142.

[73] Dinarello CA. Biology of interleukin 1. FASEB J 1998;2,108–115.

epithelial cell cultures. Endocrine 2001;16,21–28.

cycle. J Clin Endocrinol Metab 1993;77,549–555.

Obstet Gynecol Reprod Biol 2005;123,198–203.

Endocrinol Metab 2004;89:2442-2451.

Anim Sci 1998;76:1405–1420.

1996;12:318–320.

Cell Biol 1996;8,685–691.

Sci USA 1995;92,855–859.

Endocrinology 1994;134,521–528.

898.

**16**

*Italy*

**Fertility Cryopreservation**

*2Department of Veterinarian Clinical Sciences*

*University of Naples Federico II*

*1Department of Biological Structures, Functions and Technology*

*3Department of Animal Sciences and Inspection of Food of Animal Origin*

Francesca Ciani1, Natascia Cocchia2, Luigi Esposito3 and Luigi Avallone1

The cryobiology is the science of low temperature biology. Fertility cryopreservation is a vital branch of reproductive science and involves the preservation of gametes (sperm and oocytes), embryos, and reproductive tissues (ovarian and testicular tissues) for use in assisted reproduction techniques (ART). The cryopreservation of reproductive cells is the process of freezing, storage, and thawing of spermatozoa or oocytes. It involves an initial exposure to cryoprotectants, cooling to subzero temperature, storage, thawing, and finally, dilution and removal of the cryoprotectants, when used, with a return to a physiological environment that will allow subsequent development. Proper management of the osmotic pressure to avoid damage due to intracellular ice formation is crucial for successful freezing

Management of non-cryopreserved reproductive cells (i.e., spermatozoa or oocytes) and tissues (i.e., testicular tissue or ovarian tissue) is problematic due to difficulties in donorrecipient synchronization and the potential for transmission of infectious pathogens, which cumulatively limits widespread application of these techniques. Cryopreserved cells and tissues can endure storage for centuries with almost no change in functionality or genetic information, making this storage a method highly attractive. Cryopreservation procedures are established on the basis of cellular physical characteristics in order to maintain viability and limit membrane damage that may occur during exposure to such non-physiological conditions as sub-zero temperatures, ice format, ion and high solute concentrations. Afterwards, there is a pressing need for the development of optimum cryopreservation methods for reproductive cells and tissues from many species. There are two major techniques for cryopreservation: freeze-thaw processes and vitrification. The major difference between them is the total avoidance of ice formation in vitrification. However, the biotechnology of the reproduction, although widely implemented, has generated protocols currently used to cryopreserve bovine sperm or oocytes, for example, that are still

ART provide an ensemble of strategies for preserving fertility in patients and commercially valuable or endangered species. Nevertheless, it is very difficult to successfully cryopreserve. Currently, there is a growing interest to understand the

suboptimal, and cannot readily be extrapolated to other species' gametes.

**1. Introduction**

and thawing procedure.