Several therapeutic strategies have been proposed (Hazout et al., 2008). The most effective is of course to treat infections of the urogenital tract. The patient must also be aware of the harmful effects of lifestyle (smoking, obesity, exposure to heat ...) on the quality of his sperm. If possible, it may be beneficial to limit exposure to pollutants. In the presence of varicoceles, embolization or surgery improve sperm quality, not really in term of numeration or morphology but clearly in terms of fragmentation of DNA (Smit et al., 2010). Finally there are several studies that have examined the effect of antioxidant treatment semen quality and sperm DNA. While it is necessary to have new controlled-randomized studies, many

In conclusion, the study of sperm DNA has highlighted abnormalities in chromatin organization, in DNA double helix and in epigenetic marks that impact male fertility and sperm role during reproduction. Sperm DNA analyses enable new diagnosis in infertilities previously classified as of idiopathic origin. These problems of DNA damages appear to influence fertility in early pregnancy by causing failure of implantation or repeated miscarriages. New treatments begin to be developed, but need to be well evaluated in prospective controlled-randomized studies. Improved lifestyle, environment and antioxidant treatments could improve semen and sperm DNA quality. However the newly developed sperm selection techniques remain limited to the quality of semen used: if all the

Finally, in accordance with the conclusions of the Position Report published by the Working Group of the ESHRE Andrology (Barratt et al., 2010), several recommendations can be made. First, it is necessary to continue to develop basic research on the chromatin organization to better understand the causes and the nature of DNA damages. It is also important to standardize analysis techniques to allow a correct interpretation of results, and also to be able to compare the different studies. The development of animal models allowing studies on the long-term effects of sperm DNA damages and ARTs, and well conducted studies paralleling DNA damages and results in ARTs (with neonatal data) will help the clinician in the guidance of patients in treatment options. Finally, the long-term monitoring

Successful implantation requires a competent blastocyst able to cross-talk with the receptive endometrium. This process includes dynamic and coordinated changes at the intricate

consequently the DNA damage (Aitken et al., 2010; Thomson et al., 2009).

preliminary publications suggest beneficial effects (Agarwal and Sekhon, 2010).

sperm have an abnormal DNA, it becomes impossible to make a selection.

of children from ARTs should be systematized.

**4. Embryo**

sperm without problems of chromatin organization. In 2001 Bartoov and his team have associated their MSOME technique with ICSI to give birth to IMSI. Sperm is then observed and selected on a morphology basis at a 10000 times magnification, as compared to a conventional ICSI magnification of 200 to 400 times. In IMSI, a Nomarski differential interference contrast is used and allows a more precise evaluation of the classical strict criteria of sperm morphology (shape of the head, midpiece...), and of the presence at the head of surface abnormalities called vacuoles. These vacuoles vary in size and number between sperm an also between patients. Several teams have shown a correlation between a higher rate of vacuolated sperm and higher DNA damages (Franco et al., 2008; Garolla et al., 2008; Oliveira et al., 2010), and IMSI was presented as a potential solution for the selection of non-fragmented sperm in order to increase the implantation rate and to reduce the miscarriage rate (Antinori et al., 2008; Bartoov et al., 2003; Bartoov et al., 2002; Berkovitz et al., 2006a; Berkovitz et al., 2006b; Berkovitz et al., 2005). These date are however controversial and the question about the origin – fragmentation or decondensation – of the vacuoles remain open. Recently, different teams aimed to answer this question. They studied a specific population of sperm with large vacuoles (more than 13% of the head surface)(Boitrelle et al., 2011; Franco Jr et al., 2011; Perdrix et al., 2011) and came to the conclusion that their presence is correlated with abnormal DNA condensation.  On the other side, no positive correlation was demonstrated between these large vacuoles and DNA fragmentation (Boitrelle et al., 2011; Perdrix et al., 2011). So it seems now clear that the presence of large vacuoles reflects a disruption of sperm chromatin condensation. Other types of vacuoles also exist (particularly small vacuoles, sometimes numerous, with an area <4% of the surface of the head). Further studies are still needed to understand if these vacuoles also reflect DNA damages.

#### **3.5 Impact of environment on sperm DNA damages**

Numerous factors affect semen oxidative balance, sperm quality and DNA damages. Tobacco has an impact on oxidative stress, by increasing the presence of leukocytes in semen (Saleh et al., 2002; Soares and Melo, 2008). Pesticides provoke sperm DNA damages, especially in cases of occupational exposure, as well as pollutants such as heavy metals or polycyclic aromatic hydrocarbons in the air (Delbes et al., 2010; Perry, 2008; Somers and Cooper, 2009). Occupational exposure of testicles to heat also has deleterious effects on male fertility (Mieusset et al., 1987; Paul et al., 2008) as well as sporadic exposure (Rockett et al., 2001). The effects of obesity on semen quality are marked not only on the concentration of sperm, but also on DNA fragmentation (Kort et al., 2006), probably as a result of a high scrotal temperature and of an important hormonal disruption (Du Plessis et al., 2010). Mobile phone port to the belt, especially when using a hands-free kit (the emission of electromagnetic waves is more important during a call) could have an adverse effect on male fertility (Desai et al., 2009). Age has also an effect on sperm quality and in particular on the quality of genetic material (Sartorius and Nieschlag, 2010). It has been demonstrated by Singh (Singh et al., 2003) that the double-strand fragmentation increases with age. Finally, other factors such as high consumption of alcohol, dietary exposure to plasticizers, stress, poor diet, are potentially impacting the maintenance of the balance ROS / antioxidants in the semen (Tremellen, 2008).

sperm without problems of chromatin organization. In 2001 Bartoov and his team have associated their MSOME technique with ICSI to give birth to IMSI. Sperm is then observed and selected on a morphology basis at a 10000 times magnification, as compared to a conventional ICSI magnification of 200 to 400 times. In IMSI, a Nomarski differential interference contrast is used and allows a more precise evaluation of the classical strict criteria of sperm morphology (shape of the head, midpiece...), and of the presence at the head of surface abnormalities called vacuoles. These vacuoles vary in size and number between sperm an also between patients. Several teams have shown a correlation between a higher rate of vacuolated sperm and higher DNA damages (Franco et al., 2008; Garolla et al., 2008; Oliveira et al., 2010), and IMSI was presented as a potential solution for the selection of non-fragmented sperm in order to increase the implantation rate and to reduce the miscarriage rate (Antinori et al., 2008; Bartoov et al., 2003; Bartoov et al., 2002; Berkovitz et al., 2006a; Berkovitz et al., 2006b; Berkovitz et al., 2005). These date are however controversial and the question about the origin – fragmentation or decondensation – of the vacuoles remain open. Recently, different teams aimed to answer this question. They studied a specific population of sperm with large vacuoles (more than 13% of the head surface)(Boitrelle et al., 2011; Franco Jr et al., 2011; Perdrix et al., 2011) and came to the conclusion that their presence is correlated with abnormal DNA condensation.  On the other side, no positive correlation was demonstrated between these large vacuoles and DNA fragmentation (Boitrelle et al., 2011; Perdrix et al., 2011). So it seems now clear that the presence of large vacuoles reflects a disruption of sperm chromatin condensation. Other types of vacuoles also exist (particularly small vacuoles, sometimes numerous, with an area <4% of the surface of the head). Further

studies are still needed to understand if these vacuoles also reflect DNA damages.

Numerous factors affect semen oxidative balance, sperm quality and DNA damages. Tobacco has an impact on oxidative stress, by increasing the presence of leukocytes in semen (Saleh et al., 2002; Soares and Melo, 2008). Pesticides provoke sperm DNA damages, especially in cases of occupational exposure, as well as pollutants such as heavy metals or polycyclic aromatic hydrocarbons in the air (Delbes et al., 2010; Perry, 2008; Somers and Cooper, 2009). Occupational exposure of testicles to heat also has deleterious effects on male fertility (Mieusset et al., 1987; Paul et al., 2008) as well as sporadic exposure (Rockett et al., 2001). The effects of obesity on semen quality are marked not only on the concentration of sperm, but also on DNA fragmentation (Kort et al., 2006), probably as a result of a high scrotal temperature and of an important hormonal disruption (Du Plessis et al., 2010). Mobile phone port to the belt, especially when using a hands-free kit (the emission of electromagnetic waves is more important during a call) could have an adverse effect on male fertility (Desai et al., 2009). Age has also an effect on sperm quality and in particular on the quality of genetic material (Sartorius and Nieschlag, 2010). It has been demonstrated by Singh (Singh et al., 2003) that the double-strand fragmentation increases with age. Finally, other factors such as high consumption of alcohol, dietary exposure to plasticizers, stress, poor diet, are potentially impacting the maintenance of the balance ROS / antioxidants in

**3.5 Impact of environment on sperm DNA damages**

the semen (Tremellen, 2008).

Infection of the genitourinary tract is an important medical cause of DNA damages, as the influx of activated leukocytes provokes an increased production of free radicals. Systemic infections may also increase oxidative stress in sperm through an increase in the concentration of leukocytes in semen (HIV) (Umapathy et al., 2001), or via a systemic oxidative stress (Tremellen, 2008). Patients who underwent surgery to treat cryptorchidism still have a production of ROS and a sperm DNA fragmentation higher than those of fertile men (Smith et al., 2007). The presence of varicoceles also increases oxidative stress and DNA damage (Chen et al., 2004; Smith et al., 2006). Finally, there is a variety of iatrogenic origins of DNA damages. Cancer treatments (chemotherapy, pelvic radiotherapy) are generally detrimental to the integrity of sperm DNA (Tremellen, 2008). Concerning ARTs, various semen manipulations may affect sperm DNA. Centrifugation and cryopreservation increased oxidative stress, and consequently the DNA damage (Aitken et al., 2010; Thomson et al., 2009).

Several therapeutic strategies have been proposed (Hazout et al., 2008). The most effective is of course to treat infections of the urogenital tract. The patient must also be aware of the harmful effects of lifestyle (smoking, obesity, exposure to heat ...) on the quality of his sperm. If possible, it may be beneficial to limit exposure to pollutants. In the presence of varicoceles, embolization or surgery improve sperm quality, not really in term of numeration or morphology but clearly in terms of fragmentation of DNA (Smit et al., 2010). Finally there are several studies that have examined the effect of antioxidant treatment semen quality and sperm DNA. While it is necessary to have new controlled-randomized studies, many preliminary publications suggest beneficial effects (Agarwal and Sekhon, 2010).

In conclusion, the study of sperm DNA has highlighted abnormalities in chromatin organization, in DNA double helix and in epigenetic marks that impact male fertility and sperm role during reproduction. Sperm DNA analyses enable new diagnosis in infertilities previously classified as of idiopathic origin. These problems of DNA damages appear to influence fertility in early pregnancy by causing failure of implantation or repeated miscarriages. New treatments begin to be developed, but need to be well evaluated in prospective controlled-randomized studies. Improved lifestyle, environment and antioxidant treatments could improve semen and sperm DNA quality. However the newly developed sperm selection techniques remain limited to the quality of semen used: if all the sperm have an abnormal DNA, it becomes impossible to make a selection.

Finally, in accordance with the conclusions of the Position Report published by the Working Group of the ESHRE Andrology (Barratt et al., 2010), several recommendations can be made. First, it is necessary to continue to develop basic research on the chromatin organization to better understand the causes and the nature of DNA damages. It is also important to standardize analysis techniques to allow a correct interpretation of results, and also to be able to compare the different studies. The development of animal models allowing studies on the long-term effects of sperm DNA damages and ARTs, and well conducted studies paralleling DNA damages and results in ARTs (with neonatal data) will help the clinician in the guidance of patients in treatment options. Finally, the long-term monitoring of children from ARTs should be systematized.

#### **4. Embryo**

Successful implantation requires a competent blastocyst able to cross-talk with the receptive endometrium. This process includes dynamic and coordinated changes at the intricate

The Actors of Human Implantation: Gametes, Embryo, Endometrium 101

Subsequent transformation of spiral arteries is a 2-steps process: the first seems to be mediated to factors secreted by uNK and covers endothelial swelling and vascular smooth muscle cells (VSMC) loss of coherence (Harris, 2011); the second is linked to the loss of endothelium, the beak down of VSMC/elastic fibers and finally the incorporation into the vessel wall of extravillous trophoblast cells, embed in a fibrinoid-rich matrix (Carter and Pijnenborg, 2011). The consequences of these changes are the widening of the vessel lumen, highly increasing the supplying blood to the intervillous space. In the placenta formation as

Successful invasion is mediated through both maternal and embryo factors. Differentiation of cytotrophoblast to ST or EVCT is accurately controlled by transcription factors, hormones, growth factors, cytokines, and O2 level. Implantation, early embryo development and placentation processes take indeed place under a low oxygen environment during the first trimester of gestation. Many pregnancy disorders such as fetal growth restriction or preeclampsia are associated to the loss of invasiveness, with the extravillous trophoblast that does not reach the myometrium and the corresponding arterial segments retaining therefore their endothelium; whereas an excessive trophoblast invasion is associated with invasive mola, placenta accrete or choriocarcinoma (Lunghi et al., 2007). Pre-eclampsia is a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema. It resolves on placenta delivery. Placental hypoxia is likely to be responsible for the maternal vascular dysfunction, through the increased placental release of anti-angiogenic factors such as soluble receptors flt1 and endoglin, both binding vascular endothelium growth factor (VEGF), placental growth factor (PLGF) and transforming growth factor (TGF)beta1/3 in the

during implantation process, immunity and angiogenesis are closely related.

maternal circulation and causing endothelial dysfunctions (Lorquet et al., 2010).

all of which sharing the hCG β amino acide sequence (Banerjee and Fazleabas, 2011):

The first known human embryo specific signal is hCG which is produced by the embryo before implantation, since it has been detected in embryo supernatants as soon as day 2 postfertilization (Ramu et al., 2011). Human chorionic gonadotropin is the major pregnancy glycoprotein hormone, from the cystine knot cytokines superfamily, and is specifically secreted by trophoblast. It is classically well known from it action as corpus luteum progesterone production rescuer but many recent studies has evidenced more and more extragonadal actions. HCG is a non-covalently linked heterodimer composed of 2 subunits α (produced by cytotrophoblast) and β (produced by syncytium). Maternal concentration and glycosylation of hCG change throughout the pregnancy since 3 majors isoforms are described,

**4.2 Embryonic signals: Example of the embryo-specific hCG**

2009).

crossroad between endocrinology, immunity and angiogenesis. Implantation process is not easily accessible *in vivo* to the research for obvious ethical and technical reasons. The only way to evaluate it is to extend data derived from animal studies, from *in vivo* studies about endometrium and embryo separately or from *in vitro* endometrium-early blastocyst coculture. Despite extensive research in this field, the implantation process remains the black box of the reproduction, even in 2012. An important wastage of embryos is observed since the majority of blastocysts will perish before or around implantation (Macklon et al., 2002; Robertson et al., 2011; Teklenburg et al., 2010a). Implantation failure would come from an inadequate gamete/embryo quality, an erroneous cross-talk at the implantation site or an inadequate peri-conceptual environment. As recently demonstrated by Robertson et al., the peri-conceptual period and environment are critical for pregnancy success: the local cytokine network of the reproductive tract integrates environmental information and provides a signaling system programming both maternal receptivity and embryo development (Robertson et al., 2011). On the other hand, data from Kashiwagi (Kashiwagi et al., 2007) and from Bauersachs (Bauersachs et al., 2009) evidence that both embryo and endometrium are closely related and are actively influenced by each other, in order to favor pregnancy or, at contrary, to reject it.

Implantation is a 3-steps process including free-floating apposition of the blastocyst then adhesion between two epitheliums: endometrium and trophoblast, and finally, the finetuned process of trophoblastic invasion and differentiation. This materno-fetal dialogue is mediated through a broad array of molecules released at the implantation site both by trophoblast and/or endometrium. During implantation process, embryo and maternal tissues talk about immunity and angiogenesis, on an autocrine, paracrine and/or juxtacrine mood (Singh et al., 2011). In definitive, implantation process is the best example of a successfully tolerated graft and controlled tumor invasion.

#### **4.1 Trophoblast differentiation**

Placentation in Humans is characterized by the formation of a highly invasive hemochorial placenta accompanied with dramatical changes in the vasculature of the uterus. Differentiation of cytotrophoblast includes villous trophoblast ensuring exchanges at the maternal-fetal interface, and extravillous cytotrophoblast (EVCT) anchoring the placenta and participating to the vascular remodeling of the wall of spiral arteries. This deep trophoblastic invasion through the entire depth of the endometrium and the inner third of the myometrium is considered the hallmark of human pregnancy. Trophoblast deriving from trophectoderm, clothes the terminal villi (the outermost branches of the villous trees) that descend from the chorionic plate and fix to the basal plate by anchoring villi (Carter, 2011). The chorionic villi are then the structural and functional unit of the placenta, floating within the maternal blood present in the intervillous chambers. Villous cytotrophoblast that mantles the chorionic villi, aggregates and fuses to form the syncytiotrophoblast (ST), allowing efficient communication and signal exchanges. It ensures the endocrine function of placenta, releasing hormones involved in the homeostasis of pregnancy such as chorionic gonadotropin and placental lactogen. In the other hand, EVCT refers to the invading trophoblast that exits the villi and colonizes the uterine wall and spiral arteries, the lumen of which is plugged by trophoblastic cells during the first 8 weeks of gestation (Fournier et al., 2011).

crossroad between endocrinology, immunity and angiogenesis. Implantation process is not easily accessible *in vivo* to the research for obvious ethical and technical reasons. The only way to evaluate it is to extend data derived from animal studies, from *in vivo* studies about endometrium and embryo separately or from *in vitro* endometrium-early blastocyst coculture. Despite extensive research in this field, the implantation process remains the black box of the reproduction, even in 2012. An important wastage of embryos is observed since the majority of blastocysts will perish before or around implantation (Macklon et al., 2002; Robertson et al., 2011; Teklenburg et al., 2010a). Implantation failure would come from an inadequate gamete/embryo quality, an erroneous cross-talk at the implantation site or an inadequate peri-conceptual environment. As recently demonstrated by Robertson et al., the peri-conceptual period and environment are critical for pregnancy success: the local cytokine network of the reproductive tract integrates environmental information and provides a signaling system programming both maternal receptivity and embryo development (Robertson et al., 2011). On the other hand, data from Kashiwagi (Kashiwagi et al., 2007) and from Bauersachs (Bauersachs et al., 2009) evidence that both embryo and endometrium are closely related and are actively influenced by each other, in order to favor

Implantation is a 3-steps process including free-floating apposition of the blastocyst then adhesion between two epitheliums: endometrium and trophoblast, and finally, the finetuned process of trophoblastic invasion and differentiation. This materno-fetal dialogue is mediated through a broad array of molecules released at the implantation site both by trophoblast and/or endometrium. During implantation process, embryo and maternal tissues talk about immunity and angiogenesis, on an autocrine, paracrine and/or juxtacrine mood (Singh et al., 2011). In definitive, implantation process is the best example of a

Placentation in Humans is characterized by the formation of a highly invasive hemochorial placenta accompanied with dramatical changes in the vasculature of the uterus. Differentiation of cytotrophoblast includes villous trophoblast ensuring exchanges at the maternal-fetal interface, and extravillous cytotrophoblast (EVCT) anchoring the placenta and participating to the vascular remodeling of the wall of spiral arteries. This deep trophoblastic invasion through the entire depth of the endometrium and the inner third of the myometrium is considered the hallmark of human pregnancy. Trophoblast deriving from trophectoderm, clothes the terminal villi (the outermost branches of the villous trees) that descend from the chorionic plate and fix to the basal plate by anchoring villi (Carter, 2011). The chorionic villi are then the structural and functional unit of the placenta, floating within the maternal blood present in the intervillous chambers. Villous cytotrophoblast that mantles the chorionic villi, aggregates and fuses to form the syncytiotrophoblast (ST), allowing efficient communication and signal exchanges. It ensures the endocrine function of placenta, releasing hormones involved in the homeostasis of pregnancy such as chorionic gonadotropin and placental lactogen. In the other hand, EVCT refers to the invading trophoblast that exits the villi and colonizes the uterine wall and spiral arteries, the lumen of which is plugged by trophoblastic cells

pregnancy or, at contrary, to reject it.

**4.1 Trophoblast differentiation**

successfully tolerated graft and controlled tumor invasion.

during the first 8 weeks of gestation (Fournier et al., 2011).

Subsequent transformation of spiral arteries is a 2-steps process: the first seems to be mediated to factors secreted by uNK and covers endothelial swelling and vascular smooth muscle cells (VSMC) loss of coherence (Harris, 2011); the second is linked to the loss of endothelium, the beak down of VSMC/elastic fibers and finally the incorporation into the vessel wall of extravillous trophoblast cells, embed in a fibrinoid-rich matrix (Carter and Pijnenborg, 2011). The consequences of these changes are the widening of the vessel lumen, highly increasing the supplying blood to the intervillous space. In the placenta formation as during implantation process, immunity and angiogenesis are closely related.

Successful invasion is mediated through both maternal and embryo factors. Differentiation of cytotrophoblast to ST or EVCT is accurately controlled by transcription factors, hormones, growth factors, cytokines, and O2 level. Implantation, early embryo development and placentation processes take indeed place under a low oxygen environment during the first trimester of gestation. Many pregnancy disorders such as fetal growth restriction or preeclampsia are associated to the loss of invasiveness, with the extravillous trophoblast that does not reach the myometrium and the corresponding arterial segments retaining therefore their endothelium; whereas an excessive trophoblast invasion is associated with invasive mola, placenta accrete or choriocarcinoma (Lunghi et al., 2007). Pre-eclampsia is a pregnancy-specific syndrome characterized by hypertension, proteinuria and edema. It resolves on placenta delivery. Placental hypoxia is likely to be responsible for the maternal vascular dysfunction, through the increased placental release of anti-angiogenic factors such as soluble receptors flt1 and endoglin, both binding vascular endothelium growth factor (VEGF), placental growth factor (PLGF) and transforming growth factor (TGF)beta1/3 in the maternal circulation and causing endothelial dysfunctions (Lorquet et al., 2010).

#### **4.2 Embryonic signals: Example of the embryo-specific hCG**

The first known human embryo specific signal is hCG which is produced by the embryo before implantation, since it has been detected in embryo supernatants as soon as day 2 postfertilization (Ramu et al., 2011). Human chorionic gonadotropin is the major pregnancy glycoprotein hormone, from the cystine knot cytokines superfamily, and is specifically secreted by trophoblast. It is classically well known from it action as corpus luteum progesterone production rescuer but many recent studies has evidenced more and more extragonadal actions. HCG is a non-covalently linked heterodimer composed of 2 subunits α (produced by cytotrophoblast) and β (produced by syncytium). Maternal concentration and glycosylation of hCG change throughout the pregnancy since 3 majors isoforms are described, all of which sharing the hCG β amino acide sequence (Banerjee and Fazleabas, 2011):

The Actors of Human Implantation: Gametes, Embryo, Endometrium 103

the maternal-fetal interface, showing a suppressive phenotype, whereas Treg cells are not increase in the circulation of pregnant women. In part, they are attracted by hCG at the fetal–maternal interface during early pregnancy, via a LHR signalling (Leber et al., 2010; Schumacher et al., 2009). Finally, hCG treatment of activated dendritic cells results in an upregulation of MHC class II, IL-10 and IDO expression, reducing the ability to stimulate T cell proliferation. Impact of dendritic cells during implantation has recently been review by

Interestingly, immunologic properties of hCG are likely to differ as far as urinary versus recombinant molecules are concerned. These data are particularly important to keep in mind if immunomodulation of hCG would apply in clinical practice (Carbone et al., 2010;

Beside immune tolerance, a successful implantation requires also an extensive vascular remodeling of maternal arteries at the placental bed. Recent data demonstrate angiogenic effects of hCG via its interaction with endometrial epithelial cells which are able to produce angiogenic VEGF under hCG-LHR binding. Moreover, hCG highly increases angiogenesis of new mature vessels, via the stimulation endothelial cells proliferation as well as the migration of smooth muscle cells leading to the maturation of vessels, an important step for

Taken together the immune and vascular roles of hCG during early pregnancy, it is not surprising that this hormone has been studied during pregnancy disorders such as pre-

Implantation of the embryo into the maternal endometrium represents a unique biological process example of an immunological (tolerance of an allograft) and biological (adhesion of two epithelia) paradox (Perrier D'hauterive et al., 2002). The success of implantation depends on a receptive endometrium, a functionally normal blastocyst and a synchronized cross-talk between embryonic and maternal tissues. Though sexual steroids control the process (Paulson, 2011), a cascade of cytokines, growth factors and adhesion molecules are the private paracrine mediators of the uterine receptivity. Particularly, progesterone is crucial to establishment of endometrial receptivity, modulating subsequently the appearance or disappearance of a wide

Endometrium undergoes cyclic morphological and functional changes, including growth, differentiation and desquamation. Altogether, these cyclic physiological modifications lead to the preparation of a receptive endometrium able to tolerate the foetal allograft and to control invasiveness of trophoblastic cells, allowing implantation process during the midsecretory phase. Control of gene expression is crucial to this process, and inappropriate epigenetic modifications occasioning an altered chromatin structure and transcriptional activity may result in aberrant expression of receptive endometrial pattern. Epigenetics is likely to be associated with the initial regeneration and then proliferation of endometrium,

Blois (Blois et al., 2011).

Kajihara et al., 2011).

**5. The endometrium**

placentation (Berndt et al., 2009; Berndt et al., 2006).

eclampsia (Kalinderis et al., 2011; Norris et al., 2011).

network of molecules responsible of the uterine receptivity.

angiogenesis, decidual reaction and angiogenesis (Munro et al., 2010).

**5.1 The window of implantation and the decidua**

modulating its own production and highly promoting invasiveness of trophoblast. HhCG 2D structure is different from native hCG, appears to be independent in numerous biological functions, overall promoting placental implantation/invasiveness and is likely to signal through a different receptor than LHR (TGF-beta Receptor?). H-hCG is also critical for choriocarcinoma growth and malignancy (Cole, 2010; Fournier et al., 2011; Guibourdenche et al., 2010).

Data from literature and from our own work show that through native hCG (and H-hCG concerning invasiveness of EVCT), embryo profoundly intervenes in its own implantation and favors immunological tolerance and active angiogenesis that are crucial for successful implantation (Banerjee and Fazleabas, 2011; Tsampalas et al., 2010).

At the first steps of implantation, hCG is able to prolong the WOI, by inhibiting decidual Insulin like Growth Factor (IGF)-Binding Protein and IGFI (both markers of complete decidualization) (Fluhr et al., 2008b), and in association with interleukin 1 (IL-1), to favor adhesion via the increase in trophinin expression on endometrial epithelium (Sugihara et al., 2008). Concerning trophoblast functions hCG (and its hyperglycosylated variant) has been demonstrated to promote trophoblastic cells migration, and to reduce endometrial barrier, through its action on matrix metalloprotease protein 9 (MMP9, increased), TIMP-1, -2, -3 (reduced), GM-CSF, IL-11 (Chen et al., 2011; Fluhr et al., 2008a; Paiva et al., 2011). We also observed that hCG is able to increase leukemia inhibitory factor (LIF) production by endometrial epithelial cells *in vitro*, a cytokine known to be crucial for implantation in mice (Perrier d'Hauterive et al., 2004; Stewart, 1994). These data has been confirmed by different teams (Licht et al., 2002; Sherwin et al., 2007).

HCG levels coincide with the development of trophoblast tolerance. Indeed, it offers many immunological properties. For example, hCG increases the number of uterine natural killer cells that play a key role in the establishment of pregnancy (Kane et al., 2009). HCG also intervenes in the development of local immune tolerance through apoptosis via Fas/Fas-Ligand (Kayisli et al., 2003). It also modulates the Th1/Th2 balance toward the Th2 pathway (Koldehoff et al., 2011). During pregnancy, the balance of Th1 (cell-mediated immunity) and Th2 (humoral immunity) cytokines is characterized by an initial prevalence (but not an exclusivity) of Th2 cytokines, followed by a progressive shift towards Th1 predominance late in gestation, that when is abnormal, may initiate and intensify the cascade of inflammatory cytokine production involved in adverse pregnancy outcomes (Challis et al., 2009). In a more general way, Khan *et al.* showed that the administration of hCG to nonobese diabetic mice (NOD) before the beginning of the clinical symptoms reduced the increase in glycaemia, reversed establishment of insulitis, and inhibited the development of Th1 autoimmune diabetes (Khan et al., 2001). A recent large proteomic study demonstrated the influence of several molecules produced by the trophoblast that regulate the mother's immune tolerance. Among these molecules, hCG inhibits T lymphocytes (Dong et al., 2008).

The transient tolerance, evident during gestation is at least partially achieved via the presence of regulatory T cells that are expended during pregnancy (Dimova et al., 2011; Ernerudh et al., 2011; Fainboim and Arruvito, 2011; Nevers et al., 2011; Xiong et al., 2010) at the maternal-fetal interface, showing a suppressive phenotype, whereas Treg cells are not increase in the circulation of pregnant women. In part, they are attracted by hCG at the fetal–maternal interface during early pregnancy, via a LHR signalling (Leber et al., 2010; Schumacher et al., 2009). Finally, hCG treatment of activated dendritic cells results in an upregulation of MHC class II, IL-10 and IDO expression, reducing the ability to stimulate T cell proliferation. Impact of dendritic cells during implantation has recently been review by Blois (Blois et al., 2011).

Interestingly, immunologic properties of hCG are likely to differ as far as urinary versus recombinant molecules are concerned. These data are particularly important to keep in mind if immunomodulation of hCG would apply in clinical practice (Carbone et al., 2010; Kajihara et al., 2011).

Beside immune tolerance, a successful implantation requires also an extensive vascular remodeling of maternal arteries at the placental bed. Recent data demonstrate angiogenic effects of hCG via its interaction with endometrial epithelial cells which are able to produce angiogenic VEGF under hCG-LHR binding. Moreover, hCG highly increases angiogenesis of new mature vessels, via the stimulation endothelial cells proliferation as well as the migration of smooth muscle cells leading to the maturation of vessels, an important step for placentation (Berndt et al., 2009; Berndt et al., 2006).

Taken together the immune and vascular roles of hCG during early pregnancy, it is not surprising that this hormone has been studied during pregnancy disorders such as preeclampsia (Kalinderis et al., 2011; Norris et al., 2011).

#### **5. The endometrium**

102 Embryology – Updates and Highlights on Classic Topics

Data from literature and from our own work show that through native hCG (and H-hCG concerning invasiveness of EVCT), embryo profoundly intervenes in its own implantation and favors immunological tolerance and active angiogenesis that are crucial for successful

At the first steps of implantation, hCG is able to prolong the WOI, by inhibiting decidual Insulin like Growth Factor (IGF)-Binding Protein and IGFI (both markers of complete decidualization) (Fluhr et al., 2008b), and in association with interleukin 1 (IL-1), to favor adhesion via the increase in trophinin expression on endometrial epithelium (Sugihara et al., 2008). Concerning trophoblast functions hCG (and its hyperglycosylated variant) has been demonstrated to promote trophoblastic cells migration, and to reduce endometrial barrier, through its action on matrix metalloprotease protein 9 (MMP9, increased), TIMP-1, -2, -3 (reduced), GM-CSF, IL-11 (Chen et al., 2011; Fluhr et al., 2008a; Paiva et al., 2011). We also observed that hCG is able to increase leukemia inhibitory factor (LIF) production by endometrial epithelial cells *in vitro*, a cytokine known to be crucial for implantation in mice (Perrier d'Hauterive et al., 2004; Stewart, 1994). These data has been confirmed by different

HCG levels coincide with the development of trophoblast tolerance. Indeed, it offers many immunological properties. For example, hCG increases the number of uterine natural killer cells that play a key role in the establishment of pregnancy (Kane et al., 2009). HCG also intervenes in the development of local immune tolerance through apoptosis via Fas/Fas-Ligand (Kayisli et al., 2003). It also modulates the Th1/Th2 balance toward the Th2 pathway (Koldehoff et al., 2011). During pregnancy, the balance of Th1 (cell-mediated immunity) and Th2 (humoral immunity) cytokines is characterized by an initial prevalence (but not an exclusivity) of Th2 cytokines, followed by a progressive shift towards Th1 predominance late in gestation, that when is abnormal, may initiate and intensify the cascade of inflammatory cytokine production involved in adverse pregnancy outcomes (Challis et al., 2009). In a more general way, Khan *et al.* showed that the administration of hCG to nonobese diabetic mice (NOD) before the beginning of the clinical symptoms reduced the increase in glycaemia, reversed establishment of insulitis, and inhibited the development of Th1 autoimmune diabetes (Khan et al., 2001). A recent large proteomic study demonstrated the influence of several molecules produced by the trophoblast that regulate the mother's immune tolerance. Among these molecules, hCG inhibits T lymphocytes (Dong et al., 2008). The transient tolerance, evident during gestation is at least partially achieved via the presence of regulatory T cells that are expended during pregnancy (Dimova et al., 2011; Ernerudh et al., 2011; Fainboim and Arruvito, 2011; Nevers et al., 2011; Xiong et al., 2010) at

implantation (Banerjee and Fazleabas, 2011; Tsampalas et al., 2010).

2011; Guibourdenche et al., 2010).

teams (Licht et al., 2002; Sherwin et al., 2007).

cells.

modulating its own production and highly promoting invasiveness of trophoblast. HhCG 2D structure is different from native hCG, appears to be independent in numerous biological functions, overall promoting placental implantation/invasiveness and is likely to signal through a different receptor than LHR (TGF-beta Receptor?). H-hCG is also critical for choriocarcinoma growth and malignancy (Cole, 2010; Fournier et al.,

> Implantation of the embryo into the maternal endometrium represents a unique biological process example of an immunological (tolerance of an allograft) and biological (adhesion of two epithelia) paradox (Perrier D'hauterive et al., 2002). The success of implantation depends on a receptive endometrium, a functionally normal blastocyst and a synchronized cross-talk between embryonic and maternal tissues. Though sexual steroids control the process (Paulson, 2011), a cascade of cytokines, growth factors and adhesion molecules are the private paracrine mediators of the uterine receptivity. Particularly, progesterone is crucial to establishment of endometrial receptivity, modulating subsequently the appearance or disappearance of a wide network of molecules responsible of the uterine receptivity.

#### **5.1 The window of implantation and the decidua**

Endometrium undergoes cyclic morphological and functional changes, including growth, differentiation and desquamation. Altogether, these cyclic physiological modifications lead to the preparation of a receptive endometrium able to tolerate the foetal allograft and to control invasiveness of trophoblastic cells, allowing implantation process during the midsecretory phase. Control of gene expression is crucial to this process, and inappropriate epigenetic modifications occasioning an altered chromatin structure and transcriptional activity may result in aberrant expression of receptive endometrial pattern. Epigenetics is likely to be associated with the initial regeneration and then proliferation of endometrium, angiogenesis, decidual reaction and angiogenesis (Munro et al., 2010).

The Actors of Human Implantation: Gametes, Embryo, Endometrium 105

of VEGF, an angiogenic cytokine produced by epithelial endometrial and stromal cells (Berndt et al., 2006). As demonstrated at the next section, some other molecules from a trophoblastic origin, are able to actively stimulate maternal vascular remodelling as well.

In parallel, immune cells increasingly infiltrate endometrium from post-ovulation to menstruation in absence of pregnancy. In case of pregnancy, an higher increased number is observed after fertilisation up to mid gestation, since 30% of decidual cells are leukocytes, including 75% of uterine natural killers (uNK) (Plaisier, 2011; Zhang et al., 2011). It has been proposed that uNK could exert, directly or indirectly, either positive or negative control over implantation process. These cells secrete an array of cytokines important for adequate local immune regulation, angiogenesis, placental development, and establishment of pregnancy. Other immune cells or systems such as dendritic cells, T regulatory cells, macrophages, Th1/Th2 equilibrium orchestrate immune tolerance, trophoblast invasion and angiogenesis associated with embryo implantation (Blois et al., 2011; Denney et al., 2011).

ART programs, and particularly egg/embryo donation protocols, has clearly contributed to our knowledge of endometrial physiology. They gave us the opportunity to separately evaluate oocyte and endometrium, and to discover that endometrial receptivity can be controlled artificially with exogenous estrogen and progesterone, with a certain success (Paulson, 2011). Although it ensures obtaining a sufficient number of oocytes and thus embryos, ovarian hyper stimulation with exogenous gonadotropins is likely to be associated with modified endometrial development that may impact endometrial receptivity and success of implantation. These phenomenons would result from the inducing of an embryoendometrium asynchrony (histologic endometrial advancement), the up-regulation of Preceptor expression, and a negative correlation between implantation and premature progesterone elevation, impacting fresh implantation rates in normal and high responders

(Shapiro et al., 2011a, b), despite some other controversial results (Levi et al., 2001).

A better understanding of the uterine–embryo interaction and of the "seed and soil" regulations during embryo implantation is mandatory to increase the efficacy of ART. Implantation failure, recurrent miscarriage and preeclampsia have several recognized causes in common, but in most cases, the precise etiology remains obscure. Recent data in reproductive immunology identify the importance of the local immune environment and suggest to the clinician the need to develop tools to explore these endometrial perturbations

Indeed the semantic distinction between implantation failure, abortion and preeclampsia might in fact be more quantitative than qualitative (Chaouat, 2008). An important subset of implantation defects is the consequence of a deregulation of the interleukin systems, tumor necrosis factor (TNFα), interferon system as well as the uNK cell-mediated networks. Both deficient and excess expression of cytokines and immune cells number and activation play indeed detrimental key roles in implantation, since these actors can have both positive and

For example, in human reproduction, a proper balance in the IL-12, IL-18, IL-15 and a correct uNK activation stat result in successful implantation and pregnancy. Conversely,

**5.2 Clinical relevance of endometrial immunity**

(Tuckerman et al., 2010).

negative effects.

Whereas the implantation can occur in any human tissue, the endometrium is one of rare in which the embryo cannot implant, except during one limited period called the *window of implantation* (WOI), at the time when progesterone reaches peak serum concentrations (day 20-day24), allowing embryo to implant only under optimal circumstances. During this period, it offers a high receptivity to the embryo, resulting in a successful pregnancy (Lessey, 2011). This physiological state of the endometrium at the mid-secretory phase allows blastocyst 3-steps implantation process: apposition, then adhesion followed by trophoblast invasion and then induction of localized changes in the stroma called decidualization (Rashid et al., 2011).

WOI ends with the decidual transformation, where endometrium becomes a well vascularized tissue and stromal cell differentiate into specialized decidual cells (Teklenburg et al., 2010b). Factors secreted by decidual cells (such as LEFTY-A) compromise endometrial receptivity, ending the WOI. Oedema, increased vascular permeability, proliferation and transformation of fibroblast into cuboid secreting cells, invasion of leucocytes and angiogenesis are the principal mechanisms enabling decidua to resist to oxidative stress, to allow interaction within immune cells and to restrain the invasiveness of trophoblasts.

Apprehending molecular mechanisms of endometrial receptivity and then decidual transformation would provide a better understanding implantation process and its pathological implications. Failure to express adequate decidualization pattern is likely to induce early pregnancy lost or predispose to obstetrical complications such as implantation failure, recurrent miscarriages, pre-eclampsia, fetal growth restriction... (Blois et al., 2011; Brosens, 2011; Plaisier, 2011).

Actual research aims to dissect the molecular basis for the changes occurring during the WOI at the genomic and proteomic level (Berlanga et al., 2011; Diaz-Gimeno et al., 2011; Horcajadas et al., 2007). Despite a lot of experiments aiming to evidence relevant and selective biomarkers of uterine receptivity, there is to date no molecular fingerprint of the WOI. This is due partly to the discovery of an increasingly higher number of potential new markers according to the evolution of research, partly to the structural composition of endometrium, where the specialized epithelial cells that encounter the embryo are distinct from the glandular part. Finally, it is important to note the absence of consensus to which biomarkers to use for endometrial receptivity, and none of studied mediators (Mucine 1, Lselectin ligand, integrins, heparin-binding epidermal growth factor-like growth factor) has been explored in sufficient detail to validate its usefulness in clinical practice (Lessey, 2011).

The modification occurring in the endometrium during the WOI is closely related to angiogenesis and to immune system, allowing the tolerance of the fetal allograft and an adequate vascular remodelling during plancentation for a successful pregnancy until birth. Angiogenesis refers to the formation of new vessels from existing ones, by elongation, intussusception or sprouting of endothelial cells. One of the key local adaptations to pregnancy is the stimulation of maternal vessel network at the embryo implantation site. Normal fetal development requires extensive angiogenesis and important vascular remodelings allowing adequate supply of nutrients as well as gas and metabolite exchanges. Abnormal uterine blood supply is associated with higher perinatal morbidity and mortality caused by preterm delivery, preeclampsia, or intrauterine growth restriction. Stimulation of angiogenesis in many organs (including uterus) is mediated through enhanced expression

Whereas the implantation can occur in any human tissue, the endometrium is one of rare in which the embryo cannot implant, except during one limited period called the *window of implantation* (WOI), at the time when progesterone reaches peak serum concentrations (day 20-day24), allowing embryo to implant only under optimal circumstances. During this period, it offers a high receptivity to the embryo, resulting in a successful pregnancy (Lessey, 2011). This physiological state of the endometrium at the mid-secretory phase allows blastocyst 3-steps implantation process: apposition, then adhesion followed by trophoblast invasion and then induction of localized changes in the stroma called

WOI ends with the decidual transformation, where endometrium becomes a well vascularized tissue and stromal cell differentiate into specialized decidual cells (Teklenburg et al., 2010b). Factors secreted by decidual cells (such as LEFTY-A) compromise endometrial receptivity, ending the WOI. Oedema, increased vascular permeability, proliferation and transformation of fibroblast into cuboid secreting cells, invasion of leucocytes and angiogenesis are the principal mechanisms enabling decidua to resist to oxidative stress, to allow interaction within immune cells and to restrain the invasiveness of trophoblasts.

Apprehending molecular mechanisms of endometrial receptivity and then decidual transformation would provide a better understanding implantation process and its pathological implications. Failure to express adequate decidualization pattern is likely to induce early pregnancy lost or predispose to obstetrical complications such as implantation failure, recurrent miscarriages, pre-eclampsia, fetal growth restriction... (Blois et al., 2011;

Actual research aims to dissect the molecular basis for the changes occurring during the WOI at the genomic and proteomic level (Berlanga et al., 2011; Diaz-Gimeno et al., 2011; Horcajadas et al., 2007). Despite a lot of experiments aiming to evidence relevant and selective biomarkers of uterine receptivity, there is to date no molecular fingerprint of the WOI. This is due partly to the discovery of an increasingly higher number of potential new markers according to the evolution of research, partly to the structural composition of endometrium, where the specialized epithelial cells that encounter the embryo are distinct from the glandular part. Finally, it is important to note the absence of consensus to which biomarkers to use for endometrial receptivity, and none of studied mediators (Mucine 1, Lselectin ligand, integrins, heparin-binding epidermal growth factor-like growth factor) has been explored in sufficient detail to validate its usefulness in clinical practice (Lessey, 2011). The modification occurring in the endometrium during the WOI is closely related to angiogenesis and to immune system, allowing the tolerance of the fetal allograft and an adequate vascular remodelling during plancentation for a successful pregnancy until birth. Angiogenesis refers to the formation of new vessels from existing ones, by elongation, intussusception or sprouting of endothelial cells. One of the key local adaptations to pregnancy is the stimulation of maternal vessel network at the embryo implantation site. Normal fetal development requires extensive angiogenesis and important vascular remodelings allowing adequate supply of nutrients as well as gas and metabolite exchanges. Abnormal uterine blood supply is associated with higher perinatal morbidity and mortality caused by preterm delivery, preeclampsia, or intrauterine growth restriction. Stimulation of angiogenesis in many organs (including uterus) is mediated through enhanced expression

decidualization (Rashid et al., 2011).

Brosens, 2011; Plaisier, 2011).

of VEGF, an angiogenic cytokine produced by epithelial endometrial and stromal cells (Berndt et al., 2006). As demonstrated at the next section, some other molecules from a trophoblastic origin, are able to actively stimulate maternal vascular remodelling as well.

In parallel, immune cells increasingly infiltrate endometrium from post-ovulation to menstruation in absence of pregnancy. In case of pregnancy, an higher increased number is observed after fertilisation up to mid gestation, since 30% of decidual cells are leukocytes, including 75% of uterine natural killers (uNK) (Plaisier, 2011; Zhang et al., 2011). It has been proposed that uNK could exert, directly or indirectly, either positive or negative control over implantation process. These cells secrete an array of cytokines important for adequate local immune regulation, angiogenesis, placental development, and establishment of pregnancy. Other immune cells or systems such as dendritic cells, T regulatory cells, macrophages, Th1/Th2 equilibrium orchestrate immune tolerance, trophoblast invasion and angiogenesis associated with embryo implantation (Blois et al., 2011; Denney et al., 2011).

ART programs, and particularly egg/embryo donation protocols, has clearly contributed to our knowledge of endometrial physiology. They gave us the opportunity to separately evaluate oocyte and endometrium, and to discover that endometrial receptivity can be controlled artificially with exogenous estrogen and progesterone, with a certain success (Paulson, 2011). Although it ensures obtaining a sufficient number of oocytes and thus embryos, ovarian hyper stimulation with exogenous gonadotropins is likely to be associated with modified endometrial development that may impact endometrial receptivity and success of implantation. These phenomenons would result from the inducing of an embryoendometrium asynchrony (histologic endometrial advancement), the up-regulation of Preceptor expression, and a negative correlation between implantation and premature progesterone elevation, impacting fresh implantation rates in normal and high responders (Shapiro et al., 2011a, b), despite some other controversial results (Levi et al., 2001).

#### **5.2 Clinical relevance of endometrial immunity**

A better understanding of the uterine–embryo interaction and of the "seed and soil" regulations during embryo implantation is mandatory to increase the efficacy of ART. Implantation failure, recurrent miscarriage and preeclampsia have several recognized causes in common, but in most cases, the precise etiology remains obscure. Recent data in reproductive immunology identify the importance of the local immune environment and suggest to the clinician the need to develop tools to explore these endometrial perturbations (Tuckerman et al., 2010).

Indeed the semantic distinction between implantation failure, abortion and preeclampsia might in fact be more quantitative than qualitative (Chaouat, 2008). An important subset of implantation defects is the consequence of a deregulation of the interleukin systems, tumor necrosis factor (TNFα), interferon system as well as the uNK cell-mediated networks. Both deficient and excess expression of cytokines and immune cells number and activation play indeed detrimental key roles in implantation, since these actors can have both positive and negative effects.

For example, in human reproduction, a proper balance in the IL-12, IL-18, IL-15 and a correct uNK activation stat result in successful implantation and pregnancy. Conversely,

The Actors of Human Implantation: Gametes, Embryo, Endometrium 107

Implantation process and placenta formation are closely associated to angiogenesis and immune tolerance, at the crossroad of endocrinology. Whereas embryo could implant in every human tissues and particularly in Fallopian tubes with sometimes a very huge morbidity (Shaw and Horne, 2011), uterus is tailored to tolerate the fetal allograft and to allow controlled invasiveness of trophoblast only during the WOI, ensuring the success of the intricate cascade of implantation. Blastocyst implantation requires a competent embryosupported by the fertilization of a good quality oocyte by a top spermatozoa – able to crosstalk with a receptive endometrium, and the dialogue must talk about immunity and angiogenesis. Implantation failure and some pathologies of pregnancy are associated to

Implantation rate in IVF treatment certainly benefits from research about all these fields in reproductive medicine, able to increase selection, diagnostic and treatment of the different actors of implantation: gametes, endometrium, embryo and the most difficult issue: the

Agarwal, A., Gupta, S., and Sharma, R. K., 2005, Role of oxidative stress in female

Agarwal, A., Gupta, S., Sekhon, L., and Shah, R., 2008, Redox considerations in female

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Aitken, R. J., and Roman, S. D., 2008, Antioxidant systems and oxidative stress in the testes:

Aitken, R. J., De Iuliis, G. N., and McLachlan, R. I., 2009, Biological and clinical significance of DNA damage in the male germ line: Int J Androl, v. 32, no. 1, p. 46-56. Aitken, R. J., and De Iuliis, G. N., 2010, On the possible origins of DNA damage in human

Aitken, R. J., De Iuliis, G. N., Finnie, J. M., Hedges, A., and McLachlan, R. I., 2010, Analysis

Antinori, M., Licata, E., Dani, G., Cerusico, F., Versaci, C., d'Angelo, D., and Antinori, S.,

Assou, S., Haouzi, D., De Vos, J., and Hamamah, S., 2010, Human cumulus cells as

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of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria: Hum Reprod, v. 25, no. 10,

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**6. Conclusion: Implantation process**

dialogue between each other.

9, no. 6, p. 367-376.

p. 2415-2426.

531-538.

Adv Exp Med Biol, v. 636, p. 154-171.

**7. References**

defect of this close dialogue at one or many steps of the cascade.

reproduction: Reprod Biol Endocrinol, v. 3, p. 28.

imbalances in these parameters correlate with implantation failure or early pregnancy loss (Ledee, 2005). Their expression controls the local uNK recruitment and subendometrial angiogenesis as reflected by the vascular flow index (VFI) determined by three-dimensional ultrasound (Ledee et al., 2008a; Ledee et al., 2011b).

The excellent correlation between IL-15 mRNA expression and the sub endometrial vascular flow index (VFI) suggests that this cytokine and the uNK cells that produce it participate to the local control of angiogenesis. Patients with a low sub-endometrial VFI and low IL-15 mRNA are patients with insufficient uNK recruitment and/or inadequate uNK-derived angiogenic-related proteins. In contrast, some patients with implantation failure exhibit very high VFI and, at the same time, high IL-15 and IL-18 mRNAs and CD56+ cell count. A Th-1 excess could possibly be involved in implantation failure (Kwak-Kim et al., 2005).

Abnormal subendometrial vascularization assessed by ultrasound may be the consequence of distinct cytokine dysregulation patterns. These may cause implantation failure, abortion or preeclampsia, through abnormal (insufficient or excessive) recruitment of uNK cells or through inadequate endothelial vascular remodeling before implantation. 3-D ultrasonography with vocal analysis may inform on the uterine preparation to a constructive dialogue with the conceptus through an adequate trophicity and angiogenesis. This point on echographic evaluation of endometrium opens up new horizons for the evaluation of the endometrium, well beyond the simple measurement of thickness that improperly correlate with the outcome of IVF, since without absolute cutoff (Noyes et al., 1995; Paulson, 2011). Nevertheless, focusing on vascularization at the time of implantation is absolutely mandatory from a physiological point of view (Ledee, 2005). In daily clinical practice, echographic evaluation of subendometrial VFI and measurement of the IL-15 and IL-18 mRNAs together with CD56+ counts may be useful to identify those women at risk of implantation failure (Ledee et al., 2011b).

Moreover, Tumor necrosis factor like WEAK inducer of apoptosis (TWEAK) is a transmembrane protein which, when cleaved, functions as a soluble cytokine. It is highly expressed by different immune cells and triggers multiple roles, including control of angiogenesis. TWEAK and IL-18 mRNA expression are correlated in patients with implantation failures. Basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. Actually, as demonstrated by Petitbarat et al., TWEAK doesn't act on IL-18 expression but is likely to control IL-18 related cytotoxicity on uNK cells when IL-18 is over-expressed (Petitbarat et al., 2010).

Using large-scale microarray analysis, endometrial gene expression at the time of the implantation window was compared in fertile control patients (FC) and women displaying previous IVF/ICSI repeated implantation failure (IF) or recurrent unexplained miscarriages (RM). Biological functions and gene networks were explored using the Ingenuity Pathways Analysis software. The number of differentially expressed genes revealed the extent of changes within the preconceptional endometrium as a function of either fertility or infertility. The main similarities of differentially expressed genes between IF and RM relate to immune and hematological system development abnormalities, especially deregulation of the differentiation and development of T lymphocytes and blood cells. As the endometrium is now thought to be a biosensor for the quality of the embryo, such differential expression may have direct consequences on the initial embryo-uterus dialogue (Chaouat et al., 2011; Ledee et al., 2011c).

#### **6. Conclusion: Implantation process**

Implantation process and placenta formation are closely associated to angiogenesis and immune tolerance, at the crossroad of endocrinology. Whereas embryo could implant in every human tissues and particularly in Fallopian tubes with sometimes a very huge morbidity (Shaw and Horne, 2011), uterus is tailored to tolerate the fetal allograft and to allow controlled invasiveness of trophoblast only during the WOI, ensuring the success of the intricate cascade of implantation. Blastocyst implantation requires a competent embryosupported by the fertilization of a good quality oocyte by a top spermatozoa – able to crosstalk with a receptive endometrium, and the dialogue must talk about immunity and angiogenesis. Implantation failure and some pathologies of pregnancy are associated to defect of this close dialogue at one or many steps of the cascade.

Implantation rate in IVF treatment certainly benefits from research about all these fields in reproductive medicine, able to increase selection, diagnostic and treatment of the different actors of implantation: gametes, endometrium, embryo and the most difficult issue: the dialogue between each other.

#### **7. References**

106 Embryology – Updates and Highlights on Classic Topics

imbalances in these parameters correlate with implantation failure or early pregnancy loss (Ledee, 2005). Their expression controls the local uNK recruitment and subendometrial angiogenesis as reflected by the vascular flow index (VFI) determined by three-dimensional

The excellent correlation between IL-15 mRNA expression and the sub endometrial vascular flow index (VFI) suggests that this cytokine and the uNK cells that produce it participate to the local control of angiogenesis. Patients with a low sub-endometrial VFI and low IL-15 mRNA are patients with insufficient uNK recruitment and/or inadequate uNK-derived angiogenic-related proteins. In contrast, some patients with implantation failure exhibit very high VFI and, at the same time, high IL-15 and IL-18 mRNAs and CD56+ cell count. A Th-1

Abnormal subendometrial vascularization assessed by ultrasound may be the consequence of distinct cytokine dysregulation patterns. These may cause implantation failure, abortion or preeclampsia, through abnormal (insufficient or excessive) recruitment of uNK cells or through inadequate endothelial vascular remodeling before implantation. 3-D ultrasonography with vocal analysis may inform on the uterine preparation to a constructive dialogue with the conceptus through an adequate trophicity and angiogenesis. This point on echographic evaluation of endometrium opens up new horizons for the evaluation of the endometrium, well beyond the simple measurement of thickness that improperly correlate with the outcome of IVF, since without absolute cutoff (Noyes et al., 1995; Paulson, 2011). Nevertheless, focusing on vascularization at the time of implantation is absolutely mandatory from a physiological point of view (Ledee, 2005). In daily clinical practice, echographic evaluation of subendometrial VFI and measurement of the IL-15 and IL-18 mRNAs together with CD56+ counts may be useful to identify those women at risk of

Moreover, Tumor necrosis factor like WEAK inducer of apoptosis (TWEAK) is a transmembrane protein which, when cleaved, functions as a soluble cytokine. It is highly expressed by different immune cells and triggers multiple roles, including control of angiogenesis. TWEAK and IL-18 mRNA expression are correlated in patients with implantation failures. Basic TWEAK expression influences the IL-18 related uNK recruitment and local cytotoxicity. Actually, as demonstrated by Petitbarat et al., TWEAK doesn't act on IL-18 expression but is likely to control IL-18 related cytotoxicity on uNK cells

Using large-scale microarray analysis, endometrial gene expression at the time of the implantation window was compared in fertile control patients (FC) and women displaying previous IVF/ICSI repeated implantation failure (IF) or recurrent unexplained miscarriages (RM). Biological functions and gene networks were explored using the Ingenuity Pathways Analysis software. The number of differentially expressed genes revealed the extent of changes within the preconceptional endometrium as a function of either fertility or infertility. The main similarities of differentially expressed genes between IF and RM relate to immune and hematological system development abnormalities, especially deregulation of the differentiation and development of T lymphocytes and blood cells. As the endometrium is now thought to be a biosensor for the quality of the embryo, such differential expression may have direct consequences on the initial embryo-uterus dialogue (Chaouat et al., 2011;

excess could possibly be involved in implantation failure (Kwak-Kim et al., 2005).

ultrasound (Ledee et al., 2008a; Ledee et al., 2011b).

implantation failure (Ledee et al., 2011b).

Ledee et al., 2011c).

when IL-18 is over-expressed (Petitbarat et al., 2010).

The Actors of Human Implantation: Gametes, Embryo, Endometrium 109

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**6**

*USA*

**The Role of Macrophages in the Placenta**

*UMDNJ – New Jersey Medical School,*

*Newark, New Jersey,*

*Department of Cell Biology and Molecular Medicine,*

Grace Pinhal-Enfield, Nagaswami S. Vasan and Samuel Joseph Leibovich

Placenta formation occurs through a complex and coordinated effort between the fetus's extraembryonic tissues and the gravid endometrial tissues. Many macrophages are present in the placenta throughout pregnancy and have been detected as early as day 10 of pregnancy (Chang et al., 1993). Placental macrophages include Hofbauer cells of the fetal chorionic villi and decidual macrophages of the maternal decidua basalis (Figure 1) (Bulmer & Johnson, 1984). Functions of placental macrophages include the production of substances that regulate local immune reactions (such as factors that regulate maternal immunological tolerance and fetal protection) and that promote angiogenesis in the placenta during its development (Mues et al., 1989; Sevala et al., 2007). Although they represent a significant presence on both the maternal and fetal sides of the placenta, placental macrophage functions have not been completely elucidated and still remain a

Fig. 1. The mesenchymal core of this fetal tertiary chorionic villus contains a Hofbauer cell,

seen as a large, pale-staining placental macrophage with an eccentric nucleus.

**1. Introduction**

significantly studied area of investigation.

### **The Role of Macrophages in the Placenta**

Grace Pinhal-Enfield, Nagaswami S. Vasan and Samuel Joseph Leibovich

*UMDNJ – New Jersey Medical School, Department of Cell Biology and Molecular Medicine, Newark, New Jersey, USA*

#### **1. Introduction**

126 Embryology – Updates and Highlights on Classic Topics

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