Brazilian Journal of Medical and Biological Research (2006) 39: 1373-1385 - interactions 1373 ISSN 0100-879X Review

Blastocyst-endometrium interaction: intertwining a network

W.A. Castro-Rendón1,2, 1Semillero de Investigación SIMBIOSIS, J.F. Castro-Álvarez1,2, 2Grupo Reproducción - Corporación Biogénesis, C. Guzmán-Martinez2 Universidad de Antioquia, Medellín, Colombia and J.C. Bueno-Sanchez1,2

Abstract

Correspondence The successful implantation of the blastocyst depends on adequate Key words W.A. Castro-Rendón interactions between the embryo and the uterus. The development of • Embryo implantation Grupo Reproducción the embryo begins with the fertilized ovum, a single totipotent cell • Sede de Investigación Universitaria which undergoes mitosis and gives rise to a multicellular structure • Cll 62 #52-59, Laboratorio 534 named blastocyst. At the same time, increasing concentrations of • Prostaglandins Universidad de Antioquia • Aspirin Medellín ovarian steroid hormones initiate a complex signaling cascade that Colombia stimulates the differentiation of endometrial stromal cells to decidual Fax: +57-4-210-6470 cells, preparing the uterus to lodge the embryo. Studies in humans and E-mail: [email protected] in other mammals have shown that cytokines and growth factors are produced by the pre-implantation embryo and cells of the reproduc- tive tract; however, the interactions between these factors that con- verge for successful implantation are not well understood. This review Received November 3, 2005 Accepted June 21, 2006 focuses on the actions of interleukin-1, leukemia inhibitory factor, epidermal growth factor, heparin-binding epidermal growth factor, and vascular endothelial growth factor, and on the network of their interactions leading to early embryo development, peri-implantatory endometrial changes, embryo implantation and trophoblast differen- tiation. We also propose therapeutical approaches based on current knowledge on cytokine interactions.

Introduction stimulating factors (CSF), among others (1), supported by an adequate hormonal microen- Some decades ago cytokines were known vironment and the recognition of the fetus by as intercellular messengers with a unique the maternal . The embryo function in the immune system. Subse- plays an active role in the modulation of the quently, experimental evidence showed that immunoendocrine activity during endome- they participate in many events of immu- trial implantation, particularly by participat- noendocrine interaction, characteristic of ing in (in the murine model) or enhancing (as complex signaling networks. Thus, the sig- in humans) the endometrial inflammatory nals from both the embryo and the mother reaction known as , thus regu- during early embryo development converge lating the production of maternal soluble around the actions of leukemia inhibitory factors that might be necessary for the inva- factor (LIF), interleukin-1 (IL-1) and colony- sion of the trophoblast for embryo lodging

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into the endometrium (2). These typical embryo begins when the oocyte is fertilized changes of decidualization can be induced by a spermatozoid. When the penetration of with oil, air bubbles or concanavalin A and the male gamete into the oocyte is con- have also been observed in deciduomata (3) cluded, maternal and paternal pronuclei mi- and ectopic . grate to the center of the cell and the nuclear The decidual inflammatory reaction dif- membranes fuse to allow the recombination fers from the classical description of inflam- of the genetic material. This is the first stage mation that occurs in response to injury. In of embryo development, named . Sub- particular, in , the recruitment of sequently mitotic divisions of the zygote leukocytes is mainly dominated by natural give rise to the stages of two, four and eight killer (NK) cells that in other contexts are cells, until the (5). Then, the morula recognized as cytotoxic cells which partici- undergoes a process of cell polarization, pate in the antitumoral and antiviral immune where the outermost cells form the trophec- response. On the other hand, the transforma- toderm (external embryo layer). Simulta- tion of the uterine stromal cells into decidual neously, the expression and activation of cells is demonstrated by produc- Na+/K+ ATPase pumps transport sodium and tion, increased IL-15 concentrations and ac- water towards the interior of the morula that tivation of the arachidonic acid pathway. generates a cavity known as the blastocele These factors enhance the decidual reaction (6). Meanwhile, a group of pluripotent cells in response to embryo invasion. In general, denoted the is established in the decidual environment is able to regulate a pole of the embryo, between the trophecto- embryo differentiation, mainly the tropho- derm and the blastocele. These changes give ectodermic layer, which is dependent on rise to the blastocyst, the final step of early epithelial contact and embryo development. proteins from the stroma in addition to cyto- Previous studies have provided informa- kines (4). These trophoectodermic/tropho- tion about the regulatory effects of the IL-1 blast cell subpopulations are producers of system in human pre-implantatory embryos, hormones and cytokines whose endocrine as well as in embryos of other species (Table action can cross the maternal-fetal interface 1). The IL-1 system is composed of two and directly influence the systemic physi- agonists (IL-1α and IL-1ß), one inhibitor ological changes that characterize gestation. designated IL-1 receptor antagonist (IL-l ra), In this intricate network of interactions and two receptors known as IL-1 receptors cytokines act as intermediary links of the type I (IL-1R tI) and type II. Interestingly, maternal-fetal relationship including the only IL-1R tI transduces a signal in response events related to decidualization, implanta- to IL-1 (7). Single from human tion, placentation, embryogenesis, and fetal preimplantation embryos cultured in vitro growth. The aim of this review is to identify express the mRNAs for IL-1ß, IL-1R tI and certain basic aspects of cytokine actions in IL-1 ra (7). In bovine embryos the addition maternal-fetal interaction and to suggest new of IL-1ß after in vitro fertilization enhances possibilities for immunoendocrine regula- the recovery of under conditions tion, which could be useful to understand of culture at high density (25-30 embryos/ some disorders of early in hu- drop) but not at low density (10 embryos/ mans and in other animal species. drop). Thus, IL-1ß can modulate embryo development towards the blastocyst stage Early embryo development (8). Human embryos cultured in vitro pro- The establishment of the pre-implantatory duce high concentrations of IL-1α and IL-

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1ß (>60 and >80 pg/mL, respectively), and level at the stage of hatched blastocyst (10). the presence of these cytokines has been The aim of assisted reproductive tech- correlated with successful implantation after niques and embryo culture is to obtain em- transfer to the uterine cavity (9). Kruessel et bryos of good quality with the ability to al. (10) demonstrated the expression of IL- implant into the uterus. In this context, co- 1ß, IL-1R tI and IL-1 ra mRNA by RT-PCR culture systems of embryos on cell mono- in single-cell preimplantation mouse em- layers enhance early embryo development. bryos. They did not detect the corresponding Different types of cells such as uterine fibro- mRNAs at the stage of 2-cell-embryos, and blasts, human tubular cells, endometrial epi- only IL-1 ra was expressed at the 8-cell- thelial cells, and Vero cells (a cell line de- stage in a few embryos. Thus, the stage rived from green rhesus monkey kidney) where IL-1ß, IL-1R tI and IL-1 ra could be have been used successfully. These mono- detected was the compact morula stage. layers produce embryotrophic factors that Therefore, this expression was increased by make it possible to obtain a higher yield of ongoing maturation and reached its highest blastocysts. Some studies suggest that low

Table 1. Cytokines, prostaglandins, and growth factors implicated in embryo-endometrium interaction.

Compounds Biological actions Reference

LIF Blastocyst implantation Stewart et al., 1992 (40) Early embryogenesis as an embryotrophin Lavranos et al., 1995 (16) Decidualization Chen et al., 2000 (41) LIFR Decidualization and placentation Ni et al., 2002 (31) IL-1 Successful implantation Sheth et al., 1991 (9) Induces LIF production Arici et al., 1995 (43) Induces COX-2 Huang et al., 1998 (26) Endometrial changes McMaster et al., 1992 (23) GM-CSF and CSF-1 production Garcia-Lloret et al., 1994 (57) IL-1 ra Failure of implantation Simon et al., 1994 (49) IL-1R tI Embryo development Kruessel et al., 1997 (10) EGF Blastocyst differentiation Kaye, 1997 (1) Angiogenic factor Moller et al., 2001 (37) Trophoblastic differentiation Kliman et al., 1990 (55) Induces LIF production Arici et al., 1995 (43) HB-EGF Blastocyst growth, zona-hatching and Das et al., 1994 (39) trophoblast outgrowth Leach et al., 1999 (36) Implantation reaction Wang et al., 1994 (46) IL-15 Decidual marker Okada et al., 2000 (19) IGFBP-1 Decidual marker Dimitriadis et al., 2002 (20) TNF-α Endometrial changes von Wolff et al., 1999 (24) GM-CSF and CSF-1 production Garcia-Lloret et al., 1994 (57) PGE2 Endometrial changes Jabbour et al., 2003 (33) Multiple female reproductive failures Lim et al., 1997 (28) Reese et al., 2001 (29) Differentiation of NK cells Linnemeyer et al., 1993 (35) VEGF Angiogenic factor Moller et al., 2001 (37) GM-CSF Trophoblastic differentiation Garcia-Lloret et al., 1994 (57) CSF-1 Trophoblastic differentiation Garcia-Lloret et al., 1994 (57)

LIF = leukemia inhibitory factor; LIFR = LIF receptor; IL-1 = interleukin-1; IL-1 ra = IL-1 receptor antagonist; IL- 1R tI = IL-1 receptor type I; EGF = epidermal growth factor; HB-EGF = heparin binding-EGF; IL-15 = interleukin-15; IGFBP-1 = insulin-like growth factor-binding protein 1; TNF-α = tumor necrosis factor alpha; PGE2 = prostaglandin E2; VEGF = vascular endothelial growth factor; GM-CSF = granulocyte-macrophage colony-stimulating factor; CSF-1 = colony stimulating factor-1.

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concentrations of matrigel in embryo cul- tein 130-kDa complex (LIFR-gp130). There- tures enhance blastocyst formation and in fore, signal transduction and its biological vitro hatching (11). Matrigel is an extracel- activity require heterodimerization of these lular matrix whose main structural compo- two low affinity, components LIFR and nents are proteins and growth factors that are gp130, which generate a high-affinity bind- also present in the endometrial extracellular ing site (14). The group of Tsai (15) con- matrix such as fibronectin, collagen type IV, cluded that culture media supplemented with heparan sulfate proteoglycans, entactin, ni- recombinant human LIF (r-hLIF) enhances dogen, transforming growth factor ß, fibro- the rate of two-cell embryos reaching the blast growth factor, and plasminogen tissue stage of hatched blastocysts. Thus, the regu- activator, among others. These compounds latory effect of LIF is associated with both constitute a culture medium similar to the early embryo development and viability: uterine microenvironment that is appropri- when early-stage embryos are cultured with ate for embryo growth, viability, and for 1,000 IU/mL of r-hLIF the rate of blastocyst cytokine secretion. For example, the amount formation and trophoectodermic prolifera- of the highly secreted IL-1α in Quinn’s HTF tion is enhanced (16). medium enriched with matrigel can be cor- related with the embryo’s ability to be im- The peri-implantatory endometrial planted (11). Another explanation for this changes effect could be that these monolayers elimi- nate toxic metabolites generated by the em- The endometrium is regulated by cyclic bryo and mimic the environment of the ovi- changes that modify the uterine microenvi- duct (12). It is possible that matrigel in- ronment for embryo implantation. In hu- creases IL-1ß and IL-1 ra and the expression mans the has been described of membrane receptors such as IL-1R tI, but in two consecutive phases: proliferative or this possibility has not yet been tested. ovarian and secretory or luteal. This view of Before embryo implantation the blasto- the cycle is supported by the hypothalamic- cyst must escape from the zona pellucida hypophysial-gonadal axis that gives rise to (ZP), a thick extracellular cover composed the ovarian and endometrial cycles. The be- of three glycoproteins: ZP1, ZP2, ZP3. This ginning of the ovarian phase is mediated by event is known as hatching, a dual process the action of follicle-stimulating hormone where the ZP is degraded by enzymes in which acts on several follicles that enter the both the trophectoderm and the uterus, fa- maturation process during this cycle and on cilitating blastocyst protrusion from the ZP. growth of dominant follicles. The granulosa In humans, hatching occurs prior to implan- and theca cells that surround the oocyte syn- tation between the 5th and 7th day post- thesize estrogens; these hormones influence (13) and at 3.5 days post-mating in the proliferation of the endometrial cells that mice. is characteristic of the endometrial prolifera- In humans and mice, hatching appears to tive phase (17). be regulated by some cytokines, and the A positive feedback occurs in the hypo- beneficial effect of LIF on embryo develop- thalamic-hypophysial-gonadal axis when ment, in vitro, has been very well docu- estrogens are increased in mid-cycle, sud- mented (Table 1). LIF is a glycoprotein with denly releasing luteinizing hormone and pro- a remarkable range of biological activities in moting ovulation. Granulosa and theca cells various tissues; it is a highly glycosylated form the , an endocrine struc- single chain polypeptide whose action is ture that produces and estro- mediated through a LIF receptor-glycopro- gen, hormones that are necessary to generate

Braz J Med Biol Res 39(11) 2006 Blastocyst-endometrium interactions 1377 the phenotypic changes characteristic of the expressed and located in the gastric mucosa, next phase of the endometrial cycle, i.e., the while COX-2 presents an inducible expres- luteal or secretory phase. The action of these sion related to inflammatory processes (27). hormones is to transform the epithelial and The actions of PGs are not clearly under- stromal cells into a secretory phenotype, stood, since COX-2 null mice showed a enhancing synthesis of vasoactive com- significant decrease of the weight of the pounds such as prostaglandins (PGs) and uterus when decidualization was induced by cytokines related to the communication be- intraluminal oil injection (28). The same tween mother and embryo (17,18). observations were made when celecoxib and

In the secretory phase, progesterone in- aspirin (selective inhibitors of COX-2 and duces the morphological and phenotypic COX-1, respectively) were used in wild- changes of the endometrial cells that are type mice (29). Based on these results, one associated with the production of cytokines of the mechanisms implicated in deciduali- and inflammatory mediators (18). Changes zation could be the action of PGE2 and pros- occurring during the secretory phase in the tacyclin (PGI2) through production of cyto- stroma are known as decidualization, an in- kines and growth factors (30). flammatory process characterized by the The enzymatic function of COX is to morphological transformation of the stromal oxygenate arachidonic acid in cell mem- cells from small spindle-shaped cells to large branes and generate PGH, which is the sub- plump . Decidual cells secrete strate used by tissue-specific isomerases pro- prolactin, IL-15 and insulin-like growth fac- ducing different kinds of PGs (27). Using tor-binding protein 1 (19-21). Spiral arteries immunohistochemistry, Milne et al. (25) and endometrial glands undergo character- observed the production of PGE synthetase istic changes during this phase: widening of and PGE2 in the endometrial , the walls of the blood vessels and the muscle stroma and endothelium during different layer of the arteries and increase of the se- phases of the menstrual cycle and also found cretion of inflammatory mediators and cyto- that these compounds decrease during the kines by the endometrial gland cells (22). late secretory phase. In the murine endome- Regulation of endometrial changes is not trium it has been documented that there is exclusively due to ovarian hormones: dur- expression and production of both cytosolic ing the proliferative phase, for instance, and microsomal PGE synthetase isoforms mediators that induce an inflammatory pro- produced by COX-1 and COX-2, respec- cess in the endometrium such as IL-1 (23), tively. mRNA expression and protein syn- tumor necrosis factor alpha (TNF-α) (24) thesis of these enzymes increase on day 5 and PGs are produced (25). This inflamma- post-ovulation, when murine endometrial tory process is characterized by and decidualization begins (31,32). angiogenesis of the endometrial tissue, leu- The pre-implantatory endometrium re- kocyte recruitment and production of cyto- quires PGs during decidualization, especially kines that could be participating in endome- for the vascular changes generated by its trium remodeling and blastocyst-endome- action as a vasodilator. Furthermore, new trium interaction (23). functions of PGE2 related to cell prolifera- IL-1 production during the endometrial tion in the glandular epithelium have been proliferative phase induces the release of described: epithelial cells stimulated with vasoactive lipids such as PGs (26), whose PGE2 significantly increased BrdU incorpo- synthesis is regulated by the cyclooxygenase ration (33), indicating that glandular epithe-

(COX) enzyme, which presents two isoforms: lial cell division is facilitated by PGE2, which COX-1 and COX-2. COX-1 is constitutively may also exert these actions on other cell

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populations. the endometrium (38), invades the decidual- Another peculiarity of the early pregnant ized stroma and reaches the maternal mi- uterus is the recruitment of immune cells crovasculature (13). Hence, the endometrium from peripheral blood, principally NK cells, requires structural modifications induced by which differentiate into another subpopula- progesterone and estrogens, pivotal changes tion distinct from circulating NK cells, i.e., that facilitate the three consecutive phases of uNK cells. The latter grow in size and granu- implantation: apposition, attachment and in- larity and, conversely, decrease their cyto- vasion (38). This morphological and func- toxic ability (34). Linnemeyer and Pollack tional transformation of the endometrium (35) showed that NK cell differentiation could around embryo implantation generates a pe-

be induced by PGE2 in a concentration-de- riod known as implantation window (13). In pendent manner. Thus, PGE2 could be re- this context changes involve glycocalix modi- sponsible for inducing these endometrial fications in the epithelial cells of the luminal functions but the mechanisms for the induc- surface, growth factor secretion and adhe- tion of differentiation of these cells through sion molecule expression related to blasto-

PGE2 have not been elucidated. cyst adherence and stromal invasion. Increased production of growth factors The murine blastocyst moves down the such as epidermal growth factor (EGF), hep- oviduct and falls into the uterine cavity and arin binding-epidermal growth factor (HB- at that time it orientates its mural trophecto- EGF) and vascular endothelial growth factor derm towards the epithelial surface. In hu- (VEGF) during both phases of the menstrual mans, the hatched blastocyst opposes its cycle has been observed in the endometrium polar trophectoderm to the surface of the (36,37). VEGF and its receptor have been epithelium on the fifth day of gestation, a detected in endometrial cells and blood ves- process that occurs on the fourth day in sels during the proliferative and secretory mice. Das et al. (39) proposed that the im- phases with sustained protein expression of plantation process begins as a kind of dialog VEGF during both phases, but the number of between the embryo and the endometrium, receptors increases during the secretory phase prior to active penetration into the decidua. (37). EGF has been found at high concentra- They observed a high level of HB-EGF by in tions in epithelial cells, and its receptor is situ hybridization in the luminal epithelium also increased in the secretory phase at the that surrounds the embryo, at the site of level of epithelia, stroma and blood vessels implantation, whereas HB-EGF was absent (37). HB-EGF production is enhanced in the from pseudopregnant mice. Another finding endometrial stroma during both the prolif- related to blastocyst growth and hatching erative and secretory phases in glandular was obtained with HB-EGF, which enhances epithelium and endometrial blood vessels the cell number per blastocyst by almost (36). In this complex network a balanced 40% and leads to a 2-fold increase in the cell production of inflammatory mediators percentage of hatched embryos. Another and cytokines provides an adequate endome- cytokine, LIF, is necessary at this stage, as trial environment for interaction between demonstrated in LIF-deficient females, the embryo and endometrium. whose embryos normally develop to the blas- tocyst stage but fail to implant (40). Implan- Embryo implantation: tation and normal pregnancy can occur when blastocyst-endometrium interaction these embryos are transferred from LIF-de- ficient mothers to wild-type pseudopregnant Embryo implantation can be defined as recipients, or when an exogenous supply of the process by which the embryo attaches to LIF is provided to the LIF knock-out fe-

Braz J Med Biol Res 39(11) 2006 Blastocyst-endometrium interactions 1379 males (41). Based on this approach, Vogiagis amount of LIF mRNA in a concentration- et al. (42) examined the and dependent manner. These findings suggest early pregnancy in ewes and observed an that the function of LIF is mediated by the increase in immunoreactive LIF at the time IL-1 system and by the action of the EGF of blastocyst hatching in the luminal and family. We propose that LIF can exert its glandular epithelium. In humans, LIF is ex- function in two ways: First, it may act in a pressed throughout the cycle with a striking synergistic manner with other cytokines on increase in LIF mRNA levels in the middle embryo hatching and attachment. Second, it and late secretory phases, the period during may act on the endometrial epithelia in a which embryo implantation occurs (43). In paracrine manner to increase the expression the murine uterus, from days one to eight of of specific growth factors belonging to the pregnancy, the expression of LIFR and gp130 IL-1 system and the EGF family that act on mRNAs was detected by in situ hybridiza- the trophectoderm promoting cell attach- tion, reaching the highest levels on days 5 ment and embryo development (Figure 1). and 4, respectively (44). During early mouse The IL-1 system has been considered to embryogenesis the mRNAs of LIF, LIFR be relevant in regulating communication and gp130 are undetectable in 1- or 2-cell between the blastocyst and the endometrium. embryos, but are present in the blastocyst In the human and murine endometrium the stage (45). This pattern of expression is IL-1α and IL-1ß mRNAs are expressed dur- strongly suggestive of a paracrine action of ing the menstrual or estrous cycle. In hu- LIF between the embryo and the uterus dur- ing the implantation window. Other evidence suggests that hormone actions are mediated by cytokines during the peri-implantation period. For instance, the nidatory surge of estrogens stimulates im- plantation in mice through the action of sec- ondary factors such as LIF (41). Up-regula- tion of uterine LIF expression is coincident with high levels of estradiol (E2) at ovulation and implantation. Thus, LIF can replace the nidatory effect of E2 in ovariectomized fe- male mice, inducing implantation and de- cidualization (41). Based on these findings, enhanced uterine expression of LIF could be mediated by a direct action of E2, but we rather postulate that such LIF expression can be induced by HB-EGF, which increases the level of LIF by stimulation of E2 (46) (Figure 1). In this context the action of LIF can be Figure 1. Model for the interaction between cytokines and mediators of mediated also by other cytokines: an in vitro associated with embryo implantation. The effect of estrogen (E2) is mediated by the nidatory study with decidual human cells showed action of leukemia inhibitory factor (LIF) in the endometrium, which is regulated by interleu- kin-1 (IL-1) and heparin binding-epidermal growth factor (HB-EGF). Both factors induce how the addition of 20 µL rIL-1 or E2 to the trophoblastic differentiation and angiogenesis through vascular endothelial growth factor culture medium increased LIF production in (VEGF). Prostaglandin modulates IL-1 and possibly colony stimulating factor-1 (CSF-1) a time-dependent manner (47). In another production. Both contribute to syncytiotrophoblast and anchoring cytotrophoblast differen- tiation. Aspirin (acetylsalicylic acid, ASA) acts in a selective manner on cyclooxygenase-1 assay, Arici et al. (43) demonstrated that the (COX-1) and this could have an indirect effect on prostaglandin (PG) production by induc- addition of 0.01-10 ng EGF ß1 increased the tion of COX-2.

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mans, IL-1α, IL-1ß, IL-1R tI, and IL-1 ra are characterized the luminal epithelium at the also expressed. This secretion of IL-1α, IL- time of implantation and observed a con- 1ß and IL-1 ra changes in the endometrium tinuous staining pattern of MUC-1 between according to the levels of steroid hormones, days 20 and 24 of the normal cycle (pre- an event that is clearly visible when IL-1ß dicted period of the receptive phase). This reaches a peak around the time of implanta- means that the antiadhesive properties of tion. The IL-1R tI mRNA is expressed at MUC-1 detected in other species may not be increased concentrations in human endome- valid in humans. These antiadhesive proper- trial epithelium during the luteal phase (48). ties of MUC-1 have been shown to be due to In mice it was demonstrated that IL-1 ra the large extracellular domain, but it is clear interferes with embryonic attachment; IL-1 from Aplin’s work that immunoreactivity ra injected during the peri-implantation pe- against sulfated glycosylation in apical epi- riod induces failure of implantation (49). thelia is maintained during the proliferative The pre-ovulatory increase of 17-ß estra- phase and is increased in the early secretory diol stimulates both proliferation and differ- phase. Thus, on day 20, apical staining with entiation of endometrial epithelial cells (17). a 5D4 mAb, that recognizes keratan sulfate In particular, 17-ß estradiol promotes an chains, was reduced in the luminal epithe- increase of apical protein density and a de- lium. Based on this observation, we propose crease of electronegative charges at the site that both the glycosylation with sulfated of implantation. In some species, glycocalix chains in the core protein of MUC-1 and the changes are mediated by mucin 1 (MUC-1) electronegativity of this molecule are altered expression on the epithelial surface in the during uterine receptivity. The antiadherence/ endometrium, which overlaps putative bind- adherence balance is modulated in the endo- ing sites of trophectoderm cells, mainly gly- metrium by the attached embryo, which could coconjugates and heparan sulfate-associated induce indirect actions through enzymes such proteoglycans (50). For instance, MUC-1, a as metalloprotease and heparinase. In this component of the apical glycocalix, is a way, the group of Simon (52) showed by large glycoprotein (>200 kDa) with an ex- immunofluorescence that MUC-1 was ab- tracellular domain rich in proline, serine and sent in endometrial epithelial cells subjacent threonine residues. This protein is abun- and adjacent to the attached embryo, sug- dantly present on the apical glandular epi- gesting an active regulation by the embryo thelial surface, and in the midsecretory im- in the receptivity of the human endometrium plantation phase; it is also secreted into the (52). gland lumen and in the uterine cavity. Over- On the other hand, synthesis of heparan expression of MUC-1 is able to induce steric sulfate in trophoectodermic cells increases inhibition of the adhesive properties of cul- 4- to 5-fold at the murine peri-implantation tured cells and it is known that this might stage, which indicates that the embryo ex- encourage metastatic spread. In mice and presses proteoglycans necessary for adhe- other species there is a distinctive reduction sive interactions with the endometrium, such in MUC-1 protein expression during im- as syndecan and perlecan (53). The latter, as plantation, but not in humans (50), in whom well as other proteoglycans (fibronectin and down-regulation of this molecule may per- laminin) are expressed on the external sur- mit the expression of proteins such as hepa- face of the trophectoderm and could interact ran sulfate binding proteins that mediate with the extracellular matrix (54) in the de- embryo attachment. cidualized endometrium. These compounds Blastocyst attachment in humans occurs could also bind to members of the EGF in the midsecretory phase. Aplin et al. (51) family (amphiregulin, HB-EGF, among oth-

Braz J Med Biol Res 39(11) 2006 Blastocyst-endometrium interactions 1381 ers, improving attachment and growth of the obtained from five men and five women and implantatory blastocyst. stimulated with aspirin, aspirin/phytohemag- glutinin (PHA) and PHA or culture medium. Effect of growth factors on The supernatants were used to evaluate the trophoblast differentiation effect on the fusion of BeWo cells and the production of hPL and hCG as an indicator The decidua is thought to be a source of of syncytialization (Figures 2 and 3). We important growth factors and cytokines for found that aspirin had no direct effect on placental development. Growth factors regu- GM-CSF and IL-3 production by PBMC, late PG synthesis in the endometrium, pro- although the production of hPL and syncy- moting the invasion, proliferation and changes tialization over five or more nuclei were related to endometrial vascular permeabil- increased using PBMC supernatants obtained ity. Enhanced PG production by the action from women and stimulated with aspirin and of EGF has been shown in rat endometrial aspirin/PHA (Bueno J, Cadavid L, Betancur stromal cells. EGF acts through the phos- L, Guzmán-Martinez C, Peña B and Cada- phorylation of membrane proteins as a mito- vid Á, unpublished data) (Figure 3). We gen promoting DNA and RNA synthesis. As pregnancy progresses, the increased produc- tion of EGF enhances trophoblast differen- tiation (55). Granulocyte-macrophage CSF (GM- CSF) is produced by murine decidual cells in response to invasive trophoblast. Wegmann (56) documented the expression of GM-CSF, α CSF-1, TNF- , IL-1, among other factors, Figure 2. Cell fusion was quantitated by counting the number of nuclei, the number of cells, in the murine using a panel of cDNA and the number of cells containing a single nucleus in each photographic field (black probes. This pattern of cytokine expression arrows). Cell fusion was grouped according to the number (2, 3, 4, 5, or more) of nuclei per cell. Scale bar = 15 µm. may involve growth regulatory signals for the placenta (56). Garcia-Lloret et al. (57) demonstrated that both CSF-1 and GM-CSF stimulate the differentiation of the cytotro- phoblast to the syncytiotrophoblast. This morphological differentiation was indirectly measured by the increased production of human placental lactogen (hPL) and human chorionic gonadotropin (hCG). Additionally, they found that GM-CSF and CSF-1 produc- tion was controlled by IL-1 and TNF-α in fibroblasts derived from the villous stroma of the placenta. These cytokines could play an important role in the development and function of the human placenta (57). Our group has studied the effect of aspi- rin on the production of GM-CSF and IL-3 Figure 3. Correlation between cell fusion and human placental lactogen production in the by peripheral blood mononuclear cells (PBMC) group of women. The correlation between cell fusion and hormonal production was evalu- ated by the Pearson test (P ≤ 0.05). RPMI 1640 medium purchased from Sigma. ASA = and the effect of these cytokines on tropho- acetylsalicylic acid; PHA = phytohemagglutinin; FSK = forskolin; EGF = epidermal growth blast differentiation. Briefly, PBMC were factor; C- = negative control.

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conclude that the trophoblast differentiation tive charges produced by keratan sulfate induced by the effect of aspirin cannot be chains in the glycoprotein structure and fi- correlated with GM-CSF or IL-3 produc- nally enhances the exposure of heparan sul- tion. Perhaps other cytokines could be in- fate-binding proteins in the epithelium. LIF duced by aspirin to improve syncytializa- could play an important role in these modifi- tion. Moreover, since it is possible that aspi- cations, improving the attachment of the rin can induce some effect on growth factor embryo to the endometrium and ensuring secretion by the trophoblast per se, we have trophoectodermic differentiation towards the performed some experiments in which aspi- fibronectin-binding phenotype influenced by rin directly induced an increase in tropho- HB-EGF. blast syncitialization, maybe through the au- We believe that the differentiation of NK tocrine action of growth factors produced by cells and other endometrial cells such as

these cells. stromal cells may be induced by PGE2. At the same time this could amplify the produc- Therapeutical approaches tion of other cytokines that participate in decidualization and embryo implantation. A recently developed r-hLIF (or emfiler- Furthermore, PG production could also act min) will be tested in human clinical trials. on trophoblast proliferation and differentia- The experimental evidence in animals has tion. The therapeutic use of aspirin prior to demonstrated the physiological role of LIF implantation could be useful for successful in implantation, but the implications of the pregnancy because it has been demonstrated use of r-hLIF in human reproductive medi- that aspirin can inhibit the action of COX-1 cine are not clear. LIF has a wide spectrum in platelets and up-regulate COX-2 produc- of action and therefore has been used for tion from other sources. This stimulus po-

many diseases; e.g., in a phase I study in- tentiates PGE2 production that could finally cluding patients with advanced lung cancer, enhance trophoblast differentiation and in- a biological effect of r-hLIF was demon- crease the vasodilatory action necessary for strated by the increase of blood progenitor adequate blood perfusion of the embryo. cells, C-reactive protein levels, and hemopoi- Although therapeutic strategies based on etic recovery after chemotherapy (58). On cytokine targets have been used in many the basis of reports from animal and human models and in diseases such as cancer, the studies, we agree with the statement made results reported are not completely conclu- by SERONO in its website (59) that LIF sive in clinical trials, maybe due to the com- could be used as a safe therapeutical alterna- plexity of the cytokine network implicated tive and as a potent inducer of human em- in each disease. Alternative pathways could bryonic implantation. be activated when a cytokine is blocked or For the adaptive modifications of the when it is used as an inducer of a specific endometrial environment, steroid hormones immune response. New therapeutical ap-

are a key factor, particularly E2 that exerts its proaches must focus on drugs able to induce actions on the production of LIF in the endo- the production of several cytokines, in order metrium. On this basis, we propose that the to maintain an adequate profile of cytokines nidatory surge of LIF during the implanta- that support the desired response. Aspirin tion window could induce a change of the could be one such compound: it is an ancient electronegative charges on the epithelial sur- drug with unexplored actions on cytokine face and the remodeling of the glycocalix to production, for instance on IL-3 production home the embryo. It is possible that glycosy- by mononuclear cells. We did not establish lation of MUC-1 changes these electronega- what kind of cytokine modulate the syncy-

Braz J Med Biol Res 39(11) 2006 Blastocyst-endometrium interactions 1383 tialization of the BeWo cells throughout through a paracrine effect of the IL-1. On the acetylsalicylic acid or what kind of mediators, other hand, they could suggest a synergistic e.g., PGE2 or leukotrienes, were indirectly effect of both IL-1 and LIF on blastocyst implicated in this immune modulation but it is implantation (Figure 1). possible that more than one cytokine was Detailed information about the cellular involved in these effects. The probable induc- and molecular mechanisms associated with tion by aspirin of cytokine production by tro- the action of cytokines on stromal cells and phoblastic cells cannot be ruled out in this on the blastocyst during implantation will context: the trophoblast has an important role have a significant impact on the understand- in the maternal-fetal dialogue and aspirin could ing of the fundamental causes of implanta- modulate bidirectionally this interaction. tion failure. Moreover, the participation of In other animal models such as the pig, the trophoblast in the paracrine dialogue the immunomodulator effect of PGE2 has between mother and fetus must be under- been shown on growth factor and cytokine stood in more detail due to its pivotal action production by lymphocytes during gestation on embryo and fetus development. (60). In humans, this same action on the lymphocytes and other cells such as NK Acknowledgments cells could be stimulated by PGE2 and could induce the production of cytokines such as The authors are grateful for the help and those of the CSF family. Thus, implantation encouragement of Professors Angela Cadavid and trophoblast invasion could be mediated (coordinator of the Reproduction Group at the by CSF-1, GM-CSF and other cytokines University of Antioquia), Juan Guillermo produced by cells localized in the decidua. Maldonado (coordinator of the CENTAURO Finally, IL-1ß induces COX-2 synthesis Group at the University of Antioquia), Anne- (26) and LIF production (47) by human en- Lise Haenni (from the Institute Jacques Monod dometrial stromal cells and in decidual cells - France), and Jorge Ossa (from the University in vitro. These results strongly suggest that of Antioquia). PGs may control blastocyst implantation

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