Approximately 0.5–1% of couples who are trying to conceive suffer from subsequent miscarriages (Regan, 1992). Eighty percent of these losses cannot be accounted for by chromosomal defects, hormonal disorders, or uterine abnormalities. Evidence is accumulating that these unexplained miscarriages might have an immunological background as the fetus is semiallogeneic to the mother and it has conclusively been shown that both fetal cells and placental tissues do express the paternally derived and thus foreign human leukocyte antigens (Houlihan et al., 1995; Hutter et al., 1996; King et al., 1996; Van Wijk et al., 1996). There is considerable evidence for the presence of a specialized immune system within the maternal part of the placenta (the decidua), which is postulated to have a role in this process (Laird et al., 2003). Embryo implantation represents the most critical step of the reproductive process in many species. It consists of a unique biological phenomenon, by which the blastocyst becomes intimately connected to the maternal endometrial surface to form the placenta that will provide an interface between the growing fetus and the maternal circulation (Denker, 1993; Aplin, 2000). During blastocyst apposition, trophoblast cells adhere to the receptive endometrial epithelium. It will subsequently anchor to the endometrial basal lamina and to the stromal extracellular matrix. This is followed by the invasive blastocyst penetration through the luminal epithelium (Achache and Revel, 2006). Within hours of ovulation, under the influence of steroid hormones secreted by the corpus luteum, the human endometrium begins a transformation from fibroblast-like cells to plump secretory decidual cells (Heuvel et al., 2004).
It has been suggested that the unique environment resulting from the transformation of endometrium to decidua plays a crucial role in uterine natural killer (uNK) cell specificity acquisition. They are phenotypically and functionally different from circulating ones. They express integrins, which allow their migration and invasion of the decidualizing endometrium (Lunghi et al., 2007). They are found in proliferative endometrium, and they increase abundantly during the secretory phase of the menstrual cycle (Eastabrook et al., 2008), with a dramatic increase between days 6 and 7 after the luteinizing hormone surge, the putative time of implantation (Tuckerman et al., 2007). They are also the major component of stromal leukocytes in the late secretory endometrium and in the first trimester decidua (Ozcimen et al., 2009).
They interact with target cells by a series of inhibitory and activating NK cell receptors constitutively expressed by uNK cells (Nakashima et al., 2008). These NK cells are a unique population of CD56brightCD16 cells, which are phenotypically different from the main population of CD56dimCD16bright peripheral blood NK cells (Eastabrook et al., 2008). It has been suggested that women with recurrent early pregnancy loss (RPL), in the first trimester, may have impaired CD56+ leukocyte response in the deciduas, whereas women with spontaneous losses (not RPL) have increased numbers of CD57 ‘classical’ NK cells (Quenby et al., 1999). Further studies on women experiencing idiopathic recurrent pregnancy loss have found abnormalities in the decidual NK (dNK) cell populations in the peri-implantation endometrium of these patients (Lachapelle et al., 1996; Yamamoto et al., 1999a). However, the results of functional studies have been less consistent. For example, although one study reported decreased dNK cytotoxicity in vitro during normal pregnancies in comparison with anembryonic gestations or in women with RPL (Chao et al., 1995), another study reported decreased dNK cytotoxicity in cells isolated from women with RPL (Vassiliadou and Bulmer, 1998). Recently, it was stated that measuring dNK from endometrial biopsies of patients with RPL does not appear to be of any prognostic value in predicting the outcomes of future pregnancies. Although the numbers of dNK cells are higher in women with a history of RPL than in normal controls, the significance of this finding is uncertain (Eastabrook et al., 2008). E-cadherin is a cell surface transmembrane glycoprotein that mediates cell–cell adhesion through homeotypic binding and is believed to be critical for the establishment and maintenance of junction in epithelial cells (Gumbiner, 1996; Huber et al., 1996). It plays an important role in embryogenesis formation (Barth et al., 1997). Suppression of E-cadherin expression is regarded as one of the main molecular events responsible for dysfunction of cell–cell adhesion. It is possible that E-cadherin possesses a dual function. In the preliminary phases, its expression at the cell surface is required to ensure adhesiveness. In contrast, E-cadherin may be subsequently downregulated to enable epithelial cells dissociation and blastocyst invasion (Achache and Revel, 2006).
The aim of this study was to investigate the uNK cells and E-cadherin in human endometrium of nonpregnant fertile women and of those with recurrent abortion and to understand their role in fetal implantation.
Materials and methods
Unexplained recurrent abortion (RA) was defined as a history of three consecutive miscarriages in the first trimester. A total number of 65 samples were collected (35 samples from women with RA and 30 samples of women with normal fertility, having more than two children, the youngest being not more than 2 years of age). Thirty-five samples from cases with unexplained first trimesteric abortion in Egyptian women aged 21–38 years with gestational period of 8 weeks±10 days were collected (based on patients history, laboratory as well as sonographic findings). The mean number of miscarriages was four (range 3–7). An embryonic pregnancy or fetal death was confirmed by both pelvic and transvaginal ultrasonography. Confirmation of absence of fetal heart beats using Doppler was performed. In cases in which fetal pole were not seen by ultrasound, chorionic gonadotrpin levels were measured quantitatively using two samples 24 h apart. All evacuations were performed under general anesthesia, followed by sterilization using betadine solution and sterile towel application. Bimanual examination, dilatation of the cervix up to number 8 Hegar, evacuation of any uterine content using ovum forceps, and curettage of the uterine wall using a blunt curette were conducted. In cases of spontaneous abortion, curettage was carried out within 24 h after diagnosis. Postoperative antibiotic was prescribed for 48 h. None of the abortions (studied cases) were pharmacologically induced; karyotypic examination was conducted and those with chromosomal abnormalities were excluded. Women with uterine abnormalities, infectious, autoimmune, other systemic, local diseases, or receiving any medication were also excluded. Of these 35 women with RA, three had subsequent live births, one developed a vesicular mole, and 31 miscarried again in the pregnancy after the biopsy. Endometrial tissues were also obtained from another 30 Egyptian women of the same age group (21–38 year) in the proliferative phase (15 cases) as well as in the secretory phase (15 cases) in the nonpregnant state, in the outpatient clinic, seeking for other gynecological diseases. Biopsies were collected using a Medgyn endosampler. They had regular menstrual cycles and had not used oral contraception or an intrauterine contraceptive device in the 2 months preceding the biopsy. All women were of proven fertility. Samples were collected from El-Kasr El-Aini Hospital, Cairo University. Informed consent was obtained from each patient. Sections were prepared and stained by hematoxylin and eosin for histopathological assessment.
Five-micrometer thick sections of formalin-fixed paraffin-embedded tissues were mounted onto ChemMate capillary gap slides (Dako, Glostrup, Denmark), dried in a slide oven at 60°C for 1 h, deparaffinized with xylene, and rehydrated with ethanol to distilled water. The staining procedures were performed on an automated immunostainer (TechMate 1000; Dako) using the biotin–streptavidin detection system (ChemMate-HRP/DAB; Dako). The primary antibody was diluted in ChemMate diluent, and incubation was done overnight at 4°C. All of the following procedures were carried out at room temperature in accordance with the ChemMate protocol. Each TechMate holder included a positive and a negative control slide. The results of this analysis revealed that the optimal procedure was epitope retrieval in microwave heating/TEG buffer with anti-CD56 and anti-CD57 antibody. We determined the cytoplasmic expression of CD56 and CD57 receptors on NK cells (Ozcimen et al., 2009). Sections were subjected to the same protocol using the antihuman E-cadherin. In normal cycle, we considered E-cadherin-positive cases as those in which endometrial glands expressed the immunostaining reaction at glandular epithelial surface and not intercellular staining, as the latter was considered as an internal control and the positivity of chorionic villi for E-cadherin immunostaining expression when cytoplasm of syncytiotrophoblastic cells of chorionic villi (ST) were positive (Floridon et al., 2000).
Statistical analysis was performed using Windows software. The relationship was obtained by the Pearson correlation test. A P value of less than 0.05 was considered significant and a P value of less than 0.001 was considered highly significant.
In this study, all biopsies from 30 nonpregnant women of proliferative phase and secretory phase at day 20 or 21, aged 21–38 years (mean age 31 years), as well as 35 obtained from unexplained RA cases, aged 21–38 years (mean age 32 years), were subjected to CD56, CD57, and E-cadherin antibody immunostaining.
NK cells were present in both phases of the normal nonpregnant cycle; (30 cases; proliferative and secretory). These uNK cells were CD57+, CD56− in 29 cases (96.6%; P=0.005) with more notable reaction in the secretory phase (day 20 or 21) than in the proliferative phase. E-cadherin staining was positive in 28 cases (93.3%). As regards the intensity, there was great deviation throughout the menstrual cycle when related to proliferative or secretory phases, although only two of 15 cases were negative in proliferative phase. It was notable that the epithelial surface of the glands showed more intense reaction in the secretory phase (marked intensity) than in the proliferative phase (Fig. 1a–d). The reverse was seen in biopsies taken from cases of RA. This study showed that there was an inverse correlation between dNK cells expressing CD57 and those expressing CD56 as two of 35 (5.7%) RA cases were CD57+ dNK cells, whereas CD56+ dNK cells were detected in 33 of 35 (94.3%) RA cases (P=−1.000). Membrane-associated E-cadherin staining was seen in the all cytotrophoblastic monolayers covering the chorionic villous core. E-cadherin showed positive correlation to NK CD57+ cells, whereas there was an inverse correlation with NK CD56+ (P=−0.001). The results are summarized in Table 1.
As regards tissues of placental bed, E-cadherin was expressed on the surface of the placenta (site of chorionic villi encroachment). However, the main placental bed was negative for E-cadherin, and none of the cases showed E-cadherin expression of trophoblastic cells around vessels. Expression within sections was classified as positive or negative, regardless of the staining intensity. In this study, we classified the biopsies taken from cases of RA into four groups: group 1: surface decidual cells+, chorionic ST+; group 2: surface decidual cells−, chorionic ST−; group 3: surface decidual cells+, chorionic ST−; and group 4: surface decidual cells−, chorionic ST+. The distribution of E-cadherin and CD56 dNK cells in chorionic villi as well as in decidual tissue is shown in Table 2 and in Fig. 1e–g. Twenty two of 35 biopsies of RA showed negative E-cadherin expression in decidual cells, chorionic villi ST, or the interface of placental bed at site of implantation (group 2). E-cadherin staining pattern differed markedly in relationship to dNK cells expressing CD56. With regard to the placental bed, CD56 dNK cells were studied in the decidual tissue as well as in the interface area. In biopsies in which CD56 dNK cells were positive in the main placental bed as well as at the interface of chorionic villi encroachment, the ST as well as decidual cell surface was E-cadherin negative in 17 of 35 cases (48.57%). None of the cases of group 1 showed immunoreaction of CD56 dNK cells at either the decidual tissue or interface area.
There is considerable evidence for the presence of a specialized immune system within the maternal part of the placenta (the decidua), which is postulated to have a role in recurrent miscarriage (Laird et al., 2003).
It can safely be assumed that every successful pregnancy must be accompanied by redirection or suppression of NK cells. Apparently, excessive immunosuppression leads to uninhibited endometrial growth with an unfavorable outcome (Emmer et al., 2000). Furthermore, the differential expression of surface receptors by dNK cells may have a role in determining reproductive success through modulation of the maternal immune system at the time of implantation and placentation (Eastabrook et al., 2008). This agrees with this study in which uNK cells were present in both phases of normal nonpregnant cycles (30 cases; proliferative and secretory phases). These uNK cells were CD57+, CD56− in 29 cases (96.6%) with more intense reaction in the secretory phase than in the proliferative phase, whereas the reverse was seen in biopsies taken from cases of RA. This study showed that there is switching of receptor expression of CD57 and CD56, as two of 35 (5.7%) RA cases had CD57− dNK cells and 33 of 35 (94.3%) of RA cases showed CD56+ dNK cells. Previous investigations on the expression of uNK cells in women with recurrent miscarriage (RM) show controversial results; our results are in agreement with Quenby et al. (1999), who observed significantly higher numbers of uNK cells in four of 12 women who miscarried in a subsequent pregnancy when compared with eight women who had a live birth in a subsequent pregnancy, which suggests that high numbers of prepregnancy uNK cells may predict miscarriage in the next pregnancy (Tuckerman et al., 2007).
In contrast, other studies have shown decreased number of decidual CD56+NK cells reported in the placental tissue from spontaneous miscarriages in RM women compared with tissue from spontaneous miscarriages in women without RM and in women requesting termination (Clifford et al., 1999; Quenby et al., 1999; Yamamoto et al., 1999b; Quack et al., 2001).
During blastocyst apposition, trophoblast cells adhere to the receptive endometrial epithelium. The blastocyst will subsequently anchor to the endometrial basal lamina and to the stromal extracellular matrix. At this point, the achieved embryo–endometrial linkage can no longer be dislocated by uterine flushing. This is followed by the invasive blastocyst penetration through the luminal epithelium (Achache and Revel, 2006). This matches the results in this study in which E-cadherin was expressed in endometrial glands (surface of glandular epithelial cells) of fertile patients; E-cadherin staining was positive in 28 of 30 cases (93.3%). The intensity of reaction was observed to increase from proliferative to secretory phase with maximum intensity in the secretory phase day 20 or 21. These cases showed CD57+dNK cells, CD56−dNK cells (96.6%).
Furthermore, it has been shown that in the first trimester intraluminal trophoblasts attached to endothelial cells as well as perivascular and endovascular trophoblasts were E-cadherin positive. In addition, a highly varying E-cadherin staining pattern is observed in vascular trophoblasts of the basal plate and in the placental bed as well as in trophoblasts related to myometrial vessels (Floridon et al., 2000). In this study, none of the 35 cases showed immunoreactions around the myometrial blood vessels. Twenty-two of 35 biopsies of RA have shown negative E-cadherin expression in decidual cells, in chorionic villi ST, or in the interface of placental bed at the site of implantation. However, we found surface decidual cells positive for E-cadherin immunostaining in eight of 35 cases. We examined the dNK cells in the placental bed (regardless of the number); we found an inverse correlation between CD56+ NK cells and E-cadherin expression at the placental bed as well as at chorionic villi ST, that is, CD56+ dNK cells might downregulate the expression of E-cadherin at the interface of placental bed as well as at chorionic villi ST in cases of RA. This is supported by in-vitro studies and animal models; there is considerable evidence that dNK cells are crucial in successful placentation. They are key mediators of maternal immune system interactions with fetal cells. They are also involved in modulating extra villous trophoblast extra villous trophoblast (EVT) invasion and in the remodeling of maternal spiral arteries. They express a wide range of surface receptors and signaling molecules, including cytokines, chemokines, and growth factors. Their functions in modulating EVT migration, invasion, and alteration from epithelial to endothelial phenotype are only beginning to be revealed. In contrast, the close temporal–spatial relationship between dNK and EVT suggests that dNK cells regulate implantation and uterine artery remodeling through receptor–ligand interactions. The differentiation of trophoblasts into the invasive extravillous phenotype is seen as a crucial part of placental angiogenesis. This process involves the switching of integrins and the downregulation of E-cadherin (Eastabrook et al., 2008).
This study confirms previous reports that dNK cells play an important role in fetal implantation. In normal fertile cycles, CD57+NK cells may have a role in the expression of E-cadherin by aiding blastocyst adhesion to the endometrium. However, CD56+NK cells are mainly expressed in women with RA. CD56+NK cells may modulate the role of E-cadherin by inhibiting its expression at chorionic trophoblasts in the first trimester, which may affect the adhesion stage of chorionic tissue to the placental bed subsequently and which may interfere with normal implantation and subsequently with maintenance of pregnancy. We recommend further investigation on the redirection of NK cells from expressing CD57 to expressing CD56 receptors, which may modulate the expression of E-cadherin in the trophoblastic cells for maintenance of pregnancy and for normal placentation in the first trimester.
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