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Ultrastructural alterations in human decidua in miscarriages compared to normal pregnancy decidua Online Publication Date: 01 January 2007 To cite this Article: Kara, F., Cinar, O., Erdemli-Atabenli, E., Tavil-Sabuncuoglu, B. and Can, A. (2007) 'Ultrastructural alterations in human decidua in miscarriages compared to normal pregnancy decidua', Acta Obstetricia et Gynecologica Scandinavica, 86:9, 1079 - 1086 To link to this article: DOI: 10.1080/00016340701505457 URL: http://dx.doi.org/10.1080/00016340701505457
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Acta Obstetricia et Gynecologica. 2007; 86: 1079 1086
ORIGINAL ARTICLE
Ultrastructural alterations in human decidua in miscarriages compared to normal pregnancy decidua
F. KARA1, O. CINAR2, E. ERDEMLI-ATABENLI3, B. TAVIL-SABUNCUOGLU3 & A. CAN3 1
Department of Obstetrics and Gynecology, Zubeyde Hanim Maternity Hospital, Ankara, Turkey, 2Ankara Etlik Obstetrics and Gynecology Hospital, Center of Assisted Reproductive Medicine, Etlik, Ankara, Turkey, and 3Department of Histology and Embryology, Ankara University School of Medicine, Sihhiye, Ankara, Turkey
Abstract Background. Pregnant endometrial stroma, an immunologically privileged site in the female reproductive system, is enriched by decidual and natural killer (NK) cells. Since the cellular microenvironment in early pregnancy from the decidual tissues of normal and miscarriage cases has gained importance, with special emphasis on cell-to-cell contacts, we aimed to document the plastic structure of the cellular milieu in normal and miscarriage decidua. Methods. Endometrial biopsies were obtained from women after legal curettage or women who had been treated by curettage after miscarriage. Samples were analysed in a light microscope (LM), a scanning electron microscope (SEM) and a transmission electron microscope (TEM). Results. Decidual cells possess several polyploidic protrusions on cell membranes. NK cells were distributed among decidual cells. Decidual cells were found to develop gap junctions in the interfaces between each other. Their cytoplasms were also found to possess well-developed protein synthesising organelles. Decidual cells obtained from miscarriages showed a moderate degree of degeneration and, in between, a decreased number of junctional complexes. Mononuclear cell infiltration was found to be significantly low. Conclusion. We conclude that decidual cells during early pregnancy build a series of miniature cell-cell contacts to assemble a proper endometrial milieu. In contrast, in miscarriage samples, those intercellular communications seem lacking, associated with an increased number of NK cells, a phenomenon which obviously alters proper implantation and leads to the induction of embryonic disgenesis and miscarriage.
Key words: Decidua cell, electron microscope, miscarriage, NK cell, ultrastructure Abbreviations: FLC: fibroblast-like cell, LGLs: large granular lymphocytes, LM: light microscope, NK: natural killer, SEM: scanning electron microscope, TEM: transmission electron microscope
Introduction The endometrium represents the mucosa, which lines the uterine cavity and consists of epithelial and stromal cells. The structure and number of both cell types change during the menstrual cycle (1 4). The transformation of the mucosal lining of the uterus from endometrium to decidua, which is called decidualization, is essential for normal implantation of the blastocyst. Many ultrastructural changes occur during this process (5,6). In humans, during implantation stromal edema is observed on day 23 of the menstrual cycle, continues for 34 days, and then conjoins with a predecidual reaction that begins
around spiral arterioles, and spreads through the upper two-thirds of the endometrium (7). Large, pale predecidua cells, differentiated from stromal fibroblast-like cell (FLC) laden with glycogen and lipid, are seen under the surface epithelium and around spiral arterioles (8). If implantation occurs, the reaction is strengthened and the endometrium becomes the decidua of pregnancy, where decidual cells become morphologically and biochemically distinct from stromal FLCs (9). Decidual cells serve in the nutrition or protection of the embryo, alternatively they protect the mother by limiting the invasiveness of the embryonic
Correspondence: Cinar Ozgur, Ankara Etlik Obstetrics and Gynecology Hospital, Center of Assisted Reproductive Medicine, Etlik, Ankara, Turkey. E-mail:
[email protected]
(Received 26 December 2006; accepted 4 June 2007) ISSN 0001-6349 print/ISSN 1600-0412 online # 2007 Taylor & Francis DOI: 10.1080/00016340701505457
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trophoblasts (10). Decidual cells can be distinguished from other members of stromal cells by light microscope, with 1 or 2 nucleoli, a granular euchromatic nucleus, and a pale cytoplasm (11,12). At the implantation site, leucocytes, particularly large granular lymphocytes (LGLs), which together compose 20 40% of endometrial stroma cells, and cytolytic granules of LGL may play a role in regulating the extent of trophoblastic invasion (13 15). Some 15% of determined pregnancy is terminated by miscarriage, which is the most common complication of pregnancies (16). Although 50% of miscarriages in women with a history of recurrent loss are correlated with an abnormal embryonic karyotype, hormones, environmental inducers, immunological reasons and cellular imperfections might also play a role in creating an unsuitable implantation environment for blastocysts (16,17). It was shown that decidual LGLs from 50% of women suffering a spontaneous pregnancy loss lack cytotoxic activity (18,19). The number of peripheral NK cells decreases in pregnant women compared with nonpregnant women (20,21), and no live infants were born when the ratio of maternal peripheral NK cells was 18% (20). ‘Uterine receptivity’ as a concept, is described as a series of cellular and molecular events in the uterine milieu forming the endometrium to be ready for implantation that eventually leads to a successful blastocyst implantation (22). Since the harmony of cells is vital to achieve this requirement, cellular structure and intercellular communications should play a pivotal role, and, therefore, must be carefully examined. In this study, we analysed the stromal cells of the decidua from normal healthy pregnant endometrium, and from endometrium after miscarriages. Briefly, no significant difference was noted between the 2 groups at light microscopic level (LM), while a series of ultrastructural differences were noted in both scanning (SEM) and transmission electron microscopic (TEM) examinations. Among those, besides fusiform decidual cell shape, shrunken cytoplasm with spindle-shaped nuclei and a reduced number of disorganised organelles, a decrease in the number of decidual NK cells and an increase in the number of lymphocytes were noted in the miscarriage group.
Materials and methods Tissue collection, fixation and electron microscopy Tissue samples from first trimester endometrial curettages obtained from healthy women after surgi-
cal evacuations, as a legal treatment of unwanted pregnancy, were used as the control group (n 8), and the curettage materials from miscarriages (n 7) were considered as the experiment group. The normal and experiment groups were selected from pregnant women who had not developed any significant disease, such as pre-eclampsia, eclampsia, preterm labour, or any drug, such as prostaglandin, acetyl salicylic acid, antibiotics or anesthetic, administration history. Only embryonic pregnancies were included in the study. Surgical evacuation was performed at least 30 min after the diagnosis was confirmed by ultrasonography. Following the surgical evacuation of embryonic tissues from the uterus, decidual tissues were obtained using a Novak curette. Samples were aseptically rinsed by an isotonic salt solution, and immediately transferred to the laboratory. Tiny blocks of endometrial tissue were cut and divided into 3 groups for LM, SEM and TEM observations. Specimens for LM were fixed with 3.5% paraformaldehyde solution (Merck Co., Darmstadt, Germany) for 24 h at 48C (23). Prior to cryosectioning, for cryoprotection PFA-fixed samples were immersed in 20% (0.58 M) and 30% (0.88 M) sucrose solutions (Merck Co.) for 12 h at 48C, respectively, and then blocked in Cryomatrix† (Shandon Scientific Ltd., Pittsburgh, USA). Cryosections (8 mm thick), stored at 48C, were stained with Harris haematoxylin and eosin (HE) (Sigma, St. Louis, MI, USA) for general orientation to sections. Slides were observed under a Nikon Eclipse E 600 microscope (Nikon, USA), and photographed by a Nikon Coolpix 5000 (Nikon) digital camera. For SEM observations, samples were fixed with phosphate-buffered Trump’s solution (pH 7.3), containing 3.7% formaldehyde and 1% glutaraldehyde, for 24 h at 48C. Then tissue blocks were frozen in liquid nitrogen and fractured into small pieces in order to expose the stromal decidual tissue to the SEM probe. Fractured pieces were immediately transferred into the high pressure SEM chamber. Samples were analysed in 12 kEV using a backscattered electron detector in a LEO 438 VP scanning microscope (LEO Electron Microscopy Ltd., Cambridge, UK). For TEM analyses, samples were fixed with phosphate buffered (pH 7.3) 2.5% glutaraldehyde and 2% paraformaldehyde mixture solution for 2 h at room temperature. The samples were washed with phosphate-buffered saline solution (PBS) (pH 7.3), and fixed with 1% osmium tetraoxide for 2 h as secondary fixation. After washing, the samples were embedded in Araldite 6005, and cut with Leica EM FCS (Vien, Austria) ultramicrotome. Semi-thin
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Ultrastructure of human decidua sections (1 mm) were stained by Toluidine blueAzure II to select region of interest for the following procedures. Thin sections (6070 nm) were stained with uranyl acetate and lead citrate, and examined and photographed using a LEO 906 E TEM (80 kV; Oberkochen, Germany). Toluidine blue-Azure II stained semi-thin sections were analysed under a Nikon Eclipse E 600 microscope (Nikon), and photographed by a Nikon Coolpix 5000 (Nikon) digital camera, to evaluate the number of NK cell in the normal and miscarriage groups. The proportions of NK cells among stromal cells were calculated directly after analysing the digitised micrographs. NK cell percentages in the 2 groups were compared using the computer-based SigmaStat software package (version 3.0; Jandel Scientific Corp., San Rafael, CA, USA). The difference between the 2 groups was tested using the Mann-Whitney Rank Sum statistical test. Level of significance was selected when p B0.05.
Results Light microscopy Initially, endometrial luminal epithelium and stroma with numerous cells and glands were identified in HE-stained slides (Figure 1) and Toluidine bluestained semi-thin sections (Figure 2). In some cases from each group, secretory activity of glandular epithelium was found prominent; glands were lined with single-layered columnar epithelium. Swollen tubular glands with irregularly aligned glandular cells were noted (Figures 1 and 2C, D). The glandular cell nuclei were located in the basal
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portion of the cell. The supranuclear cytoplasm bulged like a dome into the glandular lumen (Figure 2C). The cytoplasm had a foamy structure. Following the release of secretory products, pinched off apical cytoplasmic membranes were noticed. Although high columnar epithelium was noted in many cases, single-layered cuboidal epithelium forming the glands was also observed in some cases (Figure 1). The round heterochromatin cell nuclei located in the central portion of the cell were considered as evidence of non-secretory activity. At least 3 cell types were observed in decidua. Lymphoid cells with high nucleus/cytoplasm ratio, heterochromatic nuclei and round cytoplasm; FLCs with fusiform cytoplasm and decidual cells with lipid or/and glycogen stored cytoplasm were noted (Figure 1, inset). These round cells were characterised with swollen cytoplasm, large euchromatic nucleus, 2 or 3 nucleoli, and located around the glands and spiral arterioles. No considerable differences were observed when endometrial specimens from normal and miscarriage cases were compared in HE-stained samples scoped by LM. NK cell numbers were assessed in Toluidine blue-stained sections. A statistically insignificant (p 0.054) decrease was noted in the miscarriage group compared to the normal group.
Scanning electron microscopy Endometrial curettage samples were analysed in SEM in order to compare the decidua from normal and miscarriage cases by analysing ultrastructural features of cell surfaces. No significant differences
Figure 1. Micrographs obtained from H-E stained sections of decidual tissues from normal (A) and miscarriage (B) groups. Tortuous (A) and straight (B) glands were observed in both groups. Glandular epithelium (Gl. Ep.) and/or luminal epithelium (Lm. Ep.) was formed by cuboidal and/or columnar cells. Ar: arterioles. Basically, decidual stroma includes 3 cell types (inset); decidual cells (arrows), FLCs (arrow head) and lymphoid cells (asterisk). Bar: 200 mm for (A) and (B); 50 mm for inset.
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Figure 2. Toluidine blue stained semi-thin sections of decidual tissues from normal (A, C) and experiment (B, D) group. (A) and (B): stromal; (C) and (D): epithelial and sub-epithelial compartments of decidua. Ar: arteriole. Arrows show NK cells including intracytoplasmic granules. Higher number of NK cells was observed in normal group, especially located at interarteriolar and subepithelial region, compared to miscarriage group. Arrowhead: apical portions of epithelial cells, including secretion material, bulged into the lumen. Bar: 50 mm.
were noted between the decidual tissues and the normal and miscarriage cases at the SEM level. Basically, two dissimilarities were noted: (i) the structure of the decidual granules; and (ii) the number of NK cells. Cytoplasmic extensions, called decidual granules, were detected at the surface of the decidual cells in both groups. The number of these extensions did not correlate with the day of the pregnancy, thus, did not change during the first trimester of the normal pregnancy. While elongated and dome-shaped decidual granules were noted in the normal group, irregularly-formed and shorter granules predominated in some cases in the miscarriage group, suggesting the early termination of decidual milieu in the miscarriage group (Figure 3). Interestingly enough, in normal control samples, NK cells occasionally encountered among stromal cells (Figure 3A) as characterised by their spiny cell
surfaces, miscarriage samples have a lower number of NK cells (Figure 3B).
Transmission electron microscopy In the control group, decidual cells were noticed as large, round cells possessing 1 or 2 large euchromatic nuclei and evident nucleoli, well-developed rough endoplasmic reticulum, and a network of Golgi complex. Clathrin-coated vesicles close to cell membrane and a few condensed mitochondria were also noted. These cells closely adhered to one another by junctional complexes. Gap junctions, which support intercellular ion transport and information, were well-developed (Figure 4A, B). NK/LGL cells with dark, heterochromatin, indented nuclei and cytoplasmic electron dense granules, were also predominant in decidua. A close contact and interaction between decidual cells and NK cells was also
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Ultrastructure of human decidua
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Figure 3. (A) SEM image from control group. Arrows in the inset show NK cells among the stromal cells of decidual tissue. (B) Abortion group. Bar: 10 mm.
observed (Figure 4C). In the miscarriage group, most of the decidual cells had fusiform, smaller cytoplasm with spindle-shaped nuclei, and unclear nucleoli. Euchromatic nuclei occasionally showed
chromatin clumps under nucleolemma. The number of organelles reduced; some of the cells displayed many signs of degeneration/apoptosis (Figure 4D, E). Cellular shrinkage, loss of rough endoplasmic
Figure 4. In control group; (A) electron micrograph of 2 neighboring decidua cells with euchromatic nuclei (N), significant rough endoplasmic reticulum (RER) and Golgi complex (G), arrows indicate cytoplasmic communications of 2 decidual cells; (B) junctional complexes such as gap junction (arrowhead) and macula adherence (desmosome) in higher magnification of same 2 decidua cells, M: mitochondria, v: clathrin-coated vesicle; (C) NK (LGL) cell with cytoplasmic dense granules (asterisk) in close proximity (arrowhead) to a desidua cell (DC). In abortion group; (D) infiltration of a lymphocyte (Ly) between a decidua cell (DC) and an apoptotic one (Ap); (E) an apoptotic decidua cell and NK cell; (F) 2 plasma cell with well developed rough endoplasmic reticulum (RER). Bars: 1,560 nm for (A) and (C-F); 335 nm for (B).
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reticulum, and chromatin condensation in the nucleus was detected indicating apoptotic process. Interestingly, the number of NK cell decreased in decidua in miscarriage cases compared to the control group. The communication and/or the adhesion of the decidual cells and between decidual cell and NK cell were impaired. On the other hand, an infiltration of lymphocytes and plasma cells among decidual cells was noticed (Figure 4E, F). Discussion Miscarriages are caused by several factors which mainly originate from embryonic and/or parental features. While embryonic factors include chromosomal abnormalities, abnormal germ cells, defective implantation of trophoblast, the parental factors relate to maternal age, hormonal-immunological conditions, and preparation of an appropriate decidual microenvironment (16). Although many mechanisms are concerned in miscarriages, the alterations in the decidua need to be evaluated. For this purpose, we designed a comparative study that aimed to evaluate the human decidua from normal pregnancy and miscarriage cases at microscopic level. Ultrastructural features of normal decidual cells (7,11,12) and surface/glandular epithelium (2,24,25) were previously demonstrated in human and other mammalian species by SEM and TEM. Similarly, we observed that decidual cells are large, round cells, possessing 1 or 2 large euchromatic nuclei, well-developed rough endoplasmic reticulum and Golgi complex in the control group. Clathrincoated vesicles and gap junctions were also noted. However, we did not observe any apoptotic or necrotic cell in normal decidua. Kisalus et al. (26) reported that, in addition to similar structural findings of decidua cell, they did not detect any cell death signs, such as pyknosis, karyorrhexis or mitochondrial degeneration, in normal human decidua explants. In the miscarriage group, besides normal decidual cells, most of the decidual cell shape changed to fusiform and shriveled cytoplasm with spindle-shaped nuclei and unclear nucleoli. Decreased numbers of disorganised organelles was considered as a sign of apoptosis in some cells. Uterine NK (uNK) cells have specific membrane receptors, such as CD69, c-kit, CD56bright (15,21), and show different phenotypes compared to peripheral NK cells (27 29), and the number of NK cells increase during early pregnancy (30,31). NK cells comprise over 70% of endometrial leukocytes in first trimester decidua (32). On the other hand, T cells comprise 40% of leucocytes in the proliferative
endometrium, but they significantly decrease in the first trimester decidua (33,34). Embryo and embryonic constituents are not assaulted by high numbers of NK cells in decidua, because NK cells express inhibitory receptors, and, thus, inhibit the catalytic activity (35). Yamamoto et al. showed that the proportions of CD56 CD16 CD3 NK cells in decidual lymphocytes in recurrent miscarriage were lower than in normal pregnancies (p B0.002), and they concluded that the lower percentages of CD56 CD16 CD3 cells in recurrent miscarriage cases may show an inappropriate accumulation of NK cells in the decidua, and this finding may be a factor involved in recurrent spontaneous miscarriage (36). When they analysed the relationship between chromosomal abnormality and miscarriage, they found that the percentages of CD56 CD16 CD3 NK cells were similar in normal pregnancies and chromosomally abnormal miscarriages. However, the percentages of CD56 CD16 CD3 NK cells of chromosomally normal miscarriages were lower than those of chromosomally abnormal (p 0.0025). Moreover, the percentages of CD56 16 NK cells in miscarriages with normal chromosomes were lower than those in normal pregnancies or miscarriages with abnormal chromosomes (p 0.0037, p 0.0025) (37). On the other hand, various studies have shown different results in terms of the alterations of NK cell number in normal and miscarriage cases. For instance, Vassiliadou and Bulmer (37,38) demonstrated an insignificant decrease in the number of CD45 NK cells, and an insignificant increase in the number of endometrial granulated lymphocytes, and a significant rise in the amount of CD57 ‘classical’ NK cells. The percentage of NK cells between these 2 populations was similar, although women with recurrent pregnancy loss had a greater percentage of CD16 CD56dim NK cells, and a smaller percentage of CD16 CD56bright cells, which are normally the dominant endometrial NK population compared to control patients (39). Dosiou et al. reported that there was likely no difference in the percentage of total uNK cells in endometrial leukocytes of women with recurrent pregnancy loss compared with women without recurrent pregnancy loss (40). It was noted that no significant differences in CD56 NK cell were found between recurrent miscarriage and control women (41), and similarly, no significant discrepancies in NK cell percentage were found among miscarriage with normal chromosome karyotype, miscarriage with abnormal karyotype, and induced miscarriage (42). Our electron microscopic observations indicate that the number of decidual NK cells seem to decrease in the miscarriage group
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Ultrastructure of human decidua compared to the normal group. As the electron microscopic level is not appropriate to make a quantitative comparison, NK cell number was counted in a light microscope on Toluidine bluestained semi-thin sections. Although the number of NK cell was higher in controls than in miscarriages, it was not statistically significant. We found a reduction in the number of decidual NK cells, and an enhancement in the number of lymphocytes in the miscarriage group. This observation indicates that defective immunological milieu do not allow the maintenance of the pregnancy. Decidual cells that synthesise and secrete various proteins are exclusive cells due to their role in preparing a suitable bed for embryo implantation. Because of the alterations in decidual cells, including poorly organised organelles, chromatin clumps in the nucleus, changes in the cellular organisation, and the variations in the proportion of lymphocyte populations in the endometrium in miscarriage cases, we suggest that decidual stroma is unable to maintain the permanence of pregnancy, and, thus, cause the first trimester miscarriages.
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Acknowledgements
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The authors are grateful to Huseyin Solmaz for technical assistance. This study was supported by Scientific Research Commission 98.09.00.043 and TUBITAK NATO-A2 for Ozgur Cinar.
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