382

Gynecological Endocrinology

2012

28

5

382

385

© 2012 Informa UK, Ltd.

10.3109/09513590.2011.633127

0951-3590

1473-0766

Gynecological Endocrinology, 2012; 28(5): 382–385Copyright © 2012 Informa UK, Ltd.ISSN 0951-3590 print/ISSN 1473-0766 onlineDOI: 10.3109/09513590.2011.633127

To investigate the existence and the distribution of decidual apoptosis in normal pregnancies and miscarriages (sponta-neous and recurrent), a comparative immunofluorescent tissue labelling of normal control (n = 12) and miscarried pregnancies (n = 24) was designed. Evaluation of the existence and distribu-tion of decidual apoptosis in normal pregnancies and miscar-riages, characterization of the apoptotic cell types and the involvement of caspase-dependent pathways was analyzed with TUNEL, anti-active caspase-3, anti-pancytokeratin and anti-CD45 antibodies. Normal decidua showed few apoptotic cells, whereas decidua from recurrent miscarriages had a significantly higher number of apoptotic cells preferentially localized to the sub-epithelial and periarteriolar regions, where the onset of decidu-alization occurs. Apoptosis occurred via a caspase-dependent pathway. Neither immune nor epithelial cells were positively stained for any apoptotic markers. The increased number of apoptotic cells, which are strictly restricted to the periarteriolar stroma particularly in recurrent miscarriages leads us to suggest that decidual apoptosis could result a series of cellular dysfunc-tions that may threaten the course of pregnancy.

Keywords: Apoptosis, caspase, decidua, miscarriages

IntroductionApoptosis can be induced by intracellular and extracellular signals that activate the executive proteins, caspases − a cysteine protease family − via various pathways [1,2]. Concerning the endometrial apoptosis, the number of apoptotic cells gradually increases from the early proliferative to late secretory phase [3,4]. Caspase-dependent endometrial apoptosis significantly increased in the late secretory phase [5]. Apoptosis has also been shown in the trophoblast layer of placenta in normal pregnancies, suggesting that there is a steady cell turnover at the site of implantation which is necessary for the appropriate growth and function of the placenta [6,7]. These apoptotic cells are removed from the maternal-fetal interface with an active physiological event, mainly by induced active macrophages, which may influence not only immune responses, but also the proliferation and differentiation of surrounding cells [8].

Miscarriage is the most common complication of pregnancy, with 15% of pregnancies ending by miscarriages. Chromosomal and hormonal abnormalities, immunological problems and envi-ronmental factors such as alcohol, smoking or drug use may play a role in the aetiology [9]. A significant increase in the apoptotic

cell number has also been implied in decidua from sporadic miscarriages as compared to normal decidua [10,11]. We have shown an increased number of natural killer (NK) cells, a defi-ciency of intercellular communication between decidual cells and many apoptotic phenotypes in miscarriages as compared to normal pregnancies [12]. However, the site and the degree of apoptosis during miscarriage still remain unclear. The aim of the present study was to evaluate the level of apoptosis in human decidua from sporadic and recurrent miscarriages as compared to normal pregnancies, with specific regard to the localization and distribution of apoptotic markers.

MethodsPatient selection, tissue collection and fixation

Decidua basalis tissue samples from healthy women (n = 12) curettage as a legal treatment for unwanted pregnancy were used as the control group, whereas endometrial curettage samples from miscarriages (n = 24) composed the experimental group. The study was approved by the local ethical committee and informed consent was obtained from each woman before specimen preparation. The miscarriage group was subdivided into sporadic (<3 miscarriages, n = 14) and recurrent (≥3 miscarriages, n = 10) categories (Table I). The control and experimental groups were selected from women who had not developed any significant pregnancy-related disease such as pre-eclampsia, eclampsia, preterm labor, or systemic disease such as malignancy, autoimmune disease, hypertension, diabetes, and who had not reported the use of any drugs including prostaglandin, acetyl salicylic acid, antibiotics or anesthetics. Only embryonic pregnancies were included in the study. Specimens were fixed with 3.5% (w/v) paraformaldehyde solution (Merck Co., Darmstadt, Germany), immersed in 0.58 M and 0.88 M sucrose solutions (Merck Co.) and cut in a cryomicrotome. To test whether the decidual tissues do not include trophoblastic cells, Cdx2 staining (a trophoblast marker) was applied, and positive tissues were excluded from the study.

Apoptosis detection

TUNEL assay (Roche, Germany) was applied to slides to detect apoptosis. Slides were washed with phosphate buffered solution (PBS) and then incubated with 10 µM of TRITC-phalloidin (Sigma, St. Louis, MI, USA) for staining of filamentous actin (F-actin). The number of apoptotic cells per thousand cells was defined as the apoptotic index (AI), and two researchers counted the cell numbers and the mean values of these two counts were taken into account.

OBSTETRICS

Potential role of decidual apoptosis in the pathogenesis of miscarriages

Ozgur Cinar1, Fadil Kara2 & Alp Can3

1Etlik Zubeyde Hanim Women’s Health Teaching and Research Hospital, Center for Assisted Reproductive Medicine, Ankara, Turkey, 2Department of Obstetrics and Gynecology, Dr. Sami Ulus Maternity and Children Hospital, Ankara, Turkey, and 3Department of Histology and Embryology, Ankara University School of Medicine, Ankara, Turkey

Correspondence: Professor Ozgur Cinar MD, Etlik Zubeyde Hanim Women’s Health Teaching and Research Hospital, Center for Assisted Reproductive Medicine, Yeni Etlik Cad. No:55, Ankara, 06010 Turkey. E-mail: ozgur.cinar@yahoo.com

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Decidual apoptosis 383

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Determination of apoptotic mechanism

To test whether the decidual apoptosis was caspase-dependent, immunoreactivity of cleaved caspase-3 protein was analyzed by labeling with a rabbit polyclonal anti-cleaved caspase-3 (Asp 175) antibody (Cell Signaling, Beverly, MA), then with a 1:100 dilu-tion of an affinity-purified Cy3-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA).

Localization of apoptosis

Apoptotic cells were further analyzed to determine whether they belonged to the immune system. Dual staining was performed using TUNEL and a monoclonal anti-CD45 antibody that serves as a specific pan-leukocyte marker [13]. Slides were incubated with a monoclonal anti-CD45 antibody (Sigma), washed in PBS and incubated in a Cy3-conjugated goat anti-rabbit IgG (Jackson Immunoresearch Laboratories). Subsequently, TUNEL staining was applied to the slides as described above.

Fluorescent and/or differential interference contrast high reso-lution (2048 × 2048 pixels) digital images were obtained using a Zeiss-LSM 510 confocal laser scanning microscope (Zeiss, Jena, Germany) equipped with 488 nm argon ion, 543 nm green He-Ne and 633 nm red He-Ne lasers.

Statistical analysis

Five microscopic areas from each slide at 20× magnification were evaluated, and the number of TUNEL-positive cells was counted and transferred to the computer. Results were compared using the SPSS software package (version 15.0; SPSS Inc, Chicago, IL). Data not distributed normally were transformed with logarithmic transformation. The differences among the groups in terms of ages, pregnancy weeks or AIs were analyzed using one-way ANOVA test. To analyze specific differences among the groups, appropriate post hoc tests were implemented. The significance level was set at p ≤ 0.05. AIs were calculated and presented as mean ± SD.

ResultsTUNEL assay revealed a varying number of apoptotic cells in the control, sporadic and recurrent groups. A low percentage of TUNEL-positive cells were detected in the control group (AI = 16 ± 23); more were detected in the sporadic group (AI = 59 ± 110), but the difference was not statistically significant (p = 0.756). A significantly greater percentage (p < 0.001) was detected in the recurrent group (AI = 348 ± 202) (Figure 1A).

Figure 1. (A) Apoptotic indices of normal pregnant, sporadic and recurrent miscarriages. No significant difference was noted between the normal pregnant group (n = 12) and the sporadic miscarriage group (n = 14) while there is a significant difference (*) between the recurrent miscarriages (n = 10) and the other two groups (p < 0.001). (B) Mean age of control, sporadic and recurrent groups did not significantly differ from each other. (C) Mean pregnancy weeks of the patients were not different between sporadic (8.9) and recurrent (8.9) groups.

Table I. Patient selection was based on their miscarriage history.

Control Sporadic Recurrent

Patient number

Number of previous

miscarriages

Curettage time

(week) AgePatient number

Number of previous

miscarriagesCurettage

time (week) AgePatient number

Number of previous

miscarriagesCurettage

time (week) AgeMean 7.1 27.6 8.9 27.4 8.9 28.1 1 0 7 32 1 0 6 27 1 3 10 26 2 0 7 27 2 1 10 28 2 4 8 29 3 0 6 25 3 0 11 37 3 4 6 24 4 0 8 28 4 2 7 35 4 3 11 26 5 0 9 38 5 2 9 36 5 5 7 31 6 0 6 32 6 0 8 22 6 4 7 32 7 0 7 22 7 1 10 22 7 3 10 28 8 0 7 21 8 1 11 19 8 4 11 27 9 0 6 28 9 0 9 27 9 4 11 29 10 0 6 26 10 2 9 25 10 4 8 29 11 0 8 25 11 1 7 23 12 0 8 27 12 0 8 24 13 1 10 30 14 1 9 29

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384 O. Cinar et al.

Gynecological Endocrinology

The confidence interval of each group was calculated as (1;31), (−4;122), (202;493) in control, sporadic and recurrent group, respectively.

Regarding the patient age, no significant difference was noted between the groups (27.6 ± 4.7; 27.4 ± 5.6; 28.1 ± 2.4 in control, sporadic and recurrent, respectively) (Figure 1B). Similarly, mean pregnancy week in recurrent group (8.9 ± 1.9) was almost the same (p = 1.000) to that of the sporadic group, which indicates that increased apoptosis in recurrent group cannot be the conse-quence of the pregnancy weeks.

TUNEL-positive cells in all groups were exclusively localized to the decidual stroma (Figure 2A–C), specifically to the sub-epithelial and periarteriolar regions (Figure 2C).

Decidual tissues were labeled with an anti-active caspase-3 antibody to search caspase-dependent pathway. While only few positive cells were observed in the control group (Figure 2D), sub-epithelial and peri-vascular immunoreactivity was significant in the recurrent group (Figure 2E and F).

In order to determine whether the apoptotic cells were immune cells, double staining was performed using TUNEL and an anti-CD45 antibody as a pan-leukocyte marker. Some stromal cells

were stained positively with anti-CD45 antibody, but these cells were not TUNEL-positive (Figure 2G), directly indicating that the TUNEL-positive cells were not immune cells. Furthermore, TUNEL-positive cells did not correspond to the pan-cytokeratin-positive cells suggesting that apoptotic cells were originated from neither epithelial glandular nor trophoblastic cells.

The similarity of the distribution of TUNEL-positive and caspase-3-positive cells led us to ask whether a caspase-3 pathway is involved in the decidual apoptotic mechanism. For this purpose, co-staining was performed using TUNEL and the anti-cleaved caspase-3 antibody. TUNEL was located in the nucleus, whereas caspase-3 staining was found throughout the cytoplasm. The majority of TUNEL-positive cells were found to be caspase-3-positive whereas only a small population of caspase-3-positive cells was TUNEL-positive (Figure 2H).

DiscussionThe ultrastructural alterations of decidual cells in miscarriages as compared to normally cycling women were previously analyzed [12] and it was noted that many apoptotic cells were restricted to

Figure 2. FITC-conjugated TUNEL assay (green signal) counterstained with TRITC-phalloidin (red signal showing cellular F-actin) in decidua from normal pregnancy (A); in sporadic miscarriage (B); and in recurrent miscarriage (C). Few apoptotic cells were detected in normal pregnancy (A) and sporadic miscarriage (B), while many apoptotic cells were observed in the recurrent group (C); these were exclusively localized to the in stroma. Active caspase-3 immunoreactivity (green signal) is sparse in decidua of normal pregnancy (D). In contrast, caspase-3 positive cells are widespread in sub-epithelial (E) and periarteriolar (F) stroma in recurrent miscarriage cases. Counterstain: TRITC-phalloidin (red signal showing cellular F-actin). Double staining of TUNEL with anti-CD45 antibody shows that some stromal cells are CD-45 positive (red signals in G) but these cells are not TUNEL-positive (green signals in G). Double staining of TUNEL (green signals) with caspase-3 (red signals) shows that only a small population of caspase-3-positive cells is TUNEL-positive (arrows), whereas TUNEL-positive cells are caspase-3-positive (H). L: Glandular lumen; E: Glandular epithelium; S: Stroma. Scale bar: 50 µm for A, B, C; 20 µm for D, E, F, G, H.

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Decidual apoptosis 385

Copyright © 2012 Informa UK, Ltd.

the decidual stroma in the miscarriage group. The present study aimed to find out whether there is a relationship between the frequency of miscarriage and the level of the decidual apoptosis and also to determine the apoptotic cell types and apoptotic pathway. For this purpose, decidual apoptosis in normal and miscarriage-derived endometria was assessed with various apoptosis detection techniques.

Jones et al. [14] detected a variation in apoptosis between the cycling endometrium and the first trimester decidua in which the latter was found to contain few proliferating cells, to express high levels of bcl-2, an anti-apoptotic protein, and to show no evidence of apoptosis. Apoptotic indices for first trimester normal pregnan-cies and sporadic miscarriages as determined by TUNEL assay in cytotrophoblasts, syncytiotrophoblasts and decidua were found to be 29, 2 and 27% in normal pregnancies, and 51, 76 and 68% in sporadic miscarriages, respectively [11]. This increase in the number of apoptotic cells in the miscarriage group was similar to our results in the recurrent group. However, we did not detect any apoptotic stromal cells in the decidua of normal pregnancies. In another study, the expression of the anti-apoptotic protein bcl-2 was analyzed in normal, sporadic and recurrent miscarriage cases. Bcl-2-positive cells were found as aggregates and dispersed in the stroma and glandular epithelia of all three groups [15]. Double immunostaining with an anti-CD56 antibody revealed that the majority of stromal bcl-2-positive cells were CD56-positive large granular lymphocytes. Since bcl-2 is considered to be an anti-apoptotic marker, its expression suggests the presence of non-apoptotic cells. Similarly, we did not detect apoptosis in the glandular epithelium or stromal leukocytes. Plaisier et al. did not report any difference in the caspase-3 activity between control and sporadic miscarriages [16]. They found almost 5% apoptotic cells in decidual stroma from miscarriage, which was similar to that of our findings (6%). However, they reported 7% apoptotic cells in control cases as well in contrast to 1.6% in our controls. Correspondingly, one of our striking finding is the strong corre-lation between the number of apoptotic cell and the recurrent nature of miscarriage.

Hammer and Dohr [17] investigated whether apoptosis occurs in the maternal decidua during the first trimester of preg-nancy by applying a TUNEL assay. It was noted that apoptotic nuclei, mainly present in CD45-positive leukocytes, could be detected in the trophoblast-invaded basal decidua as well as in the non-invaded parietal decidua. It was concluded that some of the maternal leukocytes were activated and induced to apoptosis by trophoblasts that express FasL. In contrast to this finding, we did not observe any apoptotic cells in the decidua during normal pregnancy, and TUNEL staining did not correspond to CD45-positive leukocytes in the recurrent miscarriage cases. Therefore, our finding might be specific to decidual malfunction and might thus provide insight to the pathophysiology of recurrent miscarriages.

In the current study, decidual apoptosis occurred via a caspase-dependent pathway was confirmed by the positive staining of TUNEL. On the other hand, the majority of caspase-3-positive cells were not co-labeled with TUNEL, indicating that many apop-totic cells were at the onset of the apoptotic pathway and had not yet reached the final stage of DNA damage. Although the findings of the current study revealed a relationship between the degree of

apoptosis in the decidua and the number of miscarriages, the role of apoptosis as a cause or result of miscarriage remains unclear.

Small sample is the major disadvantage of this study; further large sampled studies are needed to show pathophysiology of apoptosis and relationship with recurrent miscarriage.

AcknowledgementsThe authors are grateful to Dr. Y. Sanislioglu for his help in statis-tical analysis.

Declaration of interest: This work was supported by the Ankara University Biotechnology Institute.

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