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Placental apoptosis in recurrent miscarriage
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MODEL
Kaohsiung Journal of Medical Sciences (2017) xx, 1e4
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
Original Article
Placental apoptosis in recurrent miscarriage Tarek A. Atia* Histology and Cell Biology, College of Applied Medical Sciences, Prince Sattam Bin Abdul Aziz University, Saudi Arabia Received 7 March 2017; accepted 20 June 2017
KEYWORDS Apoptosis; Immunohistochemistry; Placenta; RM
Abstract Apoptosis is an interactive and dynamic biological process involved in all phases of embryogenesis. We aimed to study the effect of placental apoptosis on recurrent miscarriage (RM). Placental tissue samples were collected from 40 women with RM (study group) and 30 women with sporadic spontaneous abortion (control group). Samples were prepared and stained immunohistochemically with markers for both the apoptotic protein (p53) and antiapoptotic Bcl-2 antibodies. Our results showed that expression of the apoptotic (p53) protein was significantly increased in the placental tissues of the RM group (p Z 0.003). By contrast, the expression of anti-apoptotic (Bcl-2) antibodies was significantly increased in the placental tissues of the control group (p Z 0.025). We concluded that placental apoptosis plays a crucial role in pregnancy continuation. However, increased p53 expression in placental tissue in early pregnancy could negatively affect pregnancy continuation. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Introduction Recurrent miscarriage (RM) is a common pregnancy complication. It is defined as three or more consecutive pregnancy losses that occur before the fetus reaches viability, i.e., prior to 24 gestational weeks [1]. The etiology of RM is variable, where unbalanced placental apoptosis plays an important role [2]. Normal placental Conflicts of interest: The author declares no conflicts of interests. * Corresponding author. Histology and Cell Biology, College of Applied Medical Sciences, Prince Sattam Bin Abdul Aziz University, P.O. Box 422, Riyadh 11942, Saudi Arabia. E-mail address: [email protected].
development undergoes several sequences of cell division and differentiation, followed by invasion of the embryonic trophoblast cells into the decidua and remodeling of the vasculature to increase blood flow into both the placenta and the fetus. The placenta then goes through a series of tissue remodeling with apoptotic changes, which result in regular loss of trophoblast cells [3]. Apoptotic changes have been detected in the maternalefetal interfaces of the placenta during normal as well as complicated pregnancies [4]. Additionally, apoptosis promotes maternal immune tolerance to the paternal antigens expressed by the trophoblast cells [5,6]. Apoptosis is an interactive and dynamic biological process involved in all phases of embryogenesis. It comprises elimination of undesirable cells to maintain normal tissue
http://dx.doi.org/10.1016/j.kjms.2017.06.012 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012
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MODEL
2 function. Apoptosis could be activated through either intrinsic (mitochondrial mediated) or extrinsic (death receptor mediated) apoptotic pathways [7]. In intrinsic pathways, mitochondrial response to cellular stress, such as DNA damage, usually initiates apoptotic signals. P53, a tumor suppressor protein, could activate the mitochondrial pathway, which in turn could activate pro-apoptotic Bcl-2 family members. In addition, certain death receptors and the mitochondrial pathway may act to amplify signals triggered by the death receptor pathway, suggesting that crossover can occur between the two pathways [8,9]. In our study, we investigated the effect of placental expression of apoptotic protein as well as anti-apCoptotic antibodies on pregnancy continuation.
Patients and methods Patients After obtaining approval from the Hospital Ethics Committee, this prospective caseecontrol study was carried out between April 2016 and October 2016 in the Obstetrics and Gynecology Department of Al-Azhar University Hospitals, Cairo, Egypt. It included 40 women with RM as a study group and 30 women with sporadic spontaneous miscarriage (spontaneous abortion of undetected cause, after having at least one normal pregnancy) as a control group. Members of the control group were matched for age with those with RM. Written informed consent was obtained from all women who were enrolled in the study. Placental samples were taken from all cases by dilatation and evacuation without any prior pharmaceutical induction and within the first 24 h post-diagnosis. The following data were collected from all women enrolled in the study: age, parity, body mass index (BMI), and number of previous abortions and maternal diseases. Next, a thorough clinical examination was performed, which aimed to exclude common disorders already known as aggravating factors for an increased risk of abortion. Following the evacuation, specimens from both groups were fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin wax, and sectioned and mounted onto APES coated slides.
Immunohistochemical staining An immunohistochemical assay for anti-apoptotic antibody Bcl-2 and apoptotic protein p53 expression were performed on formalin fixed, paraffin-embedded tissue sections using the peroxidase labeled avidinebiotin method. Commercially available antibodies for both the Bcl-2 antibody [100: SC-509] and the p53 antibody [(BP 53.12): sc-81168] for apoptotic cells (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) were used to recognize these antibodies, according to the manufacturer’s instructions. Sequential slides with 4 mm-thick tissue sections were first dewaxed followed by hydration in a sequential treatment of xylene, ethanol, and water. Then, the slides were incubated in heated citrate buffer (0.01 M citric acid, pH 6.0) to retrieve the antigens. Endogenous peroxidase activity and non-specific bindings were blocked with 3% H2O2. The slides were then incubated with primary antibodies
T.A. Atia (dilution 1:200) overnight at 4 C, followed by the addition of a biotinylated secondary antibody and streptavidinehorseradish peroxidase. The peroxidase reaction was developed with 3,30 -diaminobenzidine (DAB; Sigma Chemical Co.), which resulted in a brown-colored product. Finally, the slides were counterstained with hematoxylin, dehydrated in alcohol, and mounted.
Data registration and statistical analysis Both the number and the optical density of the immunopositive Bcl-2 and p53 cells were evaluated. Digital images of 10 randomly selected, high-power fields [hpf] (400) were both captured and analyzed using a Carl Zeis microscope and Zen Image software (2012, blue edition). Computerized image analysis (Optical Density) was used to accurately measure the strength of the immunohistochemical reactions instead of visual analysis, with the reactions designated as either (þ, or weak), (þþ or moderate), or (þþþ or strong reaction). The data were presented as a mean standard deviation (SD). Continuous variables were compared using the paired and independent student t-tests. Values of p < 0.05 were considered statistically significant.
Result Maternal demographic data Seventy women were enrolled in this study: 40 women with RM (study group) and 30 women with sporadic spontaneous miscarriages (control group). Both groups showed no significant differences with respect to age (p Z 0.7), parity (p Z 0.9), BMI (p Z 0.4), and gestational age (p Z 0.4) at the time of miscarriage; however, the number of abortions was significantly higher among the study group than among the control group (p Z 0.001) (Table 1).
Apoptotic p53 protein and anti-apoptotic Bcl-2 expression Apoptotic changes in placental tissues were examined by detecting the expression of the p53 protein in comparison to expression of the anti-apoptotic (Bcl-2) antibody. In the placental tissues of the RM group (Fig. 1A), the mean number of apoptotic/p53 immunopositive cells was significantly higher than that of the control group (Fig. 1B) (mean Z 39 8.831761 vs. 18 6.839428) respectively (p Z 0.003) (Table 2). By Table 1 Maternal demographic data; where BMI Z Body Mass Index. Study group Control group P (n Z 40) (n Z 30) value Maternal age (years SD) 22.5 3.2 23.7 2.1 Parity 2.7 1.4 2.6 2.4 24.1 3.3 23.8 2.6 BMI (kg/m2) Gestational age at 12.6 2.1 11.3 3.3 time of abortion Number of abortions 5.1 ± 0.7 0.00
0.7 0.9 0.4 0.4 0.001
Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012
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Placental apoptosis in recurrent miscarriage
3
Figure 1. Immunohistochemistry staining the expression of apoptotic protein (p53) in the trophoblast cells of the chorionic villi of RM cases (A), which is significantly higher than that of sporadic abortion cases (B) [P Z 0.003] [400].
contrast, the mean number of anti-apoptotic/Bcl-2 immunopositive cells was significantly higher in the placental tissues of the control group (Fig. 2B) than in the RM cases (Fig. 2A) (mean Z 30 10.95648 vs. 16 7.258788) respectively (p Z 0.025). Interestingly, the optical densities of both the p53 and bcl2 expression showed non-significant variation between the investigated groups.
Discussion Balanced cell proliferation and death in the maternalefetal interface are important processes for normal embryogenesis. Conversely, either unbalanced cell death or apoptosis could disturb fetal development and may cause fetal loss. In
addition, the interaction between pro- and anti-apoptotic pathways can either regulate, stimulate, or inhibit cellular apoptosis [10]. In the current study, the placental apoptotic and antiapoptotic cellular activities in RM and sporadic abortion cases were investigated using p53 and Bcl-2, respectively. The expression of the apoptotic p53 protein was significantly increased [p Z 0.003], with a significant reduction of the expression of the anti-apoptotic Bcl-2 antibody [p Z 0.025] in the placental tissues of the RM group, as compared to those in the control cases. Over-expression of p53 in the placental tissues of patients with RM has been reported by other investigators [5,6]. However, Bcl-2 expression was down-regulated in RM, with a significant
Table 2 Statistical analysis showing the mean, SD and P-values of immuno-positive Bcl-2 and p53 cell population and their optical density in RM (study group) vs Sporadic Spontaneous Abortion cases (control group). Immuno-positive p53- cell count P53- Optical density Immuno-positive Bcl-2 cells count Bcl-2 cell Optical density
Study group (n Z 40)
Control group (n Z 30)
P-value
39 8.831761 0.40260025 0.22640533 16 7.258788 0.42585494 0.13987284
18 6.839428 0.455682228 0.20412076 30 10.95648 0.49377978 0.11251472
0.003 0.618 0.025 0.262
Figure 2. Immunohistochemistry staining the expression of anti-apoptotic protein (Bcl-2) in the trophoblast cells of the chorionic villi of RM cases (A), which is significantly decreased than that of the sporadic abortion cases (B) [P Z 0.025] [400]. Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012
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MODEL
4 reduction of anti-apoptotic Bcl-2 expression [11]. These findings agreed with our results. We have speculated that Bcl-2 over-expression with a reduction of p53 expression is essential for normal placental development and pregnancy continuation. Therefore, p53 over-expression in placental tissues could result in the failure of placentation [10], while decreased Bcl-2 expression could decrease the survival of chorionic villi [11]. In addition, abnormal expression of some apoptotic related genes, such as TNF-a and TGF-b, and other factors could affect the expression of p53 and/or Bcl2, resulting in abortion [12]. Recently, computerized methods have become more widely used to evaluate the strength of immunohistochemical reactions versus the conventional method [13,14]. The convenient visual method, based on the Allred-score method [15], used an average score of 0e3, where a score of 0 equals a negative reaction and scores of 1e3 equal weak, moderate, and strong reactions, respectively, to evaluate the reaction’s intensity. However, some investigators noticed certain variations when comparing the strength of the immunohistochemical reaction by using computerized methods versus the convenient visual method. These variations could affect the accuracy of the reaction’s strength [13,14,16]. In conclusion, balanced placental apoptosis plays a crucial role in pregnancy continuation. Therefore, apoptotic p53 protein over-expression, together with decreased anti-apoptotic Bcl-2 expression in placental tissue during early pregnancy, could lead to pregnancy failure.
Acknowledgment The author is grateful to Drs. M. Abdelzaher and M. Ali from the Gynecology and Obstetrics Department at Al-Azhar University for their assistance in both the data and sample collection processes.
References [1] Wang L, Tang H, Xiong Y, Tang L. Differential expression profile of long noncoding RNAs in human chorionic villi of early recurrent miscarriage. Clin Chim Acta 2017;464:17e23. [2] Rull K, Tomberg K, Ko ˜ks S, Ma ¨nnik J, Mo ¨ls M, Sirotkina M, et al. Increased placental expression and maternal serum levels of apoptosis-inducing TRAIL in recurrent miscarriage. Placenta 2013;34:141e8. [3] Mills CD. M1 and M2 macrophages: oracles of health and disease. Crit Rev Immunol 2012;32:463e88.
T.A. Atia [4] Ishihara N, Matsuo H, Murakoshi H, Laoag-Fernandez JB, Samoto T, Maruo T. Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation. Am J Obstet Gynecol 2002;186:158e66. [5] Mayhew TM. Villous trophoblast of human placenta: a coherent view of its turnover, repair and contributions to villous development and maturation. Histol Histopathol 2001; 16:1213e24. [6] Wei D, Wu Q, Shi H. Apoptosis and p53 expression in the placental villi of females with unexplained recurrent spontaneous abortion. Exp Ther Med 2014;7:191e4. [7] Merkis C, Cristofolini A, Sanchis E, Koncurat M. Expression of death cellular receptors FAS/CD95 and DR4 during porcine placentation. Int J Morphol 2010;28:829e34. [8] Guenther S, Vrekoussis T, Heublein S, Bayer B, Anz D, Knabl J, et al. Decidual macrophages are significantly increased in spontaneous miscarriages and over-express FasL: a potential role for macrophages in trophoblast apoptosis. Int J Mol Sci 2012;13:9069e80. [9] Shang W, Shu MM, Liu M, Wang AM, Lv B, Zhao Y, et al. Elevated expressions of p53, CDKNA1, and Bax in placental villi from patients with recurrent spontaneous abortion. Eur Rev Med Pharmacol Sci 2013;17:3376e80. [10] Deftereou T-E, Lambropoulou M, Chatzaki E, Koffa M, Ypsilantis P, Pagonopoulou O, et al. Increased apoptotic activity on inflammatory human placentas in spontaneous abortions during the first and second trimester of gestation: a histochemical and immunohistochemical study. Folia Histochem Cytobiol 2012;50:118e24. _ glu O, Terek C, Ozdemir N, Ender Du [11] Kelten C, Ze kIo ¨zcan E. Expression of Ki-67, Bcl-2 and Bax in the first trimester abortion materials. Turk J Pathol 2010;26:31e7. [12] Shi W-Y, Che C-Y, Liu L. Human interleukin 23 receptor induces cell apoptosis in mammalian cells by intrinsic mitochondrial pathway associated with the down-regulation of RAS/mitogen-activated protein kinase and signal transducers and activators of transcription factor 3 signaling pathways. Int J Mol Sci 2013;14:24656e69. [13] Rizzardi AE, Johnson AT, Vogel RI, Pambuccian SE, Henriksen J, Skubitz AP, et al. Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol 2012;7: 42. [14] Cahall CF, Lilly JL, Hirschowitz EA, Berron BJ. A quantitative perspective on surface marker selection for the isolation of functional tumor cells. Breast Cancer (Auckl) 2015;9:1e11. [15] Allred DC, Carlson RW, Berry DA, Burstein HJ, Edge SB, Goldstein LJ, et al. NCCN Task Force report: estrogen receptor and progesterone receptor testing in breast cancer by immunohistochemistry. J Natl Compr Canc Netw 2009;7: S1e21. [16] Cohen DA, Dabbs DJ, Cooper KL, Amin M, Jones TE, Jones MW, et al. Interobserver agreement among pathologists for semiquantitative hormone receptor scoring in breast carcinoma. Am J Clin Pathol 2012;138:796e802.
Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012
MODEL
Kaohsiung Journal of Medical Sciences (2017) xx, 1e4
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
Original Article
Placental apoptosis in recurrent miscarriage Tarek A. Atia* Histology and Cell Biology, College of Applied Medical Sciences, Prince Sattam Bin Abdul Aziz University, Saudi Arabia Received 7 March 2017; accepted 20 June 2017
KEYWORDS Apoptosis; Immunohistochemistry; Placenta; RM
Abstract Apoptosis is an interactive and dynamic biological process involved in all phases of embryogenesis. We aimed to study the effect of placental apoptosis on recurrent miscarriage (RM). Placental tissue samples were collected from 40 women with RM (study group) and 30 women with sporadic spontaneous abortion (control group). Samples were prepared and stained immunohistochemically with markers for both the apoptotic protein (p53) and antiapoptotic Bcl-2 antibodies. Our results showed that expression of the apoptotic (p53) protein was significantly increased in the placental tissues of the RM group (p Z 0.003). By contrast, the expression of anti-apoptotic (Bcl-2) antibodies was significantly increased in the placental tissues of the control group (p Z 0.025). We concluded that placental apoptosis plays a crucial role in pregnancy continuation. However, increased p53 expression in placental tissue in early pregnancy could negatively affect pregnancy continuation. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Introduction Recurrent miscarriage (RM) is a common pregnancy complication. It is defined as three or more consecutive pregnancy losses that occur before the fetus reaches viability, i.e., prior to 24 gestational weeks [1]. The etiology of RM is variable, where unbalanced placental apoptosis plays an important role [2]. Normal placental Conflicts of interest: The author declares no conflicts of interests. * Corresponding author. Histology and Cell Biology, College of Applied Medical Sciences, Prince Sattam Bin Abdul Aziz University, P.O. Box 422, Riyadh 11942, Saudi Arabia. E-mail address: [email protected].
development undergoes several sequences of cell division and differentiation, followed by invasion of the embryonic trophoblast cells into the decidua and remodeling of the vasculature to increase blood flow into both the placenta and the fetus. The placenta then goes through a series of tissue remodeling with apoptotic changes, which result in regular loss of trophoblast cells [3]. Apoptotic changes have been detected in the maternalefetal interfaces of the placenta during normal as well as complicated pregnancies [4]. Additionally, apoptosis promotes maternal immune tolerance to the paternal antigens expressed by the trophoblast cells [5,6]. Apoptosis is an interactive and dynamic biological process involved in all phases of embryogenesis. It comprises elimination of undesirable cells to maintain normal tissue
http://dx.doi.org/10.1016/j.kjms.2017.06.012 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012
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MODEL
2 function. Apoptosis could be activated through either intrinsic (mitochondrial mediated) or extrinsic (death receptor mediated) apoptotic pathways [7]. In intrinsic pathways, mitochondrial response to cellular stress, such as DNA damage, usually initiates apoptotic signals. P53, a tumor suppressor protein, could activate the mitochondrial pathway, which in turn could activate pro-apoptotic Bcl-2 family members. In addition, certain death receptors and the mitochondrial pathway may act to amplify signals triggered by the death receptor pathway, suggesting that crossover can occur between the two pathways [8,9]. In our study, we investigated the effect of placental expression of apoptotic protein as well as anti-apCoptotic antibodies on pregnancy continuation.
Patients and methods Patients After obtaining approval from the Hospital Ethics Committee, this prospective caseecontrol study was carried out between April 2016 and October 2016 in the Obstetrics and Gynecology Department of Al-Azhar University Hospitals, Cairo, Egypt. It included 40 women with RM as a study group and 30 women with sporadic spontaneous miscarriage (spontaneous abortion of undetected cause, after having at least one normal pregnancy) as a control group. Members of the control group were matched for age with those with RM. Written informed consent was obtained from all women who were enrolled in the study. Placental samples were taken from all cases by dilatation and evacuation without any prior pharmaceutical induction and within the first 24 h post-diagnosis. The following data were collected from all women enrolled in the study: age, parity, body mass index (BMI), and number of previous abortions and maternal diseases. Next, a thorough clinical examination was performed, which aimed to exclude common disorders already known as aggravating factors for an increased risk of abortion. Following the evacuation, specimens from both groups were fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin wax, and sectioned and mounted onto APES coated slides.
Immunohistochemical staining An immunohistochemical assay for anti-apoptotic antibody Bcl-2 and apoptotic protein p53 expression were performed on formalin fixed, paraffin-embedded tissue sections using the peroxidase labeled avidinebiotin method. Commercially available antibodies for both the Bcl-2 antibody [100: SC-509] and the p53 antibody [(BP 53.12): sc-81168] for apoptotic cells (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) were used to recognize these antibodies, according to the manufacturer’s instructions. Sequential slides with 4 mm-thick tissue sections were first dewaxed followed by hydration in a sequential treatment of xylene, ethanol, and water. Then, the slides were incubated in heated citrate buffer (0.01 M citric acid, pH 6.0) to retrieve the antigens. Endogenous peroxidase activity and non-specific bindings were blocked with 3% H2O2. The slides were then incubated with primary antibodies
T.A. Atia (dilution 1:200) overnight at 4 C, followed by the addition of a biotinylated secondary antibody and streptavidinehorseradish peroxidase. The peroxidase reaction was developed with 3,30 -diaminobenzidine (DAB; Sigma Chemical Co.), which resulted in a brown-colored product. Finally, the slides were counterstained with hematoxylin, dehydrated in alcohol, and mounted.
Data registration and statistical analysis Both the number and the optical density of the immunopositive Bcl-2 and p53 cells were evaluated. Digital images of 10 randomly selected, high-power fields [hpf] (400) were both captured and analyzed using a Carl Zeis microscope and Zen Image software (2012, blue edition). Computerized image analysis (Optical Density) was used to accurately measure the strength of the immunohistochemical reactions instead of visual analysis, with the reactions designated as either (þ, or weak), (þþ or moderate), or (þþþ or strong reaction). The data were presented as a mean standard deviation (SD). Continuous variables were compared using the paired and independent student t-tests. Values of p < 0.05 were considered statistically significant.
Result Maternal demographic data Seventy women were enrolled in this study: 40 women with RM (study group) and 30 women with sporadic spontaneous miscarriages (control group). Both groups showed no significant differences with respect to age (p Z 0.7), parity (p Z 0.9), BMI (p Z 0.4), and gestational age (p Z 0.4) at the time of miscarriage; however, the number of abortions was significantly higher among the study group than among the control group (p Z 0.001) (Table 1).
Apoptotic p53 protein and anti-apoptotic Bcl-2 expression Apoptotic changes in placental tissues were examined by detecting the expression of the p53 protein in comparison to expression of the anti-apoptotic (Bcl-2) antibody. In the placental tissues of the RM group (Fig. 1A), the mean number of apoptotic/p53 immunopositive cells was significantly higher than that of the control group (Fig. 1B) (mean Z 39 8.831761 vs. 18 6.839428) respectively (p Z 0.003) (Table 2). By Table 1 Maternal demographic data; where BMI Z Body Mass Index. Study group Control group P (n Z 40) (n Z 30) value Maternal age (years SD) 22.5 3.2 23.7 2.1 Parity 2.7 1.4 2.6 2.4 24.1 3.3 23.8 2.6 BMI (kg/m2) Gestational age at 12.6 2.1 11.3 3.3 time of abortion Number of abortions 5.1 ± 0.7 0.00
0.7 0.9 0.4 0.4 0.001
Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012
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Placental apoptosis in recurrent miscarriage
3
Figure 1. Immunohistochemistry staining the expression of apoptotic protein (p53) in the trophoblast cells of the chorionic villi of RM cases (A), which is significantly higher than that of sporadic abortion cases (B) [P Z 0.003] [400].
contrast, the mean number of anti-apoptotic/Bcl-2 immunopositive cells was significantly higher in the placental tissues of the control group (Fig. 2B) than in the RM cases (Fig. 2A) (mean Z 30 10.95648 vs. 16 7.258788) respectively (p Z 0.025). Interestingly, the optical densities of both the p53 and bcl2 expression showed non-significant variation between the investigated groups.
Discussion Balanced cell proliferation and death in the maternalefetal interface are important processes for normal embryogenesis. Conversely, either unbalanced cell death or apoptosis could disturb fetal development and may cause fetal loss. In
addition, the interaction between pro- and anti-apoptotic pathways can either regulate, stimulate, or inhibit cellular apoptosis [10]. In the current study, the placental apoptotic and antiapoptotic cellular activities in RM and sporadic abortion cases were investigated using p53 and Bcl-2, respectively. The expression of the apoptotic p53 protein was significantly increased [p Z 0.003], with a significant reduction of the expression of the anti-apoptotic Bcl-2 antibody [p Z 0.025] in the placental tissues of the RM group, as compared to those in the control cases. Over-expression of p53 in the placental tissues of patients with RM has been reported by other investigators [5,6]. However, Bcl-2 expression was down-regulated in RM, with a significant
Table 2 Statistical analysis showing the mean, SD and P-values of immuno-positive Bcl-2 and p53 cell population and their optical density in RM (study group) vs Sporadic Spontaneous Abortion cases (control group). Immuno-positive p53- cell count P53- Optical density Immuno-positive Bcl-2 cells count Bcl-2 cell Optical density
Study group (n Z 40)
Control group (n Z 30)
P-value
39 8.831761 0.40260025 0.22640533 16 7.258788 0.42585494 0.13987284
18 6.839428 0.455682228 0.20412076 30 10.95648 0.49377978 0.11251472
0.003 0.618 0.025 0.262
Figure 2. Immunohistochemistry staining the expression of anti-apoptotic protein (Bcl-2) in the trophoblast cells of the chorionic villi of RM cases (A), which is significantly decreased than that of the sporadic abortion cases (B) [P Z 0.025] [400]. Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012
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MODEL
4 reduction of anti-apoptotic Bcl-2 expression [11]. These findings agreed with our results. We have speculated that Bcl-2 over-expression with a reduction of p53 expression is essential for normal placental development and pregnancy continuation. Therefore, p53 over-expression in placental tissues could result in the failure of placentation [10], while decreased Bcl-2 expression could decrease the survival of chorionic villi [11]. In addition, abnormal expression of some apoptotic related genes, such as TNF-a and TGF-b, and other factors could affect the expression of p53 and/or Bcl2, resulting in abortion [12]. Recently, computerized methods have become more widely used to evaluate the strength of immunohistochemical reactions versus the conventional method [13,14]. The convenient visual method, based on the Allred-score method [15], used an average score of 0e3, where a score of 0 equals a negative reaction and scores of 1e3 equal weak, moderate, and strong reactions, respectively, to evaluate the reaction’s intensity. However, some investigators noticed certain variations when comparing the strength of the immunohistochemical reaction by using computerized methods versus the convenient visual method. These variations could affect the accuracy of the reaction’s strength [13,14,16]. In conclusion, balanced placental apoptosis plays a crucial role in pregnancy continuation. Therefore, apoptotic p53 protein over-expression, together with decreased anti-apoptotic Bcl-2 expression in placental tissue during early pregnancy, could lead to pregnancy failure.
Acknowledgment The author is grateful to Drs. M. Abdelzaher and M. Ali from the Gynecology and Obstetrics Department at Al-Azhar University for their assistance in both the data and sample collection processes.
References [1] Wang L, Tang H, Xiong Y, Tang L. Differential expression profile of long noncoding RNAs in human chorionic villi of early recurrent miscarriage. Clin Chim Acta 2017;464:17e23. [2] Rull K, Tomberg K, Ko ˜ks S, Ma ¨nnik J, Mo ¨ls M, Sirotkina M, et al. Increased placental expression and maternal serum levels of apoptosis-inducing TRAIL in recurrent miscarriage. Placenta 2013;34:141e8. [3] Mills CD. M1 and M2 macrophages: oracles of health and disease. Crit Rev Immunol 2012;32:463e88.
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Please cite this article in press as: Atia TA, Placental apoptosis in recurrent miscarriage, Kaohsiung Journal of Medical Sciences (2017), http://dx.doi.org/10.1016/j.kjms.2017.06.012