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ORIGINAL RESEARCH |
From the Divisions of Maternal-Fetal Medicine and Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Address reprint requests to: Alexander D. Allaire, MD, MSPH Department of Obstetrics and Gynecology University of North Carolina, Chapel Hill 214 MacNider Campus Box #7570 Chapel Hill, NC 27599-7570 E-mail: allaire{at}med.unc.edu
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Abstract |
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Methods: Placental samples from 31 preeclamptic women and 31 normotensive controls were analyzed using terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining. Expression of Fas, Fas ligand, Bcl-2, and Bax was assessed using immunohistochemistry.
Results: The median percent apoptotic nuclei was significantly higher for the study group than for the controls (0.49 versus 0.19; P = .001), as was the median percent apoptotic nuclei in the trophoblast nuclei (0.33 versus 0.09; P < .01). Fas ligand expression was significantly less and Fas expression significantly greater in the villus trophoblast among the study subjects compared with controls. There was no difference in the expression of Bax or Bcl-2 between groups.
Conclusion: Placental apoptosis and altered expression of Fas and Fas ligand in trophoblast might influence pathogenesis or sequelae of preeclampsia.
Apoptosis, a form of programmed cell death, has been described in placentas of normal human pregnancies and is increased in pregnancies complicated by fetal growth resection (FGR).1,2 Apoptosis differs from necrosis in that the former is an active form of cell death dependent on the internal machinery of the cell and the latter is an accidental death caused by factors outside the cell.3,4
Preeclampsia affects 7–10% of all pregnancies and is a major cause of maternal and fetal morbidity and mortality, but its etiology remains unknown.5,6 Approximately 30% of fetuses born to preeclamptic women are at less than the tenth percentile for weight and are considered growth restricted.7 Histologic studies of placental bed biopsies have shown that interstitial cytotrophoblast invasion is often shallow and endovascular invasion nearly absent.8,9 Preeclampsia has also been attributed to a breakdown in maternal immune tolerance to foreign placental antigens.
The molecular mechanisms of apoptosis in humans are complex and involve an ever-expanding list of signaling molecules. Those include immune-mediated extracellular ligands and receptors such as the Fas ligand and Fas receptor, and endogenous death signals such as the Bcl-2 family of genes, which converge to activate a central executioner, the caspase cascade.10 The Bcl-2 gene family, isolated from a B-cell lymphoma, suppresses apoptosis and is involved in oncogenesis.11 The family includes apoptosis-promoting (Bax) and apoptosis-inhibiting (Bcl-2 and Bcl-x) members.10,11 In human pregnancy, Bcl-2 has been immunolocalized to the syncytiotrophoblast of the chorionic villi, persists from the first to the third trimester of pregnancy, and decreases as gestation progresses, suggesting an effect on normal aging of placenta.12,13 It is unknown whether the regulators of apoptosis are differentially expressed placentas of preeclamptic women.
Fas is express by many cell types including lymphocytes and trophoblasts. Fas ligand belongs to the tumor necrosis family and acts through its receptor, Fas, to induce apoptosis in cells that express it.14 Fas ligand is found in several immunologically privileged sites, such as the anterior chamber of the eye and the Sertolli cells of the testis, and might promote apoptosis of activated Fas-bearing lymphocytes that infiltrate those tissues.14 Fas ligand also is expressed in human trophoblast throughout gestation.15
The objective of this study was to determine whether there is an increase in placental apoptosis in pregnancies complicated by preeclampsia compared with placentas from normal pregnancy. We also wished to determine whether immune regulators of apoptosis such as Fas ligand and Fas and members of the Bcl-2 family are differentially expressed in placentas from pregnancies complicated by preeclampsia, compared with normal controls.
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Materials and Methods |
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Immediately after delivery, a sample of placenta was collected from the maternal–fetal interface and immediately snap-frozen in liquid nitrogen and stored at -80C. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate marker nick end-labeling staining was done on 6-µm cryosections using the Apop Tag In-Situ Apoptosis Detection kit (Intergen Company, Gaithersburg, MD) as described by Smith et al.1 Tissue cryosections were fixed in formalin. Endogenous peroxidases were quenched with 3% hydrogen peroxide in 100% methanol. Residues of digoxigeninnucleotide were catalytically added to the DNA by terminal deoxynucleotidyl transferase, an enzyme that catalyzes the addition of deoxyribonucleotide triphosphate to the 3'-OH ends of double- or single-stranded DNA. Antidigoxigenin antibody peroxidase conjugate was then applied. Filtered 0.05% diaminobenzidine (Sigma Chemical Co., St. Louis, MO) with 0.02% hydrogen peroxide was then applied to the sample. The antibody localized peroxidase enzyme catalytically generates an intense brown signal from chromogenic substrates, which can be easily viewed with light microscopy. The tissue was then counterstained with Toluidine blue and the slides were examined by light microscopy. Cryosections of postweaning mouse mammary gland were used as positive controls. A negative control for each section was done by substituting distilled water for terminal deoxyribonucleotide triphosphate enzyme. Digital images of ten randomly selected high-power fields (approximately x770 final magnification) of each placenta were obtained using a Nikon Microphot FXA microscope (Nikon Corp., Tokyo, Japan) and Scion Image software (Scion Corp., Frederick, MD). Apoptotic nuclei, easily differentiated from non-apoptotic nuclei by their brown labeling, were counted. The total nuclei and apoptotic nuclei in ten high-power fields for each sample were determined manually using Scion Image imaging software. The percentage apoptotic nuclei (number apoptotic nuclei per total number nuclei multiplied by 100) was calculated for each sample. The percentage of apoptotic nuclei also was calculated for placental trophoblast, stroma, and endothelial cells. Only nuclei with brown staining and morphologic criteria of apoptosis were considered apoptotic. Apoptotic nuclei can be identified by chromatin condensation resulting in a nuclear appearance of a single or multiple dark bodies. Areas of necrosis and inflammation were avoided in the analysis.
A mean of 3608 cells was counted in each sample. All sections were analyzed by two observers (ADA and KAB) who were masked to patient groups.
Sixty-two cryopreserved placental samples then were analyzed for expression of Fas, Fas ligand, Bax, and Bcl-2 using immunohistochemistry, as described.16 The antibodies used included monoclonal anti-Bcl-2 (Dako Corp., Carpinteria, CA), polyclonal rabbit anti-Bax (Dako Corp.), rabbit polyclonal anti-Fas (N-18) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and rabbit polyclonal anti-Fas ligand (N-20) (Santa Cruz Biotechnology Inc.). After immunostaining, sections were counterstained with hematoxylin. Those stained sections were evaluated on a Nikon microscope as above, by a masked observer (ADA). The semiquantitative immunohistochemical scoring system (HSCORE) was calculated using the equation:
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where i = intensity of staining with a value of 1, 2, or 3 (weak, moderate, or strong, respectively), and Pi is the percentage of stained trophoblast, stroma, or endothelial cells of each intensity. Previous studies that used the HSCORE have determined that technique yields low inter- and intraobserver variation and is a suitable semiquantitative method for comparing immunostaining results.17 To demonstrate satisfactory intraobserver variability all 62 specimens were read twice for expression of Fas (trophoblast), Fas ligand (trophoblast), and Bax (trophoblast and stroma).
Assuming a median apoptotic index of 0.14% with a standard deviation of 0.05 in controls, to detect a 0.05% increase in percent apoptosis with a power of 0.9 and P = .05, it was calculated that 22 subjects would be needed in each group.1,2 Data were considered non-parametric and samples were matched by gestational age, so the Wilcoxon matched pairs signed rank test was used for continuous variables. McNemar test for correlated proportions was used for categorical variables when the number of discrepancies in outcomes between matched pairs was at least 20; otherwise, McNemar exact test was used. Pearson correlation coefficients were calculated for intraobserver and interobserver variations. Statistical analysis was done with Stata Statistics/Data Analysis software (Stata Corporation, College Station, TX).
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Results |
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. Preeclamptic women and controls were similar in age, gestational age at delivery, parity, smoking status, and race-ethnicity. However, preeclamptic women delivered infants with significantly lower birth weights and were significantly more likely to have labor induced and to deliver infants at birth weights less than the tenth percentile, compared with controls.
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). The difference in apoptosis between preeclamptics and controls was limited to the trophoblast (Table 2). When analysis is limited to the 22 matched pairs with both infant birth weights greater than the tenth percentile, the percentage of apoptotic nuclei in placentas from preeclamptic women was significantly higher than the percentage of apoptotic nuclei in controls (median percent apoptotic nuclei 0.20 [interquartile range 0.10, 0.32] and 0.48 [interquartile range 0.21, 0.73]; P = .007). Intraobserver correlation coefficients were 0.99 and the interobserver correlation coefficient was 0.99 for calculated percentage of apoptosis; P < .001 for all correlations.
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) are presented in Table 3. Intraobserver correlation coefficients for observed HSCOREs were 0.77 (Fas, trophoblast), 0.65 (Fas ligand, trophoblast), 0.88 (Bax, trophoblast), and 0.65 (Bax, stroma); P < .001 for all correlations. Fas ligand expression in the villus trophoblast was less in preeclamptic women’s placentas than controls’ placentas. Fas expression in the villus trophoblast was greater in study subjects than controls. There was no statistically significant difference in percentage of apoptosis or Fas and Fas ligand expression in stroma and endotheliums between groups. There also was no statistically significant difference in expression of Bcl-2 or Bax in the villus trophoblast, stoma, or endothelium.
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Discussion |
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We also identified specific differences between the study and control groups in the expression of two immune regulators of apoptosis, Fas and Fas ligand. The Fas–Fas ligand system is believed to affect the death of inflammatory cells in immune-privileged sites such as the cornea of the eye and the testis.10 Fas and Fas ligand are membrane proteins that belong to the tumor necrosis factor receptor family of proteins. Fas is expressed widely by many tissues, whereas Fas ligand is expressed only by circulating lymphocytes and in immune-privileged sites. At those privileged sites, Fas-expressing peripheral T cells bind to Fas ligand in tissues expressing it. Fas delivers a signal to induce apoptosis in the peripheral lymphocyte.14 In that setting, circulating T cells that express Fas ligand can induce other T cells and peripheral tissues to undergo apoptosis through binding Fas on their membranes.10 Prior research has suggested that Fas ligand is normally expressed in human trophoblast throughout gestation and induces circulating activated T cells to undergo apoptosis.15 The expression of Fas ligand by the placenta might affect survival of the fetal allograft through the induction of apoptosis of circulating maternal leukocytes, allowing cytotrophoblasts to invade into the myometrium while escaping immune recognition. The Fas-expressing invading trophoblasts also might undergo apoptosis from Fas ligand–expressing T cells, limiting the extent of invasion. Alteration in the balance of mutual induction of apoptosis might affect diseases associated with abnormal placentation (eg, preeclampsia, FGR, placenta acreta/percreta, choriocarcinoma).
Preeclampsia has been attributed to a breakdown in maternal immune tolerance to foreign placental antigens.19 Trophoblasts from controls expressed Fas ligand more intensely than trophoblasts from preeclamptic women. Trophoblasts from preeclamptic women also expressed more Fas than trophoblasts from controls’ placentas. That suggests that the increase in placental apoptosis might result from alteration in the Fas–Fas ligand system, a concept supported by recent research that found impaired apoptosis in circulating leukocytes in women with preeclampsia compared with normotensive controls.20
There were a few limitations to the design of this study owing to our desire to include gestational age-matched controls. Our normal control group included many women with pregnancies complicated by preterm labor and delivery. Apoptosis increases as pregnancy progresses, so such age matching was important.1 Although we found an increase in apoptosis in the case group compared with controls, it is not clear whether factors associated with preterm labor could alter the pattern of apoptosis-related factors in controls. A second limitation was the use of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate marker nick-end labeling staining for identifying of apoptosis. Although this technique can show apoptosis reliably, there has been some concern that necrosis might cause false positive staining.1,2 The percentage of apoptosis using cryosections in our controls was similar to those of other investigators using light microscopy on paraffin-imbedded tissue, so we do not believe this is a concern. Conclusions were strengthened by the increase in expression of Fas and decrease in expression of Fas ligand in placentas from preeclamptic women. Our control group differed from the preeclamptic group in birth weight, which was expected given the association of growth restriction with preeclampsia.7 This study simply might have detected the previously described association between FGR and placental apoptosis.2 Conversely, FGR and preeclampsia might share a placental etiology of a common end point of placental apoptosis. We controlled for that potential confounding by showing an increase in placental apoptosis associated with preeclampsia in a subgroup of matched pairs not affected by FGR. To completely control for the potential confounding it would have been necessary to match controls for birth weight and gestational age.
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Footnotes |
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Received December 10, 1999. Received in revised form February 23, 2000. Accepted March 16, 2000.
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2. Smith SC, Baker PN, Symonds EM. Increased placental apoptosis in intrauterine growth restriction. Am J Obstet Gynecol 1997;177: 1395–401.[Medline]
3. Kerr IFR, Harmon BV. Definition and incidence of apoptosis: An historical perspective. In: Tomei LD, Cope FO, eds. Apoptosis: The molecular basis of programmed cell death. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1991.
4. Sayill J. Review: Apoptosis in disease. Eur J Clin Invest 1994;24: 715–23.[Medline]
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6. American College of Obstetricians and Gynecologists. Hypertension in pregnancy. ACOG technical bulletin no. 219, 1996.
7. Eskenazi B, Fenster L, Sydney S, Elkin EP. Fetal growth retardation in infants of multiparous and nulliparous women with preeclampsia. Am J Obstet Gynecol 1993;169:1112–8.[Medline]
8. Gerretsen G, Huisjes H, Elema JD. Morphological changes of the spiral arteries in the placenta bed in relation to preeclampsia and fetal growth retardation. Br J Obstet Gynaecol 1981;88:876–81.[Medline]
9. Zhou Y, Damsky CH, Chiu K, Roberts JM, Fisher SJ. Preeclampsia is associated with abnormal expression of adhesion molecules by invasive cytotrophoblasts. J Clin Invest 1993;91:950–60.
10. Hetts SW. To die or not to die: An overview of apoptosis and its role in disease. JAMA 1998;297:300–7.
11. Adams JM, Cory S. The Bcl-2 protein family; Arbiters of cell survival. Science 1998;281:1322–5.
12. Sakuragi N, Atsuo H, Coukos G. Differentiation-dependent expression of the Bcl-2 protooncogene in the human trophoblast lineage. J Soc Gynecol Invest 1994;1:164–72.[Medline]
13. Kim CJ, Choe YJ, Yoon BH, Kim CW, Chi JG. Patterns of bcl-2 expression I placenta. Pathol Res Pract 1995;191:1239–44.[Medline]
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15. Bamberger AM, Schulte HM, Thuneke I, Edrman I, Mamberger CM, Asa S. Expression of apoptosis inducing Fas ligand (FASL) in human first and third trimester placenta and choriocarcinoma cells. J Clin Endocrinol Metab 1997;82:3173–5.
16. Lessey BA, Damjanovich L, Cooutifaris C, Castelbaum A, Albela SM, Buck CA. Integrin adhesion molecules in the human endometrium. Correlation with the normal and abnormal menstrual cycle. J Clin Invest 1992;90:188–95.
17. Budwit-Novotny DA, McCarty KS, Cox EB, Soper JT, Mutch DG, Creasman WT, et al. Immunohistochemical analyses of estrogen receptor in endometrial adenocarcinoma using a monoclonal antibody. Cancer Res 1986;46:5419–25.
18. DiFederico E, Genbacev O, Fisher SJ. Preeclampsia is associated with widespread apoptosis of placental cytotrophoblasts within the uterine wall. Am J Pathol 1999;155:293–301.
19. Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: Current concepts. Am J Obstet Gynecol 1998;179:1359–75.[Medline]
20. von Dadelszen P, Watson RWG, Noorwali F, Marshall JC, Parodo J, Farine D, et al. Maternal neutrophil apoptosis in normal pregnancy, preeclampsia and normotensive intrauterine growth restriction. Am J Obstet Gynecol 1999;181:408–14.[Medline]
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