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Blood SC5b-9 complement levels increase at parturition during term and preterm labor
Journal of Reproductive Immunology 109 (2015) 24–30
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Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Blood SC5b-9 complement levels increase at parturition during term and preterm labor Enrique Segura-Cervantes a , Javier Mancilla-Ramirez b , Luis Zurita c , Yuriria Paredes d , José Luis Arredondo e , Norma Galindo-Sevilla a,∗ a b c d e
Departamento de Infectología e Inmunología Perinatal, Instituto Nacional de Perinatologia (INPer), Mexico Escuela Superior de Medicina, IPN, Mexico División Académica de Ciencias de la Salud, UJAT, Mexico Departmento de Biología Celular, INPer, Mexico Unidad de Investigación Clínica, INP, Mexico
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Article history: Received 20 September 2014 Received in revised form 17 February 2015 Accepted 19 February 2015 Keywords: Complement Pregnancy Parturition Labor Fetal membranes
a b s t r a c t We explored the hypothesis that complement, an innate and adaptive immune effector, is active in the plasma of parturient women and is deposited on fetal membranes collected after delivery. A cross-sectional study was designed to evaluate complement activity at parturition. Pregnant women (n = 97) between 15 and 41 years of age were enrolled in a hospital protocol during the perinatal period to assess both SC5b-9 complement activity in blood and complement deposition on fetal membranes during parturition. Soluble SC5b-9 complement activity in plasma fractions was measured using a standard enzyme-linked immunosorbent assay (ELISA) that included specific anti-complement antibodies. Complement deposition on membranes was analyzed using immuno-dot blots and immunohistochemistry. Soluble SC5b-9 complement complex levels were increased in the plasma of women during term labor (TL; median 3361; range 1726–5670 ng/mL), preterm labor (PL; median 2958; range 1552–7092 ng/mL), and preterm premature rupture of membranes (PPROM; median 2272; range 167–6540 ng/mL) compared with pregnant women who were not in labor (P; median 1384; range 174–4570 ng/mL; P < 0.001, Kruskal–Wallis test). Active complement, as assessed by the C9 neo-antigen in C5b-9 complexes, was deposited on fetal membranes, with no difference between term and preterm delivery. The deposition of active complement on fetal membranes was confirmed by immunohistochemistry. Women who underwent non-labor-indicated Cesarean sections did not exhibit complement deposition. Soluble SC5b-9 complement complex levels increased in the plasma of women during parturition, and complement C5b-9 complexes were deposited on fetal membranes. © 2015 Elsevier Ireland Ltd. All rights reserved.
∗ Corresponding author at: Departamento de Infectología e Inmunología Perinatal, Instituto Nacional de Perinatología, Montes Urales #800, Col. Lomas de Virreyes, Mexico City 11000, Mexico. Tel.: +52 55 55 20 99 00x103. E-mail addresses: [email protected] (E. Segura-Cervantes), [email protected] (J. Mancilla-Ramirez), [email protected] (L. Zurita), [email protected] (Y. Paredes), [email protected] (J.L. Arredondo), [email protected] (N. Galindo-Sevilla). http://dx.doi.org/10.1016/j.jri.2015.02.008 0165-0378/© 2015 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction Parturition is a physiological process that requires the synchronization of uterine contractions, cervical dilatation, and membrane rupture. Several reviews have emphasized that the physiological phenomena, molecules, and cellular mechanisms that drive the process of human parturition are not completely understood (Breuiller-Fouche and Germain, 2006). The complement system is an important effector of the immune system because it forms pores in cell membranes that lead to cell lysis; thus, the complement system is thought to play a crucial role during parturition. C4B and C1R protein levels are lower during spontaneous vaginal delivery (SVD) than during pre-labor in the same women (Yuan et al., 2012). However, the complement system and its role during parturition have not been explored as extensively as the cytokines that mediate the parturition-dependent inflammatory response. New techniques, including standard enzyme-linked immunoabsorbent assay (ELISA), now permit the evaluation of complement activity with improved sensitivity. Different methodological approaches have been used to determine complement activity in biological fluids, such as those used to assess the protein complex C5b-9 in plasma. This protein complex is a common mediator of cell lysis that is generated from any of the four complement activation pathways (Huber-Lang et al., 2006). The regulatory S protein binds to C5b-9, forming the SC5b-9 complex, which is soluble and can be detected in plasma by ELISA (Mollnes et al., 1985). We hypothesized that the complement system is active at parturition during established labor. This study aimed to investigate whether the active form of complement, C5b-9, is elevated in plasma as SC5b-9 at parturition and whether complement is deposited on fetal membranes. 2. Materials and methods 2.1. Study design To understand complement activity at parturition, a cross-sectional study was designed to assess complement activity in the plasma and complement deposition on fetal membranes. Pregnant women (n = 97) between 15 and 41 years of age who were treated as outpatients from 12 weeks of gestation until delivery at the National Institute of Perinatology (INPer, Mexico City, Mexico) were recruited for the study. Patients with cancer and patients receiving immunotherapy for transplants or autoimmunity or current steroid treatment were excluded from the study. The patients provided written informed consent during their clinical check-ups. Each patient consented to donate four fetal membrane samples (1 cm in diameter) after childbirth. Furthermore, one 3-mL sample of whole peripheral blood was obtained from each patient at delivery or during normal examinations as stated in Section 3. The study participants included pregnant women (P) at <37 weeks of gestational age. Sample membranes were also isolated from women undergoing an indicated Cesarean section at <36 weeks. Women at >37 weeks of gestational age comprised the term labor (TL) group, and women at <37 weeks
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of gestational age were assigned to one of two subgroups: the preterm labor (PL) or preterm premature rupture of membranes (PPROM) group. A flow diagram is presented in Fig. 1. The study participants were at minimal risk. The study was approved by the research and bioethical committees of the National Institute of Perinatology and was in compliance with international guidelines for human studies. 2.2. SC5b-9 protein complex assessment The C5b-9 complex binds the regulatory S protein after the C5b-7 protein complex is properly assembled in the plasma (Mollnes et al., 1985; Bhakdi and TranumIensen, 1982). SC5b-9 complex levels in the plasma are indicative of complement activation, which causes subsequent cell lysis. SC5b-9 complex levels were analyzed using a standard ELISA assay (Quidel, Mountain View, CA, USA) as previously reported (Walter et al., 2010). Participant plasma samples were routinely diluted 1:40. The samples were assayed in triplicate wells (100 L) and were incubated for 60 min according to the manufacturer’s instructions. The method was quantitative for SC5b-9 complex levels ranging from 20 to 150 ng/mL. The color was developed by incubating the samples with 100 L/well of the chromogenic substrate OPD (O-phenylenediamine dihydrochloride, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. The plates were read using an automated ELISA plate reader (Synergy 2, Bio-Tek Instruments, Winooski, VT, USA) after the enzyme reaction was terminated by the addition of 50 L of 0.1 M H2 SO4 . The absorbance was measured at 405 nm. 2.3. Fetal membrane sampling We investigated whether active complement was deposited on fetal membranes (1 cm in diameter), including the chorion and amnion. The fetal membranes were obtained within the first 30 min after childbirth and were assessed in duplicate at four points from the rupture site following a previously published protocol (McLaren et al., 1999)Ref. . One membrane sample from each duplicate was incubated in phosphate-buffered saline (PBS; pH 7.4) containing a mammalian protease inhibitor cocktail (PIC, 1:50 dilution; Sigma-Aldrich, St. Louis, MO, USA). The samples were frozen at −70 ◦ C and stored until they were used in standard immuno-dot blot assays (Janyapoon et al., 2000) of membrane complement components. The other membrane samples were used for the immunohistochemical detection of complement components. 2.4. Protein membrane extraction and immuno-dot blot assay Complement components were detected in protein fractions extracted from fetal membranes. Briefly, the membrane tissue was mechanically disrupted in a tissue grinder with 3 mL of cold PBS containing a protease inhibitor cocktail (PBS-PIC). The suspended cells were recovered and centrifuged at 400 × g. The cell pellet was
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Fig. 1. A flow diagram of the patient selection process.
disrupted by several freeze–thaw cycles. The suspended fraction was transferred to an Eppendorf tube and centrifuged at 20,000 × g for 20 min. The collected supernatant was discarded, and the pellet was re-suspended in 200 L of PBS-PIC with 1% Nonidet P-40 (a non-ionic detergent used to extract proteins from cell membranes, SigmaAldrich, St. Louis, MO, USA). The protein samples were re-centrifuged at 20,000 × g for 30 min. Then, 1 L of the supernatant was directly applied to a polyvinylidene difluoride membrane in triplicate (0.45-m PVDF membranes, Bio-Rad Labs, Hercules, CA, USA). Non-specific binding sites were blocked for 1 h in PBS (pH 7.4) containing 5% low-fat milk. The membranes were incubated overnight at 4 ◦ C with a 1:50 dilution of an HRP-conjugated anti-C9 neo-antigen monoclonal antibody (Quidel, Mountain View, CA, USA). The monoclonal antibodies used to detect the complement components in this assay were labeled with horseradish peroxidase (HRP; Sigma-Aldrich, St. Louis, MO, USA) in our laboratory according to a standard procedure (Harlow and Lane, 1988). The membranes were washed three times (5 min each) with PBS (pH 7.4)/0.05% Tween-20 to remove excess unbound antibody. The peroxidase-positive dots were detected by immersing the membrane in a developing solution (70 mM sodium acetate at pH 6.2 containing 0.3% diaminobenzidine tetrahydrochloride and 0.03% H2 O2 ) at room temperature for 5 min. The enzymatic reaction was terminated by washing the sheet. The immunoreactive protein signals were detected using an AlphaImager HP System (Cell Biosciences, Inc., Santa Clara, CA, USA).
2.5. Immunohistochemistry The presence of the C9 neo-antigen from active complement was analyzed in formalin-fixed, embedded tissue sections using the previously described HRP-conjugated mAbs (anti-C9 neo-antigen; Quidel, Mountain View, CA, USA). Briefly, fetal membranes were fixed in 4% formalin (Sigma-Aldrich, St. Louis, MO, USA), and 4-m-thick sections were mounted on paraffin slides. The paraffin was removed, and the sections were rehydrated with PBS (pH 7.4) for 30 min at room temperature. The tissue sections were then covered with 3% hydrogen peroxide to block endogenous peroxidase activity. A previously described standard procedure was used for tissue staining (Ratnoff et al., 1995). 2.6. Statistical analysis The clinical characteristics of the participants are presented using descriptive statistics, the mean ± standard deviation for continuous variables, and proportions and percentages for qualitative variables. One-way analysis of variance (ANOVA) was initially performed. When the Levine test for the equality of variances indicated significant differences, the Kruskal–Wallis test was used. The proportions were compared using Fisher’s exact test for expected values less than five. A P value <0.05 was considered statistically significant. Statistical analyses were performed using XL-STAT 2013, version 5.06 for Windows (Addinsoft, New York, NY, USA).
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3. Results 3.1. Patient characteristics The clinical parameters of the patients recruited into the study are provided in Table 1. No significant differences were noted between the pregnant and parturient groups regarding maternal age or the incidence of diabetes, uterine myomas, anemia or preeclampsia. The gestational age and the prevalence of urinary tract infections, chorioamnionitis, and cervicovaginal infections varied between the groups. 3.2. Detection of SC5b-9 in plasma To analyze complement activity in pregnant women, we determined the plasma levels of the soluble SC5b-9 complex in parturient women at different gestational ages and conditions as described above (TL, PL, and PPROM; n = 59). For comparison purposes, pregnant women (P, n = 38) at <37 weeks of gestational age who exhibited no signs of parturition were also studied. The median estimated concentration of the soluble SC5b-9 complex in parturient women was 3361 ng/mL for the TL group (range 1726–5670 ng/mL; n = 8), 2958 ng/mL for the PL group (range 1552–7092 ng/mL; n = 25), 2272 ng/mL for the PPROM group (range 167–6540 ng/mL; n = 26), and 1384 ng/mL for the control P group (range 174–4570 ng/mL; n = 38; P < 0.001, Kruskal–Wallis test; Fig. 2). 3.3. Immuno-dot blot assays We analyzed neo-antigen C9, which is expressed when the C5b-9 complex forms, to determine whether active complement was deposited on fetal membranes. Neoantigen C9 from active complement was studied in protein extracts from fetal tissue membranes from women in the TL (n = 6), PL (n = 10), and PPROM (n = 12) groups at delivery. The group of pregnant women (P) was not included in this analysis because they had not yet given birth. The tissues were analyzed using standard immunodot blot assays as previously described in Section 2. An
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intense C9 neo-antigen stain was evident on the membranes from all the groups, indicating that complement was deposited on the fetal membranes (Fig. 3A). Variable C9 neo-antigen deposits were noted in all the groups based on color intensity, which was measured by densitometry (Fig. 3B). The respective mean ± standard deviation values for the TL, PL, and PPROM groups were 5885 ± 536, 5918 ± 1040, and 6252 ± 1083 respectively at point a (P > 0.05, ANOVA); 5614 ± 250, 5443 ± 200, and 5780 ± 703 at point b (P > 0.05, ANOVA); 6862 ± 1418, 5875 ± 1064, and 5631 ± 408 at point c (P > 0.05, Kruskal–Wallis test); and 7288 ± 1487, 5748 ± 886, and 5814 ± 692 at point d (P > 0.05, Kruskal–Wallis test). The lack of differences between these values suggested that term or preterm fetal membranes exhibited complement deposition at birth regardless of the length of the pregnancy. Negative immuno-dot blot assays were observed for samples from women who were not in labor or who were delivering by Cesarean section. 3.4. Immunohistochemistry The confirmation of complement component deposits on fetal membranes was crucial for understanding their role in parturition. Selected sections from each group are presented in Fig. 4 for comparison purposes. Active complement deposits were not detected in anti-C9 neoantigen-stained fetal membranes from preterm women who delivered by Cesarean section (n = 2; Fig. 4A). C9 neo-antigen deposits were observed in the external membrane of the amniotic epithelium, the amniotic basement membrane, and the compact layer of the connective tissue of fetal membranes obtained from term women who underwent labor (Fig. 4B). Apoptotic cells were noted in the membrane samples from the TL group (n = 2). Thus, apoptotic nuclei were detected in the at-term membranes, but not in the preterm tissue (data not shown). The immunostaining pattern during preterm labor indicated strong staining in the cytoplasm of cells from the trophoblast layer (Fig. 4C, n = 3). In the fibroblast layer of the connective tissue from participants in the PPROM group (n = 4), up to 5% of the cells exhibited cytoplasmic anti-C9 neo-antigen staining (Fig. 4D).
Table 1 Clinical Characteristics of study participants. Group
Pregnant (P) n = 38
Term Labor (TL) n = 8 (>37 weeks)
Preterm labor (PL) n = 25 (<37 weeks)
Premature Rupture of Membranes (PPROM) n = 26 (<37 weeks)
Mother age (years)* Gestation age (weeks)* Gestational diabetes** Urinary tract infection** Chorioamnionitis** Cervicovaginal infection** Uterine myomas** Anemia** Preeclampsia**
28 ± 6.8 24.6 ± 8.8 2/38 1/38 0/38 0/38 3/38 1/38 3/38
28 ± 6.2 39 ± 1.4 2/8 1/8 0/8 0/8 2/8 1/8 0/8
28.3 ± 7.5 33 ± 2.5 3/25 7/25 4/25 3/25 0/25 2/25 4/25
24.5 ± 7.1 33 ± 2.4 3/26 7/26 3/26 5/26 5/26 1/26 2/26
a b c * **
ANOVA test. Kruskal Wallis test. Fisher’s exact test. Values expressed as median ± standard deviation. Frequency (percentage).
P >0.05a 0.00b 0.15c 0.01c 0.04c 0.02c 0.06c 0.47c 0.66c
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Fig. 2. Plasma SC5b-9 values based on gestational resolution. Values are presented for pregnant women between 26 and 37 weeks of gestation and for the three groups of women with resolved pregnancies: TL (term labor), PL (preterm labor), and PPROM (preterm premature rupture of membranes). The values are presented as the median (the line across the middle of the box) and the 75th and 25th percentiles (the upper and lower ends of the box; P < 0.001; Kruskal–Wallis test).
Fig. 3. Immuno-dot blot of fetal membranes using the anti-C9 neo-antigen. Membranes from patients who underwent different types of deliveries exhibited active complement deposits (A). Points (a) and (d) are equidistant from the fetal membrane rupture site. In (B), the mean ± standard deviation of the dot color intensity was determined using myImage Software V2.0 (Thermo Scientific), and data are presented for each point (a–d) for each group: term labor (TL), preterm labor (PL) and preterm premature rupture of membranes (PPROM; P > 0.05, ANOVA for points a and b and Kruskal–Wallis test for points c and d).
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Fig. 4. Fetal membranes with complement deposition. Selected sections from each group are presented for comparison purposes. (A) Fetal membrane fragments from an indicated preterm pregnancy delivered by Cesarean section did not exhibit complement deposition; (B) a sample from a term labor patient with strong cytoplasmic brown staining (arrows) from the peroxidase-labeled anti-C9-neo-antigen antibody indicated intense complement deposition; and (C) preterm labor and (D) PPROM samples reacted with anti-C9 neo-antigen (400× magnification).
4. Discussion The evidence described herein suggests that complement might be active during parturition and deposited on fetal membranes. Complement may cooperate with matrix metalloproteinases (Athayde et al., 1998; Lei et al., 1996) and apoptosis (Lei et al., 1996; Murtha et al., 2002) to weaken the membranes and facilitate the onset of labor. The results are consistent with those of previous studies, demonstrating that complement is active at parturition. The present study demonstrated the immunohistochemical localization of the C9 neo-antigen, which emerges only after complement is activated. The identification of increased complement activity in the plasma at the end of pregnancy, as demonstrated by increased SC5b-9 levels in plasma, could be used as an indicator of the delivery period for either preterm or term pregnancies. Moreover, because complement activity is modulated by endogenous regulators (e.g., decay accelerating factor (DAF/CD55) and membrane cofactor protein (MCP/CD46), anticomplement therapy could be utilized to extend pregnancy in women undergoing preterm labor. Complement inhibition therapy has been assessed in myocardial infarction (Doran et al., 1996; Hyde et al., 1998; Monsinjon et al., 2001), cerebral stroke (Huang et al., 1999), and anti-phospholipid antibody syndrome (Holers et al., 2002). These studies demonstrated positive effects regarding reduced tissue damage and improved functional recovery. In fact, anticomplement therapy has so many applications that several drugs are now under development in both preclinical and clinical trials. Based on the mechanism of action, anticomplement drugs have been
classified into five groups: serine protease inhibitors (C1INH, Rhucin/rhC1INH), therapeutic antibodies (eculizumab and pexelizumab: C5; and ofatumumab: anti-CD20), soluble complement regulators (sCR1/TP10: extracellular part of CR1, decay accelerator, and factor I cofactor; and CAB-2/MLN-2222: chimera of DAF and MCP), complement component inhibitors (compstatin/POT-4: peptidic C3 inhibitor), and receptor antagonists (PMX-53: peptidic C5aR antagonist). The last three groups comprise more promising molecules because of their molecular characteristics as small molecules with high potency and selectivity (Kulkarni and Afshar-Kharghan, 2008; Ricklin and Lambris, 2013). In the present study, the amount of SC5b-9 was measured regardless of the pathway by which the complement system was activated (classical, alternative, lectin, or extrinsic). Therefore, the values reported by other studies in women during preterm delivery and not during term delivery with spontaneous labor may differ from our data because those studies focused on the complement fragment Bb. The Bb protein is activated via the alternative complement pathway (Vaisbuch et al., 2010; Lynch et al., 2008). Historically, early studies on the classical activation pathway revealed that C3 and C4 had no predictive value in the timing of normal labor when assayed using radial immunodiffusion (Kovar and Riches, 1988). Subsequently, C3a, C4a, and C5a (products of active complement) were measured by immunoassay during pregnancy, and increased levels of these products were observed in pregnant women compared with non-pregnant women (Richani et al., 2005). More recently, mannose-binding
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lectin (MBL) pathway activity was discovered, and this pathway was investigated by ELISA during pregnancy in a longitudinal study. This study found that MBL concentrations increase during pregnancy and decrease postpartum (Van de Geijn et al., 2007). Furthermore, the C4d, C3a, SC5b9, C3, C9, and factor H levels were significantly higher in healthy pregnant women than in non-pregnant women. Conversely, C1 inhibitor levels were significantly lower in healthy pregnant women compared with non-pregnant women (Derzsy et al., 2010). These studies indicate that the complement system is active during normal pregnancy. Active complement facilitates the remodeling and generation of maternal tissue to promote fetal growth and allows the fetus to adapt to the increasing demand for space during pregnancy. Multiple studies have indicated that the role of the complement system during tissue remodeling involves binding apoptotic cells via enhanced opsonization and eliciting their subsequent removal through phagocytosis (Girardi et al., 2006; Read et al., 2007; Runic et al., 1998). Tissue remodeling is associated with apoptosis (Murtha et al., 2002) and is a common event that promotes the activation of the complement cascade to facilitate the deposition of the C5b-9 complex in the placenta, decidua, and fetal membranes. Our study revealed that the C5b-9 protein complex is primarily deposited in the amnion. In conclusion, our study showed that women exhibit increased SC5b-9 plasma levels and C9 neo-antigen deposition on fetal membranes during parturition. This deposition is indicative of complement activity during parturition. Conflict of interest statement None. Acknowledgements This work was supported by the Instituto Nacional de Perinatología (212250-07171). The primary investigator was Dr. Galindo-Sevilla. Dr. Segura-Cervantes was supported by CONACyT (92955) for the Medical, Odontological, and Health Sciences PhD Program, UNAM, Mexico City, Mexico. References Athayde, N., Edwin, S., Romero, R., Gomez, R., Maymon, E., Pacora, P., et al., 1998. A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal membranes. Am. J. Obstet. Gynecol. 179, 1248–1253. Bhakdi, S., Tranum-Iensen, I., 1982. Terminal membrane C5b-9 complex of human complement: transition from an amphiphilic to a hydrophilic state through binding of the S protein from serum. J. Cell Biol. 94, 755–759. Breuiller-Fouche, M., Germain, G., 2006. Gene and protein expression in the myometrium in pregnancy and labor. Reproduction 131, 837–850. Derzsy, Z., Prohaszka, Z., Rigo Jr., J., Füst, G., Molvarec, A., 2010. Activation of the complement system in normal pregnancy and preeclampsia. Mol. Immunol. 47, 1500–1506. Doran, J.P., Howie, A.J., Townend, J.N., Bonser, R.S., 1996. Detection of myocardial infarction by immunohistological staining for C9 on formalin fixed, paraffin wax embedded sections. J. Clin. Pathol. 49, 34–37. Girardi, G., Yarilin, D., Thurman, J.M., Holers, V.M., Salmon, J.E., 2006. Complement activation induces dysregulation of angiogenic factors
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Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Blood SC5b-9 complement levels increase at parturition during term and preterm labor Enrique Segura-Cervantes a , Javier Mancilla-Ramirez b , Luis Zurita c , Yuriria Paredes d , José Luis Arredondo e , Norma Galindo-Sevilla a,∗ a b c d e
Departamento de Infectología e Inmunología Perinatal, Instituto Nacional de Perinatologia (INPer), Mexico Escuela Superior de Medicina, IPN, Mexico División Académica de Ciencias de la Salud, UJAT, Mexico Departmento de Biología Celular, INPer, Mexico Unidad de Investigación Clínica, INP, Mexico
a r t i c l e
i n f o
Article history: Received 20 September 2014 Received in revised form 17 February 2015 Accepted 19 February 2015 Keywords: Complement Pregnancy Parturition Labor Fetal membranes
a b s t r a c t We explored the hypothesis that complement, an innate and adaptive immune effector, is active in the plasma of parturient women and is deposited on fetal membranes collected after delivery. A cross-sectional study was designed to evaluate complement activity at parturition. Pregnant women (n = 97) between 15 and 41 years of age were enrolled in a hospital protocol during the perinatal period to assess both SC5b-9 complement activity in blood and complement deposition on fetal membranes during parturition. Soluble SC5b-9 complement activity in plasma fractions was measured using a standard enzyme-linked immunosorbent assay (ELISA) that included specific anti-complement antibodies. Complement deposition on membranes was analyzed using immuno-dot blots and immunohistochemistry. Soluble SC5b-9 complement complex levels were increased in the plasma of women during term labor (TL; median 3361; range 1726–5670 ng/mL), preterm labor (PL; median 2958; range 1552–7092 ng/mL), and preterm premature rupture of membranes (PPROM; median 2272; range 167–6540 ng/mL) compared with pregnant women who were not in labor (P; median 1384; range 174–4570 ng/mL; P < 0.001, Kruskal–Wallis test). Active complement, as assessed by the C9 neo-antigen in C5b-9 complexes, was deposited on fetal membranes, with no difference between term and preterm delivery. The deposition of active complement on fetal membranes was confirmed by immunohistochemistry. Women who underwent non-labor-indicated Cesarean sections did not exhibit complement deposition. Soluble SC5b-9 complement complex levels increased in the plasma of women during parturition, and complement C5b-9 complexes were deposited on fetal membranes. © 2015 Elsevier Ireland Ltd. All rights reserved.
∗ Corresponding author at: Departamento de Infectología e Inmunología Perinatal, Instituto Nacional de Perinatología, Montes Urales #800, Col. Lomas de Virreyes, Mexico City 11000, Mexico. Tel.: +52 55 55 20 99 00x103. E-mail addresses: [email protected] (E. Segura-Cervantes), [email protected] (J. Mancilla-Ramirez), [email protected] (L. Zurita), [email protected] (Y. Paredes), [email protected] (J.L. Arredondo), [email protected] (N. Galindo-Sevilla). http://dx.doi.org/10.1016/j.jri.2015.02.008 0165-0378/© 2015 Elsevier Ireland Ltd. All rights reserved.
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1. Introduction Parturition is a physiological process that requires the synchronization of uterine contractions, cervical dilatation, and membrane rupture. Several reviews have emphasized that the physiological phenomena, molecules, and cellular mechanisms that drive the process of human parturition are not completely understood (Breuiller-Fouche and Germain, 2006). The complement system is an important effector of the immune system because it forms pores in cell membranes that lead to cell lysis; thus, the complement system is thought to play a crucial role during parturition. C4B and C1R protein levels are lower during spontaneous vaginal delivery (SVD) than during pre-labor in the same women (Yuan et al., 2012). However, the complement system and its role during parturition have not been explored as extensively as the cytokines that mediate the parturition-dependent inflammatory response. New techniques, including standard enzyme-linked immunoabsorbent assay (ELISA), now permit the evaluation of complement activity with improved sensitivity. Different methodological approaches have been used to determine complement activity in biological fluids, such as those used to assess the protein complex C5b-9 in plasma. This protein complex is a common mediator of cell lysis that is generated from any of the four complement activation pathways (Huber-Lang et al., 2006). The regulatory S protein binds to C5b-9, forming the SC5b-9 complex, which is soluble and can be detected in plasma by ELISA (Mollnes et al., 1985). We hypothesized that the complement system is active at parturition during established labor. This study aimed to investigate whether the active form of complement, C5b-9, is elevated in plasma as SC5b-9 at parturition and whether complement is deposited on fetal membranes. 2. Materials and methods 2.1. Study design To understand complement activity at parturition, a cross-sectional study was designed to assess complement activity in the plasma and complement deposition on fetal membranes. Pregnant women (n = 97) between 15 and 41 years of age who were treated as outpatients from 12 weeks of gestation until delivery at the National Institute of Perinatology (INPer, Mexico City, Mexico) were recruited for the study. Patients with cancer and patients receiving immunotherapy for transplants or autoimmunity or current steroid treatment were excluded from the study. The patients provided written informed consent during their clinical check-ups. Each patient consented to donate four fetal membrane samples (1 cm in diameter) after childbirth. Furthermore, one 3-mL sample of whole peripheral blood was obtained from each patient at delivery or during normal examinations as stated in Section 3. The study participants included pregnant women (P) at <37 weeks of gestational age. Sample membranes were also isolated from women undergoing an indicated Cesarean section at <36 weeks. Women at >37 weeks of gestational age comprised the term labor (TL) group, and women at <37 weeks
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of gestational age were assigned to one of two subgroups: the preterm labor (PL) or preterm premature rupture of membranes (PPROM) group. A flow diagram is presented in Fig. 1. The study participants were at minimal risk. The study was approved by the research and bioethical committees of the National Institute of Perinatology and was in compliance with international guidelines for human studies. 2.2. SC5b-9 protein complex assessment The C5b-9 complex binds the regulatory S protein after the C5b-7 protein complex is properly assembled in the plasma (Mollnes et al., 1985; Bhakdi and TranumIensen, 1982). SC5b-9 complex levels in the plasma are indicative of complement activation, which causes subsequent cell lysis. SC5b-9 complex levels were analyzed using a standard ELISA assay (Quidel, Mountain View, CA, USA) as previously reported (Walter et al., 2010). Participant plasma samples were routinely diluted 1:40. The samples were assayed in triplicate wells (100 L) and were incubated for 60 min according to the manufacturer’s instructions. The method was quantitative for SC5b-9 complex levels ranging from 20 to 150 ng/mL. The color was developed by incubating the samples with 100 L/well of the chromogenic substrate OPD (O-phenylenediamine dihydrochloride, Sigma-Aldrich, St. Louis, MO, USA) for 30 min at room temperature. The plates were read using an automated ELISA plate reader (Synergy 2, Bio-Tek Instruments, Winooski, VT, USA) after the enzyme reaction was terminated by the addition of 50 L of 0.1 M H2 SO4 . The absorbance was measured at 405 nm. 2.3. Fetal membrane sampling We investigated whether active complement was deposited on fetal membranes (1 cm in diameter), including the chorion and amnion. The fetal membranes were obtained within the first 30 min after childbirth and were assessed in duplicate at four points from the rupture site following a previously published protocol (McLaren et al., 1999)Ref. . One membrane sample from each duplicate was incubated in phosphate-buffered saline (PBS; pH 7.4) containing a mammalian protease inhibitor cocktail (PIC, 1:50 dilution; Sigma-Aldrich, St. Louis, MO, USA). The samples were frozen at −70 ◦ C and stored until they were used in standard immuno-dot blot assays (Janyapoon et al., 2000) of membrane complement components. The other membrane samples were used for the immunohistochemical detection of complement components. 2.4. Protein membrane extraction and immuno-dot blot assay Complement components were detected in protein fractions extracted from fetal membranes. Briefly, the membrane tissue was mechanically disrupted in a tissue grinder with 3 mL of cold PBS containing a protease inhibitor cocktail (PBS-PIC). The suspended cells were recovered and centrifuged at 400 × g. The cell pellet was
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Fig. 1. A flow diagram of the patient selection process.
disrupted by several freeze–thaw cycles. The suspended fraction was transferred to an Eppendorf tube and centrifuged at 20,000 × g for 20 min. The collected supernatant was discarded, and the pellet was re-suspended in 200 L of PBS-PIC with 1% Nonidet P-40 (a non-ionic detergent used to extract proteins from cell membranes, SigmaAldrich, St. Louis, MO, USA). The protein samples were re-centrifuged at 20,000 × g for 30 min. Then, 1 L of the supernatant was directly applied to a polyvinylidene difluoride membrane in triplicate (0.45-m PVDF membranes, Bio-Rad Labs, Hercules, CA, USA). Non-specific binding sites were blocked for 1 h in PBS (pH 7.4) containing 5% low-fat milk. The membranes were incubated overnight at 4 ◦ C with a 1:50 dilution of an HRP-conjugated anti-C9 neo-antigen monoclonal antibody (Quidel, Mountain View, CA, USA). The monoclonal antibodies used to detect the complement components in this assay were labeled with horseradish peroxidase (HRP; Sigma-Aldrich, St. Louis, MO, USA) in our laboratory according to a standard procedure (Harlow and Lane, 1988). The membranes were washed three times (5 min each) with PBS (pH 7.4)/0.05% Tween-20 to remove excess unbound antibody. The peroxidase-positive dots were detected by immersing the membrane in a developing solution (70 mM sodium acetate at pH 6.2 containing 0.3% diaminobenzidine tetrahydrochloride and 0.03% H2 O2 ) at room temperature for 5 min. The enzymatic reaction was terminated by washing the sheet. The immunoreactive protein signals were detected using an AlphaImager HP System (Cell Biosciences, Inc., Santa Clara, CA, USA).
2.5. Immunohistochemistry The presence of the C9 neo-antigen from active complement was analyzed in formalin-fixed, embedded tissue sections using the previously described HRP-conjugated mAbs (anti-C9 neo-antigen; Quidel, Mountain View, CA, USA). Briefly, fetal membranes were fixed in 4% formalin (Sigma-Aldrich, St. Louis, MO, USA), and 4-m-thick sections were mounted on paraffin slides. The paraffin was removed, and the sections were rehydrated with PBS (pH 7.4) for 30 min at room temperature. The tissue sections were then covered with 3% hydrogen peroxide to block endogenous peroxidase activity. A previously described standard procedure was used for tissue staining (Ratnoff et al., 1995). 2.6. Statistical analysis The clinical characteristics of the participants are presented using descriptive statistics, the mean ± standard deviation for continuous variables, and proportions and percentages for qualitative variables. One-way analysis of variance (ANOVA) was initially performed. When the Levine test for the equality of variances indicated significant differences, the Kruskal–Wallis test was used. The proportions were compared using Fisher’s exact test for expected values less than five. A P value <0.05 was considered statistically significant. Statistical analyses were performed using XL-STAT 2013, version 5.06 for Windows (Addinsoft, New York, NY, USA).
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3. Results 3.1. Patient characteristics The clinical parameters of the patients recruited into the study are provided in Table 1. No significant differences were noted between the pregnant and parturient groups regarding maternal age or the incidence of diabetes, uterine myomas, anemia or preeclampsia. The gestational age and the prevalence of urinary tract infections, chorioamnionitis, and cervicovaginal infections varied between the groups. 3.2. Detection of SC5b-9 in plasma To analyze complement activity in pregnant women, we determined the plasma levels of the soluble SC5b-9 complex in parturient women at different gestational ages and conditions as described above (TL, PL, and PPROM; n = 59). For comparison purposes, pregnant women (P, n = 38) at <37 weeks of gestational age who exhibited no signs of parturition were also studied. The median estimated concentration of the soluble SC5b-9 complex in parturient women was 3361 ng/mL for the TL group (range 1726–5670 ng/mL; n = 8), 2958 ng/mL for the PL group (range 1552–7092 ng/mL; n = 25), 2272 ng/mL for the PPROM group (range 167–6540 ng/mL; n = 26), and 1384 ng/mL for the control P group (range 174–4570 ng/mL; n = 38; P < 0.001, Kruskal–Wallis test; Fig. 2). 3.3. Immuno-dot blot assays We analyzed neo-antigen C9, which is expressed when the C5b-9 complex forms, to determine whether active complement was deposited on fetal membranes. Neoantigen C9 from active complement was studied in protein extracts from fetal tissue membranes from women in the TL (n = 6), PL (n = 10), and PPROM (n = 12) groups at delivery. The group of pregnant women (P) was not included in this analysis because they had not yet given birth. The tissues were analyzed using standard immunodot blot assays as previously described in Section 2. An
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intense C9 neo-antigen stain was evident on the membranes from all the groups, indicating that complement was deposited on the fetal membranes (Fig. 3A). Variable C9 neo-antigen deposits were noted in all the groups based on color intensity, which was measured by densitometry (Fig. 3B). The respective mean ± standard deviation values for the TL, PL, and PPROM groups were 5885 ± 536, 5918 ± 1040, and 6252 ± 1083 respectively at point a (P > 0.05, ANOVA); 5614 ± 250, 5443 ± 200, and 5780 ± 703 at point b (P > 0.05, ANOVA); 6862 ± 1418, 5875 ± 1064, and 5631 ± 408 at point c (P > 0.05, Kruskal–Wallis test); and 7288 ± 1487, 5748 ± 886, and 5814 ± 692 at point d (P > 0.05, Kruskal–Wallis test). The lack of differences between these values suggested that term or preterm fetal membranes exhibited complement deposition at birth regardless of the length of the pregnancy. Negative immuno-dot blot assays were observed for samples from women who were not in labor or who were delivering by Cesarean section. 3.4. Immunohistochemistry The confirmation of complement component deposits on fetal membranes was crucial for understanding their role in parturition. Selected sections from each group are presented in Fig. 4 for comparison purposes. Active complement deposits were not detected in anti-C9 neoantigen-stained fetal membranes from preterm women who delivered by Cesarean section (n = 2; Fig. 4A). C9 neo-antigen deposits were observed in the external membrane of the amniotic epithelium, the amniotic basement membrane, and the compact layer of the connective tissue of fetal membranes obtained from term women who underwent labor (Fig. 4B). Apoptotic cells were noted in the membrane samples from the TL group (n = 2). Thus, apoptotic nuclei were detected in the at-term membranes, but not in the preterm tissue (data not shown). The immunostaining pattern during preterm labor indicated strong staining in the cytoplasm of cells from the trophoblast layer (Fig. 4C, n = 3). In the fibroblast layer of the connective tissue from participants in the PPROM group (n = 4), up to 5% of the cells exhibited cytoplasmic anti-C9 neo-antigen staining (Fig. 4D).
Table 1 Clinical Characteristics of study participants. Group
Pregnant (P) n = 38
Term Labor (TL) n = 8 (>37 weeks)
Preterm labor (PL) n = 25 (<37 weeks)
Premature Rupture of Membranes (PPROM) n = 26 (<37 weeks)
Mother age (years)* Gestation age (weeks)* Gestational diabetes** Urinary tract infection** Chorioamnionitis** Cervicovaginal infection** Uterine myomas** Anemia** Preeclampsia**
28 ± 6.8 24.6 ± 8.8 2/38 1/38 0/38 0/38 3/38 1/38 3/38
28 ± 6.2 39 ± 1.4 2/8 1/8 0/8 0/8 2/8 1/8 0/8
28.3 ± 7.5 33 ± 2.5 3/25 7/25 4/25 3/25 0/25 2/25 4/25
24.5 ± 7.1 33 ± 2.4 3/26 7/26 3/26 5/26 5/26 1/26 2/26
a b c * **
ANOVA test. Kruskal Wallis test. Fisher’s exact test. Values expressed as median ± standard deviation. Frequency (percentage).
P >0.05a 0.00b 0.15c 0.01c 0.04c 0.02c 0.06c 0.47c 0.66c
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Fig. 2. Plasma SC5b-9 values based on gestational resolution. Values are presented for pregnant women between 26 and 37 weeks of gestation and for the three groups of women with resolved pregnancies: TL (term labor), PL (preterm labor), and PPROM (preterm premature rupture of membranes). The values are presented as the median (the line across the middle of the box) and the 75th and 25th percentiles (the upper and lower ends of the box; P < 0.001; Kruskal–Wallis test).
Fig. 3. Immuno-dot blot of fetal membranes using the anti-C9 neo-antigen. Membranes from patients who underwent different types of deliveries exhibited active complement deposits (A). Points (a) and (d) are equidistant from the fetal membrane rupture site. In (B), the mean ± standard deviation of the dot color intensity was determined using myImage Software V2.0 (Thermo Scientific), and data are presented for each point (a–d) for each group: term labor (TL), preterm labor (PL) and preterm premature rupture of membranes (PPROM; P > 0.05, ANOVA for points a and b and Kruskal–Wallis test for points c and d).
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Fig. 4. Fetal membranes with complement deposition. Selected sections from each group are presented for comparison purposes. (A) Fetal membrane fragments from an indicated preterm pregnancy delivered by Cesarean section did not exhibit complement deposition; (B) a sample from a term labor patient with strong cytoplasmic brown staining (arrows) from the peroxidase-labeled anti-C9-neo-antigen antibody indicated intense complement deposition; and (C) preterm labor and (D) PPROM samples reacted with anti-C9 neo-antigen (400× magnification).
4. Discussion The evidence described herein suggests that complement might be active during parturition and deposited on fetal membranes. Complement may cooperate with matrix metalloproteinases (Athayde et al., 1998; Lei et al., 1996) and apoptosis (Lei et al., 1996; Murtha et al., 2002) to weaken the membranes and facilitate the onset of labor. The results are consistent with those of previous studies, demonstrating that complement is active at parturition. The present study demonstrated the immunohistochemical localization of the C9 neo-antigen, which emerges only after complement is activated. The identification of increased complement activity in the plasma at the end of pregnancy, as demonstrated by increased SC5b-9 levels in plasma, could be used as an indicator of the delivery period for either preterm or term pregnancies. Moreover, because complement activity is modulated by endogenous regulators (e.g., decay accelerating factor (DAF/CD55) and membrane cofactor protein (MCP/CD46), anticomplement therapy could be utilized to extend pregnancy in women undergoing preterm labor. Complement inhibition therapy has been assessed in myocardial infarction (Doran et al., 1996; Hyde et al., 1998; Monsinjon et al., 2001), cerebral stroke (Huang et al., 1999), and anti-phospholipid antibody syndrome (Holers et al., 2002). These studies demonstrated positive effects regarding reduced tissue damage and improved functional recovery. In fact, anticomplement therapy has so many applications that several drugs are now under development in both preclinical and clinical trials. Based on the mechanism of action, anticomplement drugs have been
classified into five groups: serine protease inhibitors (C1INH, Rhucin/rhC1INH), therapeutic antibodies (eculizumab and pexelizumab: C5; and ofatumumab: anti-CD20), soluble complement regulators (sCR1/TP10: extracellular part of CR1, decay accelerator, and factor I cofactor; and CAB-2/MLN-2222: chimera of DAF and MCP), complement component inhibitors (compstatin/POT-4: peptidic C3 inhibitor), and receptor antagonists (PMX-53: peptidic C5aR antagonist). The last three groups comprise more promising molecules because of their molecular characteristics as small molecules with high potency and selectivity (Kulkarni and Afshar-Kharghan, 2008; Ricklin and Lambris, 2013). In the present study, the amount of SC5b-9 was measured regardless of the pathway by which the complement system was activated (classical, alternative, lectin, or extrinsic). Therefore, the values reported by other studies in women during preterm delivery and not during term delivery with spontaneous labor may differ from our data because those studies focused on the complement fragment Bb. The Bb protein is activated via the alternative complement pathway (Vaisbuch et al., 2010; Lynch et al., 2008). Historically, early studies on the classical activation pathway revealed that C3 and C4 had no predictive value in the timing of normal labor when assayed using radial immunodiffusion (Kovar and Riches, 1988). Subsequently, C3a, C4a, and C5a (products of active complement) were measured by immunoassay during pregnancy, and increased levels of these products were observed in pregnant women compared with non-pregnant women (Richani et al., 2005). More recently, mannose-binding
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lectin (MBL) pathway activity was discovered, and this pathway was investigated by ELISA during pregnancy in a longitudinal study. This study found that MBL concentrations increase during pregnancy and decrease postpartum (Van de Geijn et al., 2007). Furthermore, the C4d, C3a, SC5b9, C3, C9, and factor H levels were significantly higher in healthy pregnant women than in non-pregnant women. Conversely, C1 inhibitor levels were significantly lower in healthy pregnant women compared with non-pregnant women (Derzsy et al., 2010). These studies indicate that the complement system is active during normal pregnancy. Active complement facilitates the remodeling and generation of maternal tissue to promote fetal growth and allows the fetus to adapt to the increasing demand for space during pregnancy. Multiple studies have indicated that the role of the complement system during tissue remodeling involves binding apoptotic cells via enhanced opsonization and eliciting their subsequent removal through phagocytosis (Girardi et al., 2006; Read et al., 2007; Runic et al., 1998). Tissue remodeling is associated with apoptosis (Murtha et al., 2002) and is a common event that promotes the activation of the complement cascade to facilitate the deposition of the C5b-9 complex in the placenta, decidua, and fetal membranes. Our study revealed that the C5b-9 protein complex is primarily deposited in the amnion. In conclusion, our study showed that women exhibit increased SC5b-9 plasma levels and C9 neo-antigen deposition on fetal membranes during parturition. This deposition is indicative of complement activity during parturition. Conflict of interest statement None. Acknowledgements This work was supported by the Instituto Nacional de Perinatología (212250-07171). The primary investigator was Dr. Galindo-Sevilla. Dr. Segura-Cervantes was supported by CONACyT (92955) for the Medical, Odontological, and Health Sciences PhD Program, UNAM, Mexico City, Mexico. References Athayde, N., Edwin, S., Romero, R., Gomez, R., Maymon, E., Pacora, P., et al., 1998. A role for matrix metalloproteinase-9 in spontaneous rupture of the fetal membranes. Am. J. Obstet. Gynecol. 179, 1248–1253. Bhakdi, S., Tranum-Iensen, I., 1982. Terminal membrane C5b-9 complex of human complement: transition from an amphiphilic to a hydrophilic state through binding of the S protein from serum. J. Cell Biol. 94, 755–759. Breuiller-Fouche, M., Germain, G., 2006. Gene and protein expression in the myometrium in pregnancy and labor. Reproduction 131, 837–850. Derzsy, Z., Prohaszka, Z., Rigo Jr., J., Füst, G., Molvarec, A., 2010. Activation of the complement system in normal pregnancy and preeclampsia. Mol. Immunol. 47, 1500–1506. Doran, J.P., Howie, A.J., Townend, J.N., Bonser, R.S., 1996. Detection of myocardial infarction by immunohistological staining for C9 on formalin fixed, paraffin wax embedded sections. J. Clin. Pathol. 49, 34–37. Girardi, G., Yarilin, D., Thurman, J.M., Holers, V.M., Salmon, J.E., 2006. Complement activation induces dysregulation of angiogenic factors
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