The Journal of Maternal-Fetal & Neonatal Medicine
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Amniotic fluid markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes Marian Kacerovsky, Lubomira Tothova, Ramkumar Menon, Barbora Vlkova, Ivana Musilova, Helena Hornychova, Martin Prochazka & Peter Celec To cite this article: Marian Kacerovsky, Lubomira Tothova, Ramkumar Menon, Barbora Vlkova, Ivana Musilova, Helena Hornychova, Martin Prochazka & Peter Celec (2015) Amniotic fluid markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes, The Journal of Maternal-Fetal & Neonatal Medicine, 28:11, 1250-1259, DOI: 10.3109/14767058.2014.951628 To link to this article: http://dx.doi.org/10.3109/14767058.2014.951628
Published online: 27 Aug 2014.
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Date: 07 November 2015, At: 16:38
http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2015; 28(11): 1250–1259 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.951628
ORIGINAL ARTICLE
Amniotic fluid markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes Marian Kacerovsky1,2, Lubomira Tothova3,4, Ramkumar Menon5, Barbora Vlkova3,4, Ivana Musilova2, Helena Hornychova6, Martin Prochazka7, and Peter Celec3,4 Downloaded by ["University at Buffalo Libraries"] at 16:38 07 November 2015
1
Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic, 2Department of Obstetrics and Gynecology, Faculty of Medicine Hradec Kralove, Charles University in Prague, Prague, Czech Republic, 3Institute of Molecular Biomedicine, Comenius University in Bratislava, Bratislava, Slovak Republic, 4Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia, 5Department of Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, USA, 6Fingerland’s Department of Pathology, Faculty of Medicine Hradec Kralove, Charles University in Prague, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic, and 7Department of Obstetrics and Gynecology, Palacky University Olomouc, University Hospital Olomouc, Olomouc, Czech Republic Abstract
Keywords
Objective: To determine amniotic fluid total antioxidant capacity (TAC), ferric-reducing antioxidant power (FRAP) and thiobarbituric acid-reacting substances (TBARS), markers of oxidative stress, in pregnancies complicated by preterm prelabor rupture of membranes (pPROM) and their correlation to microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA). Methods: One-hundred thirty-eight women with singleton pregnancies complicated by pPROM were included in this study. Amniotic fluid was collected by transabdominal amniocentesis at the time of admission and amniotic fluid concentrations of TAC, FRAP and TBARS were measured. Result: The presence of MIAC and/or HCA did not show any significant differences in the amniotic fluid TAC, FRAP and TBARS concentrations. Positive correlations between gestational age at sampling and amniotic fluid TAC and FRAP concentrations were found (TAC: rho ¼ 0.32; p ¼ 0.0002; FRAP: rho ¼ 0.36; p50.0001). A negative correlation between gestation age at sampling and amniotic fluid TBARS concentrations was identified (rho ¼ –0.25; p ¼ 0.004). Conclusions: Oxidative stress is associated with pPROM as indicated by the presence of markers tested in the amniotic fluid; however, oxidative stress markers tested are not influenced by the presence of MIAC or HCA.
Infection, inflammation, preterm delivery
Introduction Preterm prelabor rupture of membranes (pPROM), defined by the rupture of fetal membranes before spontaneous onset of labor prior to 37 weeks, represents one of the main problems in perinatology [1]. Recent studies have reported a potential pathophysiology for pPROM characterized by oxidative stress-induced premature senescence (aging) of fetal membranes [2–5]. Despite this fact, approximately 75% pPROM cases are associated with microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA) [6–8]. It is still under intense debate whether MIAC is cause or consequence of pPROM [9]. Nevertheless, there is a solid body of evidence that the presence of both of these
Address for correspondence: Marian Kacerovsky, MD, PhD, Biomedical Research Center, University Hospital, Sokolska 581, 500 05 Hradec Kralove, Czech Republic. Tel: +420-777657991, + 420-495832676. E-mail: [email protected]
History Received 29 May 2014 Revised 12 July 2014 Accepted 1 August 2014 Published online 26 August 2014
pathological conditions represents the subgroup of pPROM with the worse scenario because of the highest intraamniotic and fetal inflammatory responses [10–12]. In addition, besides inflammation (characterized cytokines, chemokines, growth factors, matrix degrading enzymes and antimicrobial defense mechanisms), both MIAC and HCA contribute to oxidative stress that may also promote premature senescence of the fetal membranes. Oxidative stress is characterized by a disrupted balance between the production of pro-oxidants and antioxidants [13,14]. Oxidative stress generally arises within the cellular compartments when the production of reactive oxygen species is excessive or total antioxidant capacity (TAC) is decreased [15] resulting in damages to cellular elements. Oxidative stress induced damages play an important role in several pathological processes involving pregnancy pathology [14,16–18]. Pregnancy represents a very complex state in which mother, fetus and placenta contribute to the oxidative stress due to fetal-placental energy demand resulting in increased
Oxidative stress in pPROM
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metabolism and oxidative stress [14]. Many pathways resulting in pPROM and spontaneous preterm births are documented to be associated with increased oxidative stress [17,19] likely due to the fact that inflammation seen in these outcomes is an inevitable component of oxidative stressinduced damages. Although inflammatory mediators have been well documented in pPROM and spontaneous preterm birth with intact membranes, the markers of oxidative stress have not been measured in the amniotic fluid from pregnancies complicated by pPROM. Amniotic fluid and other biological sample studies by many laboratories including our own have reported higher F2-isoprostane concentrations, a classic marker of oxidative stress induced lipid peroxidation, in pPROM than PTB with intact membranes [17,20–22]. Cigarette smokers and subjects with MIAC in this cohort also showed higher amniotic fluid F2-isoprostanes confirming the impact of pPROM risk factors in inducing oxidative stress damages [20]. However, it still remains unclear whether the presence of MIAC and/or HCA, two of the major pathologies of pPROM, associated with increased oxidative stress. It is likely that pPROM during pregnancy may depend on the type of microbe, microbial load and the type of inflammatory response (HCA), each of which either independently or synergistically can generate distinct oxidative stress profile. The main objective of this study was to evaluate oxidative stress markers in the amniotic fluid from pregnancies complicated by pPROM either with or without MIAC and/ or HCA. For this purpose, we evaluated TAC, as ability of amniotic fluid to buffer the effect of oxidative stress; ferricreducing antioxidant power (FRAP), as a measure of antioxidant status of amniotic fluid; and thiobarbituric acid-reacting substances (TBARS), as markers of lipid peroxidation in the amniotic fluid.
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accelerate the lung maturation (two doses of 14 mg betamethasone administered intramuscularly 24 h apart), tocolytics for 48 h and antibiotics, whereas no treatment except antibiotics is initiated to delay delivery after 34 weeks. The management of pPROM in the Czech Republic is active (except 528 gestational weeks); the induction of labor or the termination of pregnancy is initiated no later than 72 h after the rupture of the membranes, depending on the gestational age of the pregnancy, the fetal status, the maternal serum levels of C-reactive protein and cervicovaginal streptococcus b colonization. Ultrasound-guided transabdominal amniocentesis was performed on admission prior to the administration of corticosteroids, antibiotics or tocolytics, and approximately 5 mL of amniotic fluid were aspirated and divided into three tubes. The first and second tubes containing non-centrifuged samples were immediately transported to the microbiology laboratory, where the first tube was used for polymerase chain reaction (PCR) testing for Ureaplasma spp., Mycoplasma hominis and Chlamydia trachomatis. The second tube was used for aerobic and anaerobic bacterial culture. The third tube was centrifuged for 15 min at 2000 g to remove cells and debris, divided into aliquots and stored at –70 C until analysis. The placentas were collected and fixed in 10% neutral buffered formalin. Tissue samples were obtained from the placenta (at least two samples), umbilical cord (typically, one sample) and placental membranes (at least two samples), and the samples were processed and embedded in paraffin. Sections of tissue blocks were stained with hematoxylin and eosin. The study was approved by the University Hospital Hradec Kralove review board committee (19 March 2008; no. 200804 SO1P), and written informed consent was obtained from all participants.
Materials and methods Sample collection
Diagnosis of MIAC
Between September 2011 and October 2013, a prospective cohort study of women with pPROM between 24 + 0 and 36 + 6 weeks’ gestation, who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic, were conducted. Women of maternal age 18 years and with a singleton pregnancy were eligible for enrollment in the study. The exclusion criteria were as follows: structural malformations or chromosomal abnormalities of the fetus, ultrasound signs of fetal growth restriction, vaginal bleeding, signs of fetal hypoxia and presence of maternal complications (i.e. hypertension, preeclampsia, diabetes mellitus and thyroid disease). Gestational ages were established by first-trimester fetal biometry. pPROM was defined as the leakage of amniotic fluid prior to the onset of labor (by at least two hours). This condition was diagnosed using a sterile speculum examination, which confirmed the pooling of amniotic fluid in the vagina, in association with a positive test for the presence of insulin-like growth factor binding protein (ACTIM PROM test; Medix Biochemica, Kauniainen, Finland) in the vaginal fluid. In the Czech Republic, women with pPROM at less than 34 weeks of gestation are treated with corticosteroids to
MIAC was defined as a positive PCR analysis for Ureaplasma spp. and/or M. hominis and/or C. trachomatis and/or growth of any bacteria in the amniotic fluid, except for coagulasenegative Staphylococcus epidermidis, which was considered a skin contaminant. Diagnosis of HCA The degree of neutrophil infiltration was evaluated separately in the free membranes (amnion and chorion–decidua), in the chorionic plate and in the umbilical cord, according to the criteria provided by Salafia et al. [23]. A diagnosis of HCA was made based on the presence of histological grades of chorion-decidua 3–4, chorionic plate 3–4, umbilical cord 1–4 and/or amnion 1–4 [23]. Histological grades of umbilical cord 1–4 were categorized as the presence of funisitis [23]. Histopathological examinations were performed by a single pathologist who was blinded to the clinical status of the patient. Amniotic fluid TPA and FRAP concentrations TAC, as a marker of total antioxidant status of amniotic fluid, was measured according to Erel [24]. Twenty microliter of
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amniotic fluid was mixed with 200 ml of acetate buffer (pH ¼ 5.8), and the initial absorbance of this mixture at 660 nm was recorded. Following the addition of 20 ml 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) in acetate buffer with hydrogen peroxide, and after 5 min incubation at room temperature, the absorbance at 660 nm was recorded again. The difference to the initial values was calculated. This method is based on decolorization of intensely green 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical by antioxidants. The bleaching rate is proportional to the antioxidant concentration in the samples. To construct the calibration curve, Trolox (a water-soluble derivative of tocopherol) was used. FRAP, as a marker of non-protein antioxidant capacity of amniotic fluid, was assessed according to Benzie & Strain [25]. This method is based on reduction of the ferric ion tripyridyl-s-triazine (TPTZ) complexes by antioxidants, in the presence of low pH. Very intense blue color is formed when Fe2+ is binded to ligands. Color intensity is proportional to the amount of antioxidants in the samples. An initial absorbance of freshly prepared FRAP reagent [200 ml mixture of acetate buffer, TPTZ, FeCl3.6H2O and water, heated to 37 C] was measured at a wavelength of 593 nm. Afterwards, 20 ml of samples and standards (prepared using FeSO4.7H2O) were added, incubated for four minutes at room temperature and specific absorbance was measured again. The difference to the initial values was calculated. TBARS, as a markers of lipid peroxidation, were measured according to Behuliak et al. [26]. Products/substances of lipid peroxidation reacts in the presence of high temperature and low pH with thiobarbituric acid. Specific pink-colored complexes are formed and measured after derivatization into butanol. Color intensity is proportional to TBARS concentration in samples. Twenty microliter of amniotic fluids was mixed with 30 ml water, 20 ml thiobarbituric and acetic acids, which formed colored complexes after incubation at 94 C for 45 min. The samples were then cooled to 4 C, and 100 ml of n-butanol was added with subsequent continuous shaking for two minutes. Phase separation was performed by centrifugation at 2000g for 10 min. Eighty microliter of upper phase was afterwards carefully removed and measured at 553 nm emission and 515 nm excitation wavelength. TBARS contents were quantified based on calibration curve made using 1,1,3,3tetramethoxypropane. Statistical analysis Demographic and clinical characteristics were compared using the unpaired t-test (presented as the mean ± SD) and the non-parametric Mann–Whitney U test and are presented as median (range). Categorical variables were compared using Fischer’s exact test and are presented as number (%). The normality of the data was tested using the D’Agostino and Pearson omnibus normality test. Because the amniotic fluid concentrations of TAC, FRAP and TBARS were not normally distributed, a non-parametric test (Mann–Whitney U test) was used for analyses. All p values were from twosided tests, and all statistical analyses were performed using
J Matern Fetal Neonatal Med, 2015; 28(11): 1250–1259
SPSS 19.0 for Mac OS X (SPSS Inc., Chicago, IL) and with GraphPad Prism 5.03 for Mac OS X (GraphPad Software, La Jolla, CA).
Results Demographical and clinical characteristic A total of 151 women with pPROM were recruited during the study period. Of these women, only 138 (91%) were included in the analyses, because aliquots of amniotic fluid samples were not available for 12 (8%) women. Results from remaining one woman (1%) were considered as outliers (amniotic fluid TAC concentration ¼ 239 lmol/L, amniotic fluid FRAP concentration ¼ 296 lmol/L and amniotic fluid TBARS concentration ¼ 0 lmol/L) and excluded from analysis. The presence of MIAC was identified in 25% (34/138) of the women. Genital mycoplasmas (Ureaplasma spp. and/or M. hominis) were responsible for 82% (28/34) of all microbial findings in the amniotic fluid. HCA and funisitis were found in 73% (101/138) and 41% (33/138) of the women, respectively. The presence of both MIAC and HCA was found in 21% (29/138) of the women. The demographic and clinical characteristics of women with respect to the presence of MIAC and HCA are listed in Table 1. Amniotic fluid concentrations of TAC and FRAC were measurable in all samples. TBARS concentrations were measurable in 93% (128/138) samples of amniotic fluid. Three women without measurable TBARS concentrations had both MIAC and HCA; remaining seven women without measurable TBARS concentrations in the amniotic fluid had the presence of HCA alone. Amniotic fluid TAC, FRAP and TBARS concentrations according to the presence of MIAC Statistically significant differences were not observed in amniotic fluid TAC, FRAP and TBARS concentrations in women with MIAC or without MIAC (amniotic fluid TAC: with MIAC: median 868 lmol/L, range 651–1216 versus without MIAC: median 914 lmol/L, range 436–1205; p ¼ 0.93; amniotic fluid FRAP: with MIAC: median 1054 lmol/L, range 773–1934 versus without MIAC: median 1110 lmol/L, range 609–1761; p ¼ 0.41; amniotic fluid TBARS: with MIAC: median 0.28 lmol/L, range 0.8–19.6 versus without MIAC: median 0.24 lmol/L, range 0.1–14.7; p ¼ 0.11; Figure 1). Amniotic fluid TAC, FRAP and TBARS concentrations according to the presence of HCA Similar to MIAC group, no differences in amniotic fluid TAC, FRAP and TBARS concentrations were seen between women with and without HCA (amniotic fluid TAC: with HCA: median 917 lmol/L, range 436–1216 versus without HCA: median 903 lmol/L, range 650–1181; p ¼ 0.63; amniotic fluid FRAP: with HCA: median 1066 lmol/L, range 609–1934 versus without HCA: median 1133 lmol/L, range 749–1675; p ¼ 0.19; amniotic fluid TBARS: with HCA: 0.24 lmol/L, range 0.2–19.6 versus without HCA: 0.24 lmol/L, range 0.01–14.5; p ¼ 0.67; Figure 2).
30.8 ± 6.7 14 (41%) 22.5 (16.5–38.0) 32 + 1 (24 + 1 to 36 + 4) 32 + 2 (24 + 5 to 36 + 6) 10 (29%) 1836 ± 674 7 (1–169) 41 (5–219) 26 (76%) 3 (9%) 0 (0%)
31.1 ± 5.7 50 (48%) 23.4 (16.8–38.6) 32 + 4 (24 + 2 to 36 + 6) 32 + 4 (24 + 4 to 36 + 6) 18 (17%) 1970 ± 545 7 (1–97) 53 (4–221) 68 (65%) 3 (3%) 2 (2%)
Absence of MIAC (n ¼ 104)
0.15 0.24 0.87 0.67 0.41 0.16 1.00
0.56
0.85 0.56 0.39 0.54
p valuea 31.4 ± 5.9 44 (44%) 23.1 (16.8–38.6) 32 + 2 (24 + 1 to 36 + 6) 32 + 5 (24 + 3 to 36 + 6) 20 (20%) 1897 ± 545 6 (1–72) 54 (5–221) 69 (69%) 5 (5%) 1 (1%)
Presence of HCA (n ¼ 101) 30.0 ± 6.0 20 (54%) 23.3 (17.9–34.7) 33 + 0 (27 + 0 to 36 + 4) 33 + 2 (27 + 2 to 36 + 6) 8 (22%) 2045 ± 553 7 (1–169) 51 (4–186) 25 (68%) 1 (3%) 1 (3%)
Absence of HCA (n ¼ 37)
0.82 0.18 0.44 0.31 1.00 1.00 0.47
0.71
0.24 0.34 0.52 0.54
p valueb
31.1 ± 6.8 11 (38%) 22.5 (16.5–38.0) 31 + 6 (24 + 2 to 36 + 6) 32 + 1 (24 + 5 to 36 + 6) 9 (31%) 1777 ± 684 6 (1–169) 48 (5–219) 22 (76%) 3 (10%) 0 (0%)
Presence of both MIAC and HCA (n ¼ 29)
31.0 ± 5.7 53 (49%) 23.4 (16.8–38.6) 32 + 4 (24 + 1 to 36 + 4) 33 + 2 (24 + 4 to 36 + 6) 19 (17%) 1980 ± 544 8 (1–97) 52 (4–221) 72 (66%) 3 (3%) 2 (2%)
Absence of both MIAC and HCA (n ¼ 109)
0.12 0.09 0.62 0.95 0.61 0.11 1.00
0.27
0.88 0.40 0.42 0.25
p valuec
pPROM, preterm prelabor rupture of membranes; MIAC, microbial invasion of the amniotic cavity; HCA, histological chorioamnionitis. Continuous variables were compared using t-test (presented as mean ± SD) or non-parametric Mann–Whitney U test [presented as median (range)]. Categorical variables were compared using Fisher’s exact test and presented as number (%). a p value: comparison between groups with and without MIAC. b p value: comparison between groups with and without HCA. c p value: comparison between groups with and without both MIAC and HCA.
Maternal age (years) Primiparous Prepregnancy body mass index Gestational age at sampling (weeks + days) Gestational age at delivery (weeks + days) Smoking during pregnancy Birth weight (g) pPROM to amniocentesis interval (h) pPROM to delivery interval (h) Vaginal delivery Apgar score 57; 5 min Apgar score 57; 10 min
Presence of MIAC (n ¼ 34)
Table 1. Demographic and clinical characteristic of women with pPROM with respect to the presence of MIAC and/or HCA.
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Figure 1. The amniotic fluid TAC concentrations (a), FRAP concentrations (b) and TBARS (c) concentrations in PPROM pregnancies complicated by MIAC. The women with MIAC did not have different medians TAC, FRAP and TBARS concentrations from the women without MIAC. The horizontal bars indicate the median values. Abbreviations: MIAC, microbial invasion of the amniotic cavity; TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
Amniotic fluid TAC, FRAP and TBARS concentrations according to the presence of both MIAC and HCA Women with the presence of both MIAC and HCA did not have a different amniotic fluid TAC, FRAP and TBARS concentrations than women in whom at least one of these conditions was ruled out (amniotic fluid TAC: with both MIAC and HCA: median 847 lmol/L, range 651–1216 versus
without both MIAC and HCA: median 918 lmol/L, range 436–1205; p ¼ 0.80; amniotic fluid FRAP: with both MIAC and HCA: median 1040 lmol/L, range 773–1934 versus without both MIAC and HCA: median 1115 lmol/L, range 609–1761; p ¼ 0.22; amniotic fluid TBARS: with both MIAC and HCA: 0.31 lmol/L, range 0.7–19.6 versus without both MIAC and HCA: 0.23 lmol/L, range 0.01–14.7; p ¼ 0.08; Figure 3).
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Figure 2. The amniotic fluid TAC concentrations (a), FRAP concentrations (b) and TBARS (c) concentrations in PPROM pregnancies complicated by HCA. The women with HCA did not have different medians TAC, FRAP and TBARS concentrations from the women without HCA. The horizontal bars indicate the median values. Abbreviations: HCA, histological chorioamnionitis; TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
Amniotic fluid TAC and FRAP concentrations according to gestational age at sampling Positive correlations were observed between amniotic fluid concentrations of TAC and FRAP and gestational age at sampling (amniotic fluid TAC: rho ¼ 0.32; p ¼ 0.0002; amniotic fluid FRAP: rho ¼ 0.36; p50.0001; Figure 4). A negative correlation between amniotic fluid TBARS concentrations and gestational age at sampling was
found (amniotic fluid TBARS: rho ¼ –0.25; p ¼ 0.004; Figure 4). The results remain significant after adjustment for smoking (TAC: p ¼ 0.001; FRAP: p50.0001; TBARS: p ¼ 0.04).
Discussion pPROM is a complex syndrome, and the pathways leading to this serious pregnancy complication still remain unclear.
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Figure 3. The amniotic fluid TAC concentrations (a), FRAP concentrations (b) and TBARS (c) concentrations in PPROM pregnancies complicated by both MIAC and HCA. The women with the presence of MIAC and did not have different medians TAC, FRAP and TBARS concentrations than the women in whom at least one of these conditions was ruled out. The horizontal bars indicate the median values. Abbreviations: MIAC, microbial invasion of the amniotic cavity; HCA, histological chorioamnionitis; TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
The clinical presentation of pPROM may differ based on the type of underlying pathways, risk exposure or the type and load of microbial pathogens and the inflammatory signature they generate [5,27]. Our ongoing studies on the amniotic fluid biomarkers of MIAC and/or HCA in pregnancies
complicated by pPROM led us to examine the role of both these conditions with relation to oxidative stress. The following are the key findings of the study: (1) Oxidative stress as documented by presence of TAC, FRAP and TBARS is associated with pregnancies
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Figure 4. Positive correlations were observed between amniotic fluid TAC concentrations (a), FRAP concentrations (b) and gestational age at sampling. A negative correlation between amniotic fluid TBARS concentrations (c) and gestational age at sampling was found. Abbreviations: TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
complicated by pPROM; (2) the presence of MIAC and/or HCA did not modify the concentrations of TAC, FRAP and TBARS in the amniotic fluid and therefore do not provide support as biomarkers of those conditions; and (3) amniotic fluid TAC and FRAP concentrations increased and TBARS concentrations decreased with advanced gestational age in pPROM pregnancies. Human cells have developed a broad spectrum of antioxidants systems that can limit oxidative stress, inactivate them and prevent oxidative stress induced damages to the cell or cellular elements [14,28]. The presence and the intensity of oxidative stress can be demonstrated by various methodologies [14]. In this manuscript, we have chosen three biomarkers reflecting the ability of the amniotic fluid to buffer the effect of oxidative stress (TAC), its antioxidant status (FRAP) and the intensity of lipid peroxidation (TBARS), a measure of oxidative stress induced damages. Amniotic fluid
TAC concentrations have been previously evaluated in amniotic fluid from uncomplicated pregnancies of smoking and non-smoking women, and pregnancies complicated by neural tube defects [29–32]. However, FRAP and TBARS have not been previously analyzed in the amniotic fluid. In this study, we found that TAC and FRAP are detectable in the amniotic fluid from pregnancies complicated by pPROM irrespective of the presence and absence of MIAC and/or HCA. The retrospective nature of sampling will not allow us to determine the origin and causality of oxidative stress markers studied in this report. But this study reaffirms an association between oxidative stress and pPROM confirming many other prior reports of similar association. This study further demonstrates that oxidative stress is associated with pPROM irrespective of MIAC and HCA status reiterating our argument that oxidative stress inducing risk factors may
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initiate pPROM and MIAC and HCA are likely consequences. This argument needs many more validations and experimental support. Cellular level oxidative stress in pPROM has recently been characterized by our group in fetal membranes [33]. These factors are similar to that observed at term, which is considered as a natural physiologic response initiating the signals for labor. Correlation of TAC and FRAP with gestational age argues that the observed findings are simply a factor of natural physiologic response and not pathologically linked to pPROM. It is also possible that TAC and FRAP do not generally reflect cellular level oxidative stress that predispose the membrane to rupture. However, the negative correlation of TBARC, an oxidative stress damage indicator (lipid peroxidation), is suggestive of an ongoing cellular level oxidative stress and oxidative damages at early gestational ages. This confirms our prior data that oxidative stress is likely an initiator of pPROM in early gestational period (534 weeks) than in late pPROM cases [5,20]. Amniotic fluid analysis of TAC, FRAP and TBARS are unchanged during pPROM regardless on MIAC and HCA status suggest that these non-specific markers of oxidative stress in amniotic fluid are unlikely indicators of tissue level/ membrane level oxidative stress status that may have contributed to pPROM. This finding does not rule of the extent of oxidative stress in pPROM regardless of MIAC and HCA status but primarily suggestive of lack of usefulness of them as tissue level response or indicators of underlying pathology. Second, we also believe that oxidative stress is one of the initiators of a cascade of events that can cause pPROM and timing of collection of amniotic fluid (after pPROM) may not reflect oxidative stress associated causal events that may have culminated in pPROM. Third, unlike inflammatory markers, measuring oxidative stress markers after pPROM may not indicate the severity of MIAC or HCA or the adverse outcome in neonates, rather measuring damages caused by oxidative stress may be a better indicative of MIAC/HCA pathology. One of the strengths of our study is that we evaluated three amniotic fluid markers related to oxidative stress in very well-defined subgroup of spontaneous preterm delivery (women with pPROM). There are no previous references in the literature describing the association between amniotic markers of oxidative stress with respect MIAC and HCA in this specific group. However, one of the limitations of this study is that we have evaluated amniotic fluid markers. So, we did not have any information about the origin of these markers. We can only speculate that these markers are released from the fetal membranes and the placenta. However, we cannot exclude the maternal contribution on their production. Therefore, it would be of great interest to evaluate their RNA/protein expression in the fetal membranes. Finally, other possible oxidative stress markers have not been evaluated in the amniotic fluid. This study further supports a role for oxidative stress in pPROM, although further studies are warranted and comparison with term and preterm birth with intact membranes are essential in determining a potential functional association of these markers in pPROM.
J Matern Fetal Neonatal Med, 2015; 28(11): 1250–1259
Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grant from the Ministry of Health, Czech Republic (NT13461-4/2012) and by REVOGENE project, ITMS 26240220067, supported by the Research & Development Operational Program funded by ERDF. Additional support came from Charles University in Prague, the Faculty of Medicine in Hradec Kralove, Czech Republic, project ‘‘PRVOUK’’ P37/10, the Faculty Hospital in Hradec Kralove (a long-term organization development plan).
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20. Menon R, Fortunato SJ, Milne GL, et al. Amniotic fluid eicosanoids in preterm and term births: effects of risk factors for spontaneous preterm labor. Obstet Gynecol 2011;118:121–34. 21. Herway C, Kanninen T, Witkin SS, et al. Ethnic disparity in amniotic fluid levels of hyaluronan, histone H2B and superoxide dismutase in spontaneous preterm birth. J Perinat Med 2013;41: 277–82. 22. Font GE, Gauthier DW, Meyer WJ, et al. Catalase activity as a predictor of amniotic fluid culture results in preterm labor or premature rupture of membranes. Obstet Gynecol 1995;85:656–8. 23. Salafia CM, Weigl C, Silberman L. The prevalence and distribution of acute placental inflammation in uncomplicated term pregnancies. Obstet Gynecol 1989;73:383–9. 24. Erel O. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin Biochem 2004; 37:112–19. 25. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Anal Biochem 1996;239:70–6. 26. Behuliak M, Palffy R, Gardlik R, et al. Variability of thiobarbituric acid reacting substances in saliva. Dis Markers 2009;26:49–53.
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27. Goldenberg RL, Culhane JF, Iams JD, Romero R. Epidemiology and causes of preterm birth. Lancet 2008;371: 75–84. 28. Pierce JD, Cackler AB, Arnett MG. Why should you care about free radicals? RN 2004;67:38–42; quiz 43. 29. Burlingame JM, Esfandiari N, Sharma RK, et al. Total antioxidant capacity and reactive oxygen species in amniotic fluid. Obstet Gynecol 2003;101:756–61. 30. Dittrich R, Schibel A, Hoffmann I, et al. Influence of maternal smoking during pregnancy on oxidant status in amniotic fluid. In vivo 2012;26:813–18. 31. Ziobro A, Bartosz G. A comparison of the total antioxidant capacity of some human body fluids. Cell Mol Biol Lett 2003;8: 415–19. 32. Vural M, Camuzcuoglu H, Toy H, Aksoy N. Amniotic fluid prolidase activity and oxidative status in neural tube defects. Fetal Diagn Ther 2010;28:34–9. 33. Menon R, Boldogh I, Hawkins HK, et al. Histological evidence of oxidative stress and premature senescence in preterm premature rupture of the human fetal membranes recapitulated in vitro. Am J Pathol 2014;184:1740–51.
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Amniotic fluid markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes Marian Kacerovsky, Lubomira Tothova, Ramkumar Menon, Barbora Vlkova, Ivana Musilova, Helena Hornychova, Martin Prochazka & Peter Celec To cite this article: Marian Kacerovsky, Lubomira Tothova, Ramkumar Menon, Barbora Vlkova, Ivana Musilova, Helena Hornychova, Martin Prochazka & Peter Celec (2015) Amniotic fluid markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes, The Journal of Maternal-Fetal & Neonatal Medicine, 28:11, 1250-1259, DOI: 10.3109/14767058.2014.951628 To link to this article: http://dx.doi.org/10.3109/14767058.2014.951628
Published online: 27 Aug 2014.
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http://informahealthcare.com/jmf ISSN: 1476-7058 (print), 1476-4954 (electronic) J Matern Fetal Neonatal Med, 2015; 28(11): 1250–1259 ! 2014 Informa UK Ltd. DOI: 10.3109/14767058.2014.951628
ORIGINAL ARTICLE
Amniotic fluid markers of oxidative stress in pregnancies complicated by preterm prelabor rupture of membranes Marian Kacerovsky1,2, Lubomira Tothova3,4, Ramkumar Menon5, Barbora Vlkova3,4, Ivana Musilova2, Helena Hornychova6, Martin Prochazka7, and Peter Celec3,4 Downloaded by ["University at Buffalo Libraries"] at 16:38 07 November 2015
1
Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic, 2Department of Obstetrics and Gynecology, Faculty of Medicine Hradec Kralove, Charles University in Prague, Prague, Czech Republic, 3Institute of Molecular Biomedicine, Comenius University in Bratislava, Bratislava, Slovak Republic, 4Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia, 5Department of Obstetrics and Gynecology, The University of Texas Medical Branch at Galveston, Galveston, USA, 6Fingerland’s Department of Pathology, Faculty of Medicine Hradec Kralove, Charles University in Prague, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic, and 7Department of Obstetrics and Gynecology, Palacky University Olomouc, University Hospital Olomouc, Olomouc, Czech Republic Abstract
Keywords
Objective: To determine amniotic fluid total antioxidant capacity (TAC), ferric-reducing antioxidant power (FRAP) and thiobarbituric acid-reacting substances (TBARS), markers of oxidative stress, in pregnancies complicated by preterm prelabor rupture of membranes (pPROM) and their correlation to microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA). Methods: One-hundred thirty-eight women with singleton pregnancies complicated by pPROM were included in this study. Amniotic fluid was collected by transabdominal amniocentesis at the time of admission and amniotic fluid concentrations of TAC, FRAP and TBARS were measured. Result: The presence of MIAC and/or HCA did not show any significant differences in the amniotic fluid TAC, FRAP and TBARS concentrations. Positive correlations between gestational age at sampling and amniotic fluid TAC and FRAP concentrations were found (TAC: rho ¼ 0.32; p ¼ 0.0002; FRAP: rho ¼ 0.36; p50.0001). A negative correlation between gestation age at sampling and amniotic fluid TBARS concentrations was identified (rho ¼ –0.25; p ¼ 0.004). Conclusions: Oxidative stress is associated with pPROM as indicated by the presence of markers tested in the amniotic fluid; however, oxidative stress markers tested are not influenced by the presence of MIAC or HCA.
Infection, inflammation, preterm delivery
Introduction Preterm prelabor rupture of membranes (pPROM), defined by the rupture of fetal membranes before spontaneous onset of labor prior to 37 weeks, represents one of the main problems in perinatology [1]. Recent studies have reported a potential pathophysiology for pPROM characterized by oxidative stress-induced premature senescence (aging) of fetal membranes [2–5]. Despite this fact, approximately 75% pPROM cases are associated with microbial invasion of the amniotic cavity (MIAC) and/or histological chorioamnionitis (HCA) [6–8]. It is still under intense debate whether MIAC is cause or consequence of pPROM [9]. Nevertheless, there is a solid body of evidence that the presence of both of these
Address for correspondence: Marian Kacerovsky, MD, PhD, Biomedical Research Center, University Hospital, Sokolska 581, 500 05 Hradec Kralove, Czech Republic. Tel: +420-777657991, + 420-495832676. E-mail: [email protected]
History Received 29 May 2014 Revised 12 July 2014 Accepted 1 August 2014 Published online 26 August 2014
pathological conditions represents the subgroup of pPROM with the worse scenario because of the highest intraamniotic and fetal inflammatory responses [10–12]. In addition, besides inflammation (characterized cytokines, chemokines, growth factors, matrix degrading enzymes and antimicrobial defense mechanisms), both MIAC and HCA contribute to oxidative stress that may also promote premature senescence of the fetal membranes. Oxidative stress is characterized by a disrupted balance between the production of pro-oxidants and antioxidants [13,14]. Oxidative stress generally arises within the cellular compartments when the production of reactive oxygen species is excessive or total antioxidant capacity (TAC) is decreased [15] resulting in damages to cellular elements. Oxidative stress induced damages play an important role in several pathological processes involving pregnancy pathology [14,16–18]. Pregnancy represents a very complex state in which mother, fetus and placenta contribute to the oxidative stress due to fetal-placental energy demand resulting in increased
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metabolism and oxidative stress [14]. Many pathways resulting in pPROM and spontaneous preterm births are documented to be associated with increased oxidative stress [17,19] likely due to the fact that inflammation seen in these outcomes is an inevitable component of oxidative stressinduced damages. Although inflammatory mediators have been well documented in pPROM and spontaneous preterm birth with intact membranes, the markers of oxidative stress have not been measured in the amniotic fluid from pregnancies complicated by pPROM. Amniotic fluid and other biological sample studies by many laboratories including our own have reported higher F2-isoprostane concentrations, a classic marker of oxidative stress induced lipid peroxidation, in pPROM than PTB with intact membranes [17,20–22]. Cigarette smokers and subjects with MIAC in this cohort also showed higher amniotic fluid F2-isoprostanes confirming the impact of pPROM risk factors in inducing oxidative stress damages [20]. However, it still remains unclear whether the presence of MIAC and/or HCA, two of the major pathologies of pPROM, associated with increased oxidative stress. It is likely that pPROM during pregnancy may depend on the type of microbe, microbial load and the type of inflammatory response (HCA), each of which either independently or synergistically can generate distinct oxidative stress profile. The main objective of this study was to evaluate oxidative stress markers in the amniotic fluid from pregnancies complicated by pPROM either with or without MIAC and/ or HCA. For this purpose, we evaluated TAC, as ability of amniotic fluid to buffer the effect of oxidative stress; ferricreducing antioxidant power (FRAP), as a measure of antioxidant status of amniotic fluid; and thiobarbituric acid-reacting substances (TBARS), as markers of lipid peroxidation in the amniotic fluid.
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accelerate the lung maturation (two doses of 14 mg betamethasone administered intramuscularly 24 h apart), tocolytics for 48 h and antibiotics, whereas no treatment except antibiotics is initiated to delay delivery after 34 weeks. The management of pPROM in the Czech Republic is active (except 528 gestational weeks); the induction of labor or the termination of pregnancy is initiated no later than 72 h after the rupture of the membranes, depending on the gestational age of the pregnancy, the fetal status, the maternal serum levels of C-reactive protein and cervicovaginal streptococcus b colonization. Ultrasound-guided transabdominal amniocentesis was performed on admission prior to the administration of corticosteroids, antibiotics or tocolytics, and approximately 5 mL of amniotic fluid were aspirated and divided into three tubes. The first and second tubes containing non-centrifuged samples were immediately transported to the microbiology laboratory, where the first tube was used for polymerase chain reaction (PCR) testing for Ureaplasma spp., Mycoplasma hominis and Chlamydia trachomatis. The second tube was used for aerobic and anaerobic bacterial culture. The third tube was centrifuged for 15 min at 2000 g to remove cells and debris, divided into aliquots and stored at –70 C until analysis. The placentas were collected and fixed in 10% neutral buffered formalin. Tissue samples were obtained from the placenta (at least two samples), umbilical cord (typically, one sample) and placental membranes (at least two samples), and the samples were processed and embedded in paraffin. Sections of tissue blocks were stained with hematoxylin and eosin. The study was approved by the University Hospital Hradec Kralove review board committee (19 March 2008; no. 200804 SO1P), and written informed consent was obtained from all participants.
Materials and methods Sample collection
Diagnosis of MIAC
Between September 2011 and October 2013, a prospective cohort study of women with pPROM between 24 + 0 and 36 + 6 weeks’ gestation, who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic, were conducted. Women of maternal age 18 years and with a singleton pregnancy were eligible for enrollment in the study. The exclusion criteria were as follows: structural malformations or chromosomal abnormalities of the fetus, ultrasound signs of fetal growth restriction, vaginal bleeding, signs of fetal hypoxia and presence of maternal complications (i.e. hypertension, preeclampsia, diabetes mellitus and thyroid disease). Gestational ages were established by first-trimester fetal biometry. pPROM was defined as the leakage of amniotic fluid prior to the onset of labor (by at least two hours). This condition was diagnosed using a sterile speculum examination, which confirmed the pooling of amniotic fluid in the vagina, in association with a positive test for the presence of insulin-like growth factor binding protein (ACTIM PROM test; Medix Biochemica, Kauniainen, Finland) in the vaginal fluid. In the Czech Republic, women with pPROM at less than 34 weeks of gestation are treated with corticosteroids to
MIAC was defined as a positive PCR analysis for Ureaplasma spp. and/or M. hominis and/or C. trachomatis and/or growth of any bacteria in the amniotic fluid, except for coagulasenegative Staphylococcus epidermidis, which was considered a skin contaminant. Diagnosis of HCA The degree of neutrophil infiltration was evaluated separately in the free membranes (amnion and chorion–decidua), in the chorionic plate and in the umbilical cord, according to the criteria provided by Salafia et al. [23]. A diagnosis of HCA was made based on the presence of histological grades of chorion-decidua 3–4, chorionic plate 3–4, umbilical cord 1–4 and/or amnion 1–4 [23]. Histological grades of umbilical cord 1–4 were categorized as the presence of funisitis [23]. Histopathological examinations were performed by a single pathologist who was blinded to the clinical status of the patient. Amniotic fluid TPA and FRAP concentrations TAC, as a marker of total antioxidant status of amniotic fluid, was measured according to Erel [24]. Twenty microliter of
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amniotic fluid was mixed with 200 ml of acetate buffer (pH ¼ 5.8), and the initial absorbance of this mixture at 660 nm was recorded. Following the addition of 20 ml 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) in acetate buffer with hydrogen peroxide, and after 5 min incubation at room temperature, the absorbance at 660 nm was recorded again. The difference to the initial values was calculated. This method is based on decolorization of intensely green 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical by antioxidants. The bleaching rate is proportional to the antioxidant concentration in the samples. To construct the calibration curve, Trolox (a water-soluble derivative of tocopherol) was used. FRAP, as a marker of non-protein antioxidant capacity of amniotic fluid, was assessed according to Benzie & Strain [25]. This method is based on reduction of the ferric ion tripyridyl-s-triazine (TPTZ) complexes by antioxidants, in the presence of low pH. Very intense blue color is formed when Fe2+ is binded to ligands. Color intensity is proportional to the amount of antioxidants in the samples. An initial absorbance of freshly prepared FRAP reagent [200 ml mixture of acetate buffer, TPTZ, FeCl3.6H2O and water, heated to 37 C] was measured at a wavelength of 593 nm. Afterwards, 20 ml of samples and standards (prepared using FeSO4.7H2O) were added, incubated for four minutes at room temperature and specific absorbance was measured again. The difference to the initial values was calculated. TBARS, as a markers of lipid peroxidation, were measured according to Behuliak et al. [26]. Products/substances of lipid peroxidation reacts in the presence of high temperature and low pH with thiobarbituric acid. Specific pink-colored complexes are formed and measured after derivatization into butanol. Color intensity is proportional to TBARS concentration in samples. Twenty microliter of amniotic fluids was mixed with 30 ml water, 20 ml thiobarbituric and acetic acids, which formed colored complexes after incubation at 94 C for 45 min. The samples were then cooled to 4 C, and 100 ml of n-butanol was added with subsequent continuous shaking for two minutes. Phase separation was performed by centrifugation at 2000g for 10 min. Eighty microliter of upper phase was afterwards carefully removed and measured at 553 nm emission and 515 nm excitation wavelength. TBARS contents were quantified based on calibration curve made using 1,1,3,3tetramethoxypropane. Statistical analysis Demographic and clinical characteristics were compared using the unpaired t-test (presented as the mean ± SD) and the non-parametric Mann–Whitney U test and are presented as median (range). Categorical variables were compared using Fischer’s exact test and are presented as number (%). The normality of the data was tested using the D’Agostino and Pearson omnibus normality test. Because the amniotic fluid concentrations of TAC, FRAP and TBARS were not normally distributed, a non-parametric test (Mann–Whitney U test) was used for analyses. All p values were from twosided tests, and all statistical analyses were performed using
J Matern Fetal Neonatal Med, 2015; 28(11): 1250–1259
SPSS 19.0 for Mac OS X (SPSS Inc., Chicago, IL) and with GraphPad Prism 5.03 for Mac OS X (GraphPad Software, La Jolla, CA).
Results Demographical and clinical characteristic A total of 151 women with pPROM were recruited during the study period. Of these women, only 138 (91%) were included in the analyses, because aliquots of amniotic fluid samples were not available for 12 (8%) women. Results from remaining one woman (1%) were considered as outliers (amniotic fluid TAC concentration ¼ 239 lmol/L, amniotic fluid FRAP concentration ¼ 296 lmol/L and amniotic fluid TBARS concentration ¼ 0 lmol/L) and excluded from analysis. The presence of MIAC was identified in 25% (34/138) of the women. Genital mycoplasmas (Ureaplasma spp. and/or M. hominis) were responsible for 82% (28/34) of all microbial findings in the amniotic fluid. HCA and funisitis were found in 73% (101/138) and 41% (33/138) of the women, respectively. The presence of both MIAC and HCA was found in 21% (29/138) of the women. The demographic and clinical characteristics of women with respect to the presence of MIAC and HCA are listed in Table 1. Amniotic fluid concentrations of TAC and FRAC were measurable in all samples. TBARS concentrations were measurable in 93% (128/138) samples of amniotic fluid. Three women without measurable TBARS concentrations had both MIAC and HCA; remaining seven women without measurable TBARS concentrations in the amniotic fluid had the presence of HCA alone. Amniotic fluid TAC, FRAP and TBARS concentrations according to the presence of MIAC Statistically significant differences were not observed in amniotic fluid TAC, FRAP and TBARS concentrations in women with MIAC or without MIAC (amniotic fluid TAC: with MIAC: median 868 lmol/L, range 651–1216 versus without MIAC: median 914 lmol/L, range 436–1205; p ¼ 0.93; amniotic fluid FRAP: with MIAC: median 1054 lmol/L, range 773–1934 versus without MIAC: median 1110 lmol/L, range 609–1761; p ¼ 0.41; amniotic fluid TBARS: with MIAC: median 0.28 lmol/L, range 0.8–19.6 versus without MIAC: median 0.24 lmol/L, range 0.1–14.7; p ¼ 0.11; Figure 1). Amniotic fluid TAC, FRAP and TBARS concentrations according to the presence of HCA Similar to MIAC group, no differences in amniotic fluid TAC, FRAP and TBARS concentrations were seen between women with and without HCA (amniotic fluid TAC: with HCA: median 917 lmol/L, range 436–1216 versus without HCA: median 903 lmol/L, range 650–1181; p ¼ 0.63; amniotic fluid FRAP: with HCA: median 1066 lmol/L, range 609–1934 versus without HCA: median 1133 lmol/L, range 749–1675; p ¼ 0.19; amniotic fluid TBARS: with HCA: 0.24 lmol/L, range 0.2–19.6 versus without HCA: 0.24 lmol/L, range 0.01–14.5; p ¼ 0.67; Figure 2).
30.8 ± 6.7 14 (41%) 22.5 (16.5–38.0) 32 + 1 (24 + 1 to 36 + 4) 32 + 2 (24 + 5 to 36 + 6) 10 (29%) 1836 ± 674 7 (1–169) 41 (5–219) 26 (76%) 3 (9%) 0 (0%)
31.1 ± 5.7 50 (48%) 23.4 (16.8–38.6) 32 + 4 (24 + 2 to 36 + 6) 32 + 4 (24 + 4 to 36 + 6) 18 (17%) 1970 ± 545 7 (1–97) 53 (4–221) 68 (65%) 3 (3%) 2 (2%)
Absence of MIAC (n ¼ 104)
0.15 0.24 0.87 0.67 0.41 0.16 1.00
0.56
0.85 0.56 0.39 0.54
p valuea 31.4 ± 5.9 44 (44%) 23.1 (16.8–38.6) 32 + 2 (24 + 1 to 36 + 6) 32 + 5 (24 + 3 to 36 + 6) 20 (20%) 1897 ± 545 6 (1–72) 54 (5–221) 69 (69%) 5 (5%) 1 (1%)
Presence of HCA (n ¼ 101) 30.0 ± 6.0 20 (54%) 23.3 (17.9–34.7) 33 + 0 (27 + 0 to 36 + 4) 33 + 2 (27 + 2 to 36 + 6) 8 (22%) 2045 ± 553 7 (1–169) 51 (4–186) 25 (68%) 1 (3%) 1 (3%)
Absence of HCA (n ¼ 37)
0.82 0.18 0.44 0.31 1.00 1.00 0.47
0.71
0.24 0.34 0.52 0.54
p valueb
31.1 ± 6.8 11 (38%) 22.5 (16.5–38.0) 31 + 6 (24 + 2 to 36 + 6) 32 + 1 (24 + 5 to 36 + 6) 9 (31%) 1777 ± 684 6 (1–169) 48 (5–219) 22 (76%) 3 (10%) 0 (0%)
Presence of both MIAC and HCA (n ¼ 29)
31.0 ± 5.7 53 (49%) 23.4 (16.8–38.6) 32 + 4 (24 + 1 to 36 + 4) 33 + 2 (24 + 4 to 36 + 6) 19 (17%) 1980 ± 544 8 (1–97) 52 (4–221) 72 (66%) 3 (3%) 2 (2%)
Absence of both MIAC and HCA (n ¼ 109)
0.12 0.09 0.62 0.95 0.61 0.11 1.00
0.27
0.88 0.40 0.42 0.25
p valuec
pPROM, preterm prelabor rupture of membranes; MIAC, microbial invasion of the amniotic cavity; HCA, histological chorioamnionitis. Continuous variables were compared using t-test (presented as mean ± SD) or non-parametric Mann–Whitney U test [presented as median (range)]. Categorical variables were compared using Fisher’s exact test and presented as number (%). a p value: comparison between groups with and without MIAC. b p value: comparison between groups with and without HCA. c p value: comparison between groups with and without both MIAC and HCA.
Maternal age (years) Primiparous Prepregnancy body mass index Gestational age at sampling (weeks + days) Gestational age at delivery (weeks + days) Smoking during pregnancy Birth weight (g) pPROM to amniocentesis interval (h) pPROM to delivery interval (h) Vaginal delivery Apgar score 57; 5 min Apgar score 57; 10 min
Presence of MIAC (n ¼ 34)
Table 1. Demographic and clinical characteristic of women with pPROM with respect to the presence of MIAC and/or HCA.
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Figure 1. The amniotic fluid TAC concentrations (a), FRAP concentrations (b) and TBARS (c) concentrations in PPROM pregnancies complicated by MIAC. The women with MIAC did not have different medians TAC, FRAP and TBARS concentrations from the women without MIAC. The horizontal bars indicate the median values. Abbreviations: MIAC, microbial invasion of the amniotic cavity; TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
Amniotic fluid TAC, FRAP and TBARS concentrations according to the presence of both MIAC and HCA Women with the presence of both MIAC and HCA did not have a different amniotic fluid TAC, FRAP and TBARS concentrations than women in whom at least one of these conditions was ruled out (amniotic fluid TAC: with both MIAC and HCA: median 847 lmol/L, range 651–1216 versus
without both MIAC and HCA: median 918 lmol/L, range 436–1205; p ¼ 0.80; amniotic fluid FRAP: with both MIAC and HCA: median 1040 lmol/L, range 773–1934 versus without both MIAC and HCA: median 1115 lmol/L, range 609–1761; p ¼ 0.22; amniotic fluid TBARS: with both MIAC and HCA: 0.31 lmol/L, range 0.7–19.6 versus without both MIAC and HCA: 0.23 lmol/L, range 0.01–14.7; p ¼ 0.08; Figure 3).
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Figure 2. The amniotic fluid TAC concentrations (a), FRAP concentrations (b) and TBARS (c) concentrations in PPROM pregnancies complicated by HCA. The women with HCA did not have different medians TAC, FRAP and TBARS concentrations from the women without HCA. The horizontal bars indicate the median values. Abbreviations: HCA, histological chorioamnionitis; TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
Amniotic fluid TAC and FRAP concentrations according to gestational age at sampling Positive correlations were observed between amniotic fluid concentrations of TAC and FRAP and gestational age at sampling (amniotic fluid TAC: rho ¼ 0.32; p ¼ 0.0002; amniotic fluid FRAP: rho ¼ 0.36; p50.0001; Figure 4). A negative correlation between amniotic fluid TBARS concentrations and gestational age at sampling was
found (amniotic fluid TBARS: rho ¼ –0.25; p ¼ 0.004; Figure 4). The results remain significant after adjustment for smoking (TAC: p ¼ 0.001; FRAP: p50.0001; TBARS: p ¼ 0.04).
Discussion pPROM is a complex syndrome, and the pathways leading to this serious pregnancy complication still remain unclear.
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Figure 3. The amniotic fluid TAC concentrations (a), FRAP concentrations (b) and TBARS (c) concentrations in PPROM pregnancies complicated by both MIAC and HCA. The women with the presence of MIAC and did not have different medians TAC, FRAP and TBARS concentrations than the women in whom at least one of these conditions was ruled out. The horizontal bars indicate the median values. Abbreviations: MIAC, microbial invasion of the amniotic cavity; HCA, histological chorioamnionitis; TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
The clinical presentation of pPROM may differ based on the type of underlying pathways, risk exposure or the type and load of microbial pathogens and the inflammatory signature they generate [5,27]. Our ongoing studies on the amniotic fluid biomarkers of MIAC and/or HCA in pregnancies
complicated by pPROM led us to examine the role of both these conditions with relation to oxidative stress. The following are the key findings of the study: (1) Oxidative stress as documented by presence of TAC, FRAP and TBARS is associated with pregnancies
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Figure 4. Positive correlations were observed between amniotic fluid TAC concentrations (a), FRAP concentrations (b) and gestational age at sampling. A negative correlation between amniotic fluid TBARS concentrations (c) and gestational age at sampling was found. Abbreviations: TAC, total antioxidant capacity; FRAP, ferric reducing antioxidant power; and TBARS, thiobarbituric acid-reacting substances.
complicated by pPROM; (2) the presence of MIAC and/or HCA did not modify the concentrations of TAC, FRAP and TBARS in the amniotic fluid and therefore do not provide support as biomarkers of those conditions; and (3) amniotic fluid TAC and FRAP concentrations increased and TBARS concentrations decreased with advanced gestational age in pPROM pregnancies. Human cells have developed a broad spectrum of antioxidants systems that can limit oxidative stress, inactivate them and prevent oxidative stress induced damages to the cell or cellular elements [14,28]. The presence and the intensity of oxidative stress can be demonstrated by various methodologies [14]. In this manuscript, we have chosen three biomarkers reflecting the ability of the amniotic fluid to buffer the effect of oxidative stress (TAC), its antioxidant status (FRAP) and the intensity of lipid peroxidation (TBARS), a measure of oxidative stress induced damages. Amniotic fluid
TAC concentrations have been previously evaluated in amniotic fluid from uncomplicated pregnancies of smoking and non-smoking women, and pregnancies complicated by neural tube defects [29–32]. However, FRAP and TBARS have not been previously analyzed in the amniotic fluid. In this study, we found that TAC and FRAP are detectable in the amniotic fluid from pregnancies complicated by pPROM irrespective of the presence and absence of MIAC and/or HCA. The retrospective nature of sampling will not allow us to determine the origin and causality of oxidative stress markers studied in this report. But this study reaffirms an association between oxidative stress and pPROM confirming many other prior reports of similar association. This study further demonstrates that oxidative stress is associated with pPROM irrespective of MIAC and HCA status reiterating our argument that oxidative stress inducing risk factors may
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initiate pPROM and MIAC and HCA are likely consequences. This argument needs many more validations and experimental support. Cellular level oxidative stress in pPROM has recently been characterized by our group in fetal membranes [33]. These factors are similar to that observed at term, which is considered as a natural physiologic response initiating the signals for labor. Correlation of TAC and FRAP with gestational age argues that the observed findings are simply a factor of natural physiologic response and not pathologically linked to pPROM. It is also possible that TAC and FRAP do not generally reflect cellular level oxidative stress that predispose the membrane to rupture. However, the negative correlation of TBARC, an oxidative stress damage indicator (lipid peroxidation), is suggestive of an ongoing cellular level oxidative stress and oxidative damages at early gestational ages. This confirms our prior data that oxidative stress is likely an initiator of pPROM in early gestational period (534 weeks) than in late pPROM cases [5,20]. Amniotic fluid analysis of TAC, FRAP and TBARS are unchanged during pPROM regardless on MIAC and HCA status suggest that these non-specific markers of oxidative stress in amniotic fluid are unlikely indicators of tissue level/ membrane level oxidative stress status that may have contributed to pPROM. This finding does not rule of the extent of oxidative stress in pPROM regardless of MIAC and HCA status but primarily suggestive of lack of usefulness of them as tissue level response or indicators of underlying pathology. Second, we also believe that oxidative stress is one of the initiators of a cascade of events that can cause pPROM and timing of collection of amniotic fluid (after pPROM) may not reflect oxidative stress associated causal events that may have culminated in pPROM. Third, unlike inflammatory markers, measuring oxidative stress markers after pPROM may not indicate the severity of MIAC or HCA or the adverse outcome in neonates, rather measuring damages caused by oxidative stress may be a better indicative of MIAC/HCA pathology. One of the strengths of our study is that we evaluated three amniotic fluid markers related to oxidative stress in very well-defined subgroup of spontaneous preterm delivery (women with pPROM). There are no previous references in the literature describing the association between amniotic markers of oxidative stress with respect MIAC and HCA in this specific group. However, one of the limitations of this study is that we have evaluated amniotic fluid markers. So, we did not have any information about the origin of these markers. We can only speculate that these markers are released from the fetal membranes and the placenta. However, we cannot exclude the maternal contribution on their production. Therefore, it would be of great interest to evaluate their RNA/protein expression in the fetal membranes. Finally, other possible oxidative stress markers have not been evaluated in the amniotic fluid. This study further supports a role for oxidative stress in pPROM, although further studies are warranted and comparison with term and preterm birth with intact membranes are essential in determining a potential functional association of these markers in pPROM.
J Matern Fetal Neonatal Med, 2015; 28(11): 1250–1259
Declaration of interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grant from the Ministry of Health, Czech Republic (NT13461-4/2012) and by REVOGENE project, ITMS 26240220067, supported by the Research & Development Operational Program funded by ERDF. Additional support came from Charles University in Prague, the Faculty of Medicine in Hradec Kralove, Czech Republic, project ‘‘PRVOUK’’ P37/10, the Faculty Hospital in Hradec Kralove (a long-term organization development plan).
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