Clinical chorioamnionitis at term is characterized by changes in the plasma concentration of CHCHD2/MNRR1, a mitochondrial protein

Abstract Objective Mitochondrial dysfunction was observed in acute systemic inflammatory conditions such as sepsis and might be involved in sepsis-induced multi-organ failure. Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 2 (CHCHD2), also known as Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1), a bi-organellar protein located in the mitochondria and the nucleus, is implicated in cell respiration, survival, and response to tissue hypoxia. Recently, the reduction of the cellular CHCHD2/MNRR1 protein, as part of mitochondrial dysfunction, has been shown to play a role in the amplification of inflammatory cytokines in a murine model of lipopolysaccharide-induced systemic inflammation. The aim of this study was to determine whether the plasma concentration of CHCHD2/MNRR1 changed during human normal pregnancy, spontaneous labor at term, and clinical chorioamnionitis at term. Methods We conducted a cross-sectional study that included the following groups: 1) non-pregnant women (n = 17); 2) normal pregnant women at various gestational ages from the first trimester until term (n = 110); 3) women at term with spontaneous labor (n = 50); and 4) women with clinical chorioamnionitis at term in labor (n = 25). Plasma concentrations of CHCHD2/MNRR1 were assessed by an enzyme-linked immunosorbent assay. Results 1) Pregnant women at term in labor with clinical chorioamnionitis had a significantly higher plasma CHCHD2/MNRR1 concentration than those in labor without chorioamnionitis (p = .003); 2) CHCHD2/MNRR1 is present in the plasma of healthy non-pregnant and normal pregnant women without significant differences in its plasma concentrations between the two groups; 3) there was no correlation between maternal plasma CHCHD2/MNRR1 concentration and gestational age at venipuncture; and 4) plasma CHCHD2/MNRR1 concentration was not significantly different in women at term in spontaneous labor compared to those not in labor. Conclusions CHCHD2/MNRR1 is physiologically present in the plasma of healthy non-pregnant and normal pregnant women, and its concentration does not change with gestational age and parturition at term. However, plasma CHCHD2/MNRR1 is elevated in women at term with clinical chorioamnionitis. CHCHD2/MNRR1, a novel bi-organellar protein located in the mitochondria and the nucleus, is released into maternal plasma during systemic inflammation.

Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 2 (CHCHD2)/Mitochondrial Nuclear Retrograde Regulator 1 (MNRR1) (also called AAG10, PARK22) is a recently characterized bi-organellar protein with pleiotropic cell functions [31].In the mitochondria, where it is predominantly located, CHCHD2/MNRR1 is a regulator of cytochrome c oxidase (COX) activity [32--34].Reduced cellular levels of CHCHD2/MNRR1 are associated with a 50% reduction in cellular oxygen consumption and with increased total reactive oxygen species (ROS) production [33].In response to stress, such as tissue hypoxia, CHCHD2/MNRR1 is recruited to the nucleus, where it is a transcriptional activator of itself and other genes involved in the cellular response to moderate hypoxia (i.e.4% oxygen) [31,33].This inter-organellar communication is particularly critical under stress conditions where the mitochondria and the nucleus are involved to ensure adequate response to stress and to maintain cellular survival [35].
Mitochondrial dysfunction has been observed in acute systemic inflammatory conditions, such as sepsis [36][37][38][39], and is proposed to be involved in sepsis-induced multiorgan failure [37,40,41].Due to its involvement in the cellular stress response, CHCHD2/MNRR1 has recently been examined in the context of infection/inflammation associated with pregnancy complications.Reduction of the cellular CHCHD2/MNRR1 protein, as part of mitochondrial dysfunction, has been shown to play a role in the amplification of inflammatory cytokine production in a murine model of lipopolysaccharide (LPS)induced systemic inflammation and preterm birth [42].Furthermore, murine macrophages treated with LPS displayed enhanced release of CHCHD2/MNRR1 (Purandare N, Grossman LI, Aras S, et al. unpublished observation).The maternal plasma concentration of CHCHD2/MNRR1 in human pregnancy has never been examined.The aim of this study was to determine whether the plasma concentration of CHCHD2/MNRR1 changed during human normal pregnancy, spontaneous labor at term, and clinical chorioamnionitis at term.

Study design and population
This cross-sectional study included women in the following groups: 1) non-pregnant women (n ¼ 17); 2) women with an uncomplicated pregnancy at various gestational ages from the first trimester until term (n ¼ 110); 3) pregnant women at term with spontaneous labor (n ¼ 50); and 4) pregnant women at term in labor with clinical chorioamnionitis (n ¼ 25).Patients with a multiple pregnancy or with multiple fetuses with congenital and/or chromosomal anomalies were excluded from the study.Samples and data were retrieved from the Perinatology Research Branch's bank of biological specimens.Many of these samples have been employed to study the biology of inflammation, hemostasis, angiogenesis regulation, and growth factor concentrations in normal and complicated pregnancies.All women provided written informed consent prior to the collection of maternal blood samples.The Institutional Review Boards of Wayne State University and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD/NIH/DHHS) approved the collection and utilization of samples for research purposes.

Clinical definitions
The inclusion criteria for normal pregnancy consisted of 1) no medical, obstetrical, or surgical complications; 2) no labor; and 3) delivery of a neonate (!37 weeks of gestation) with a birthweight appropriate for gestational age (between the 10th and 90th percentiles) [43].Normal pregnant women were enrolled from either the antenatal clinic or from the labor/delivery unit (in cases of scheduled cesarean delivery).All pregnancies were followed until delivery.Term pregnancy was defined as a gestational age !37 weeks.Labor was diagnosed by the presence of regular uterine contractions occurring at a frequency of four in 20 min for a minimum of 1 h associated with cervical dilatation and/or effacement changes [44].The diagnosis of clinical chorioamnionitis was given in the presence of maternal fever (>37.8C) together with two or more of the following symptoms: 1) maternal tachycardia (heart rate > 100 beats per minute); 2) uterine tenderness; 3) malodorous amniotic fluid; 4) fetal tachycardia (fetal heart rate >160 beats per minute); and 5) maternal leukocytosis [white blood cell (WBC) count >15,000 per mm 3 ] [4, 5,19,25,27,45].All cases of clinical chorioamnionitis had acute inflammatory lesions of the placenta consistent with amniotic fluid infection defined by the presence of acute histologic chorioamnionitis (stage 2 or more) [46][47][48] and/or acute funisitis [46,47,49].Acute histologic chorioamnionitis was diagnosed based on the presence of inflammatory cells in the chorionic plate and/or in the chorioamniotic membranes [50], and acute funisitis was diagnosed based on the presence of neutrophils in the wall of the umbilical vessels and/or in the Wharton's jelly, according to previously described criteria [50,51].

Sample collection and determination of a human CHCHD2/MNRR1 concentration
Maternal blood samples were obtained by venipuncture and collected in ethylene diamine tetra-acetic acid (EDTA)-containing tubes.Samples were centrifuged after collection and stored at À80 C. Plasma CHCHD2/MNRR1 concentrations were determined by a commercially available immunoassay (Human CHCHD2 ELISA Kit, Abbexa LTD, Cambridge, UK).Briefly, 100 lL of maternal plasma or calibrator were dispensed into separate wells of the plates and incubated at 37 C for 90 min.After the removal of the remaining sample and calibrator, the plates were washed twice with 1X wash buffer and 100 lL of detection reagent.A working solution was added to each well.Plates were then incubated at 37 C for 60 min.Then, the plates were washed three times with 1X wash buffer, 100 lL of detection reagent B solution were added to each well, and the plates were then incubated at 37 C for 30 min.Subsequently, the plates were washed five times with 1X wash buffer, and 90 lL of TMB substrate were added to each well.Plates were then mixed thoroughly and incubated at 37 C for 20 min.Finally, 50 lL of stop solution were added to each well.SpectraMax iD5 (Molecular Devices, San Jose, CA, USA) was used to read the plates and CHCHD2/MNRR1 concentrations were calculated with SoftMax Pro 7 (Molecular Devices).Each specimen was run in duplicate.The inter-and intra-assay coefficients of variation in our laboratory were 8.3% and 7.1%, respectively, with an assay sensitivity of 12.3 pg/mL.

Statistical analysis
The Kolmogorov-Smirnov test and visual plot inspection were used to assess the normality of continuous data distributions.Demographic categorical data were summarized as proportions, whereas continuous variables were summarized by medians and interquartile (IQR) ranges.Differences were examined by using a chi-square or a Fisher's exact test for categorical data and the Mann-Whitney U test for continuous data.Spearman's correlation was used to assess the relationship between two continuous variables.A two-tailed p-value of <.05 was considered statistically significant.Statistical analyses were performed with the R statistical language version 4.1.2and the IBM SPSS version 19.0 (IBM Corporation, Armonk, NY, USA).

Results
Plasma CHCHD2/MNRR1 concentrations in nonpregnant women and in pregnant women with an uncomplicated pregnancy, and the changes in concentrations with gestational age Table 1 shows the clinical characteristics of the study groups.Maternal age, frequency of nulliparity, and duration of sample storage were significantly different between the two groups.CHCHD2/MNRR1 was detectable in all samples from non-pregnant women and from pregnant women with an uncomplicated pregnancy between 8 and 42 3/7 weeks of gestation (n ¼ 127).There was no significant difference in the median (IQR range) plasma CHCHD2/MNRR1 concentration between non-pregnant women and pregnant women with an uncomplicated pregnancy [723 (584-5217) pg/mL vs. 748 (477-4668) pg/mL; p ¼ .6](Figure 1).There was no correlation between the plasma CHCHD2/MNRR1 concentration and gestational age at venipuncture among normal pregnant women (n ¼ 110, Spearman's Rho ¼ .1;p ¼ .1.Similarly, there was no correlation between the duration of sample storage and plasma CHCHD2 concentrations in the groups of non-pregnant (n ¼ 17) and normal pregnant women (n ¼ 110) (n ¼ 127, Spearman's Rho ¼ .06;p ¼ .5).Therefore, it is unlikely that the difference in the duration of sample storage would have an impact on the plasma CHCHD2/MNRR1 concentrations.See Supplementary Figure 1 for the number of samples at each gestational age and the distribution of plasma CHCHD2/MNRR1 concentrations according to gestational age at blood sampling.
Plasma CHCHD2/MNRR1 concentrations in pregnant women at term with and without labor Clinical characteristics of pregnant women at term with and without labor are displayed in Table 2.The two groups did not differ in demographics and clinical characteristics as well as in the median (IQR) plasma CHCHD2/MNRR1 concentration [no labor 1038 (527-9834) pg/mL vs. labor 819 (515-773) pg/mL; p ¼ .4](Figure 2).Plasma CHCHD2/MNRR1 concentrations in pregnant women at term with and without clinical chorioamnionitis Table 3 shows the clinical characteristics of women in labor at term according to the diagnosis of clinical chorioamnionitis.The frequency of nulliparity,   gestational age at venipuncture, and duration of sample storage were significantly different between the two groups.However, there were no significant effects of these variables on plasma CHCHD2/MNRR1 concentrations (all p values > .4).Women in labor at term with clinical chorioamnionitis had a significantly higher median (IQR) plasma CHCHD2/MNRR1 concentration than those without this condition [1722 (1201-3757) pg/mL vs. 819 (515-4773) pg/mL; p ¼ .003)](Figure 3).

Discussion
Principal findings of the study 1) CHCHD2/MNRR1 was detectable in all of the tested plasma samples collected from healthy non-pregnant and normal pregnant women; 2) no significant differences in plasma CHCHD2/MNRR1 concentrations were observed between healthy non-pregnant and uncomplicated pregnant women; 3) the maternal plasma CHCHD2/MNRR1 concentration did not change with gestational age or with spontaneous labor at term; and 4) clinical chorioamnionitis at term was associated with an elevated concentration of CHCHD2/MNRR1 in the maternal plasma.CHCHD2/MNRR1 is a member of the coiled-coil-helixcoiled-coil-helix (CHCH) domain-containing protein family, a group of evolutionarily conserved proteins [52,53], now recognized to be cellular factors with multiple functions such as respiration control, redox regulation, and maintenance of lipid homeostasis and membrane dynamics in the mitochondria [54].Growing evidence also shows that CHCH domain-containing proteins are implicated in several human diseases [54].The second member of this family, CHCHD2/MNRR1, is a biorganellar protein involved in mitochondrial-nuclear communication through a retrograde signaling.Under physiological conditions, CHCHD2/MNRR1 is located in  the mitochondria where it regulates oxidative phosphorylation by binding cytochrome c oxidase subunit 4 isoform 2 (COX4I2).In the presence of stress signals, CHCHD2/MNRR1 is recruited to the nucleus where it can initiate an adaptive response.Indeed, in the nucleus, this protein binds the oxygen-responsive element (ORE) in the promoter region of COX4I2 and of CHCHD2 itself, therefore enhancing its own transcription in a positive feedback loop [33].Additionally, under physiological conditions, CHCHD2/MNRR1 inhibits apoptosis by binding to the anti-apoptotic protein Bcl-xL and by preventing the accumulation of the pro-apoptotic protein Bax in the mitochondria [55].Lastly, CHCHD2/MNRR1 has recently been reported to be implicated in the host response to inflammatory stimuli [42].
CHCHD2/MNRR1 is physiologically present in the plasma of non-pregnant and pregnant women Plasma CHCHD2/MNRR1 concentration in humans has never been reported in the literature.Therefore, we assessed the presence of CHCHD2/MNRR1 in the peripheral blood of healthy non-pregnant and pregnant women.Our results showed that CHCHD2/MNRR1 is physiologically present in the plasma of healthy nonpregnant and normal pregnant women and that pregnancy does not significantly change the plasma concentration of this protein.These findings are consistent with the available evidence showing that CHCHD2/MNRR1 is a protein ubiquitously expressed in human cells.Specific attention has been posed on CHCHD2/MNRR1 in central nervous system cells as mutations in the CHCHD2/MNRR1 gene are implicated in neurodegenerative diseases such as Parkinson's disease [56], and Huntington's disease [57] and increased expression of CHCHD2/MNRR1 has been identified in non-small-cell lung cancer [58], in hepatitis B or C virus-associated hepatocellular carcinoma [59], and in hepatocytes of nonalcoholic steatohepatitis [60].Additionally, we have observed that CHCHD2/MNRR1 is expressed in gestational and reproductive tissues such as the myometrium [61], chorioamniotic membranes [62], and cervix [63] of pregnant women.Given the ubiquitous expression of CHCHD2/MNRR1 in human tissues, various cell types could be the potential source of CHCHD2/MNRR1 in the plasma of pregnant and non-pregnant women.However, the observation that the plasma CHCHD2/MNRR1 concentration does not change with advancing gestational age may indicate that the source of this protein in the circulation of healthy pregnant women might not be the fetus, the placenta, or the amniotic fluid and that this protein in the maternal blood is probably not developmentally regulated.
Plasma CHCHD2/MNRR1 concentrations in pregnant women at term with and without clinical chorioamnionitis Clinical chorioamnionitis has a standard clinical definition based on the studies of Gibbs et al. [19,25], which refers to the presence of maternal fever together with clinical (i.e.uterine tenderness, malodorous discharge, and maternal and fetal tachycardia) and laboratory (i.e.leukocytosis) signs.These signs are thought to be the manifestation of a local and systemic inflammatory process in response to microbial invasion of the amniotic cavity [20,21,[27][28][29]122,123].Indeed, among patients with a diagnosis of clinical chorioamnionitis at term, we demonstrated that about 54% have evidence of microbial-associated intra-amniotic inflammation and 24% have sterile intra-amniotic inflammation [23], while 22% have neither infection nor inflammation [24].Women diagnosed with clinical chorioamnionitis who have microbial-associated or sterile intra-amniotic inflammation have a dramatic upregulation of the intra-amniotic inflammatory response as assessed by the amniotic fluid concentration of cytokines [24], and such inflammation is also reflected in the maternal plasma [22].Indeed, maternal plasma concentrations of inflammation-related cytokines (i.e.interleukin (IL)-2, IL-6, IL-1b, IL-17a, interferon gamma (IFN-c), tumor necrosis factor (TNF), IL-15, IL-12/IL-23p40, IL-10, and IL-5) were found to be significantly higher in women with clinical chorioamnionitis at term than in those without clinical chorioamnionitis, reflecting a state of maternal systemic inflammation in this condition [22].Plasma concentrations of TNF-a were also reported to be above lethal levels in some women with clinical chorioamnionitis at term, resulting in maternal cardiovascular collapse and disseminated intravascular coagulation in the immediate postpartum [124].
Mitochondria are cellular organelles that provide the majority of energy required in cells through oxidative phosphorylation [186][187][188][189][190][191], and they are therefore critical to cell functions.Disruption of oxidative phosphorylation in impaired mitochondria leads to an increase in ROS production that can damage macromolecules and causes cell dysfunction in a vicious cycle [192].Additionally, impaired mitochondria are recognized to be capable of releasing endogenous danger molecules (e.g.cardiolipin, cytochrome c, mitochondrial DNA) [193,194] that are sensed as "non-self" by the innate immune system via pattern recognition receptors such as Toll-like receptor-9 and nucleotidebinding oligomerization domain (NOD)-like receptors [195][196][197][198].It was recently reported that the intact mitochondria released from cells undergoing TNFinduced necroptosis are also detected by innate immune cells as an endogenous danger signal or alarmin [199].
A growing body of evidence has shown a close relationship existing between mitochondrial dysfunction and inflammation [200][201][202][203][204][205][206].Dysfunctional mitochondria can be a source of pro-inflammatory mediators [200], and inflammatory states, including sepsis, are capable of inducing mitochondrial dysfunction.Indeed, results from animal studies in a peritonitis model of sepsis showed that this inflammatory condition resulted in mitochondrial dysfunction defined by an increase in mitochondrial ROS production and by electron chain dysfunction [203,[206][207][208]. Similarly, proinflammatory mediators such as TNF-a and IL-1b have been reported to cause mitochondrial dysfunction in various cell lines [209,210].Of these cytokines, TNF-a seems to play an important role in inflammationinduced mitochondrial dysfunction as it has been shown to inhibit oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase in bovine hepatocytes [211].
In the present study, we found that the CHCHD2/MNRR1 concentration in maternal plasma of women at term with clinical chorioamnionitis is significantly higher than that of women at term without clinical chorioamnionitis.Therefore, in the presence of systemic inflammation in women diagnosed with clinical chorioamnionitis at term, we hypothesized that damaged and stressed cells can release CHCHD2/MNRR1 into the maternal circulation.However, it remains to be determined whether this mitochondrial protein can be qualified as an alarmin and whether the plasma concentration of CHCHD2/MNRR1 has any prognostic value like other mitochondrial components in patients with sepsis and multi-organ failure [212][213][214][215][216][217][218].

Strength and limitations
This study is the first to evaluate CHCHD2/MNRR1 in the plasma of non-pregnant women, normal pregnant women, and women diagnosed with clinical chorioamnionitis at term.All cases of clinical chorioamnionitis had acute inflammatory lesions of the placenta consistent with amniotic fluid infection.However, the cross-sectional nature of the study does not allow us to provide information about the temporal relationship between changes in the CHCHD2/MNRR1 concentration and the clinical diagnoses of spontaneous labor at term or the development of clinical chorioamnionitis.Integration of these results with microbiological findings would add strength to our study.

Conclusions
This is the first study to report on the concentration of the mitochondrial protein CHCHD2/MNRR1 in human plasma.CHCHD2/MNRR1 was detected in the plasma of all women included in the study.However, progression of pregnancy does not significantly change the plasma CHCHD2/MNRR1 concentration.Similarly, spontaneous labor at term is not reflected by changes in the plasma CHCHD2/MNRR1 concentration.By contrast, clinical chorioamnionitis at term, a systemic inflammatory state, is associated with an elevation of CHCHD2/MNRR1 in maternal blood.CHCHD2/MNRR1 may represent a mitochondrial protein released into maternal blood during systemic inflammation.
Dr. Romero has contributed to this work as part of his official duties as an employee of the United States Federal Government.

Figure 2 .
Figure 2. Plasma CHCHD2/MNRR1 concentration (pg/mL) of pregnant women at term not in labor and those at term in spontaneous labor [1038 (527-9834) pg/mL vs. 819 (515-4773)].Data are reported as the median and as the interquartile range.The yaxis is in logarithmic scale.

Figure 3 .
Figure 3. Plasma CHCHD2/MNRR1 concentration (pg/mL) of pregnant women at term in labor without and with clinical chorioamnionitis [819 (515-4773) pg/mL vs 1722 (1201-3757) pg/mL].Data are reported as the median and as the interquartile range.The y-axis is in logarithmic scale.

Table 1 .
Clinical characteristics of non-pregnant and normal pregnant women.
GA: gestational age.Data are reported as the median (interquartile range) for continuous variables and as the percentage (number) for dichotomous variables.

Table 2 .
Clinical characteristics of pregnant women at term in spontaneous labor and not in labor.
BMI: body mass index; GA: gestational age.Data are reported as the median (interquartile range) for continuous variables and as the percentage (number) for dichotomous variables.

Table 3 .
Clinical characteristics of women at term in labor with and without clinical chorioamnionitis.
BMI: body mass index; GA: gestational age.Data are reported as the median (interquartile range) for continuous variables and as the percentage (number) for dichotomous variables.