Cervicovaginal Inflammatory Cytokines, Obesity and Inter-Pregnancy Interval Negatively Affect Pregnancy Duration in Pregnant Women at High-Risk for Recurrent Spontaneous Preterm Birth ()
1. Introduction
Maternal immune system regulation is crucial for maintenance of a healthy pregnancy and fetal development [1]. These processes depend on the ability of the mother’s immune system to adapt in order to tolerate the fetus through the process of immunoregulation [2]. Transition from an anti- to pro-inflammatory state in the mother and at the maternal-fetal interface may underlie the pathophysiology of preterm birth (PTB) [3].
Global prevalence of preterm birth (PTB) ranges between 5% and 18% [4] and spontaneous preterm birth (SPTB) accounts for 35% - 45% of all PTB cases [5]. Preterm infants, defined as infants who were born before <37 gestational weeks (GW), are at increased risk of morbidity and mortality and long-term disabilities [4].
The cervicovaginal (CV) space is metabolically active during pregnancy [6] and proteins in cervicovaginal fluid (CVF) are involved in immune regulation [7]. Thus, CVF is considered as a valuable source of information about female reproductive tract in pregnant and non-pregnant women [8]. Moreover, changes in CVF constituents may be observed early in pregnancy prior to any clinical symptoms [6] and these changes alone or in combination with clinical risk factors may help to identify women most at risk of PTB [7].
Cervical fetal fibronectin, α-fetoprotein, C-reactive protein (CRP) and interleukin 6 (IL-6) and cervical length can have an overall good diagnostic accuracy in identifying pregnancies at risk of SPTB [9]. However, these parameters are not effective for predicting the risk of SPTB in asymptomatic women [10]. One the other hand, the reported association between the changes in the levels of IL-6 and IL-10 early in pregnancy and decreased cervical length, suggests an imbalance of immune regulation that could impact cervical length [11].
1.1. Objectives
The current study aimed to evaluate the change of CVF cytokines’ levels during pregnancy and the relation between the extent of this change and incidence of SPTB in women at high-risk for recurrent SPTB in comparison to normal pregnant women.
1.2. Design
Comparative prospective multicenter interventional study.
1.3. Setting
Gynecology & Obstetrics Department at Benha University Hospital in conjunction with multiple private Obstetric centers and Medical Biochemistry Department, Faculty of Medicine, Benha University.
2. Patients & Methods
All women attending the Antenatal Care Unit for assurance of being pregnant were eligible for evaluation. Clinical evaluation and abdominal ultrasonographic examination was performed and women were asked to attend the clinic at start of the 2nd trimester for re-evaluation, giving CVF samples for lab examinations (Sample 1) and transvaginal ultrasonographic examination for estimation of cervical length (CL) and women with CL < 25 mm were considered at a risk for having PTB [12] and undertake cervical cerclage (CC), irrespective of having history of previous PTB.
Exclusion criteria included multiple pregnancy, fetal or uterine anomalies, history of recurrent early pregnancy loss, cervical surgeries, previous pregnancy-induced complications, manifest diabetes mellitus, autoimmune diseases, current manifest vaginal infection were excluded from the study. Also, women refused to sign the consent for study participation, lost during follow-up or gave birth outside the participating centers were excluded from the study.
Inclusion criteria included singleton fetus, attendance to OPC at beginning of the 2nd trimester, absence of exclusion criteria and signing a written consent. Women had previous SPTB were eligible for evaluation and those at high-risk for recurrent PTB were included as Study group and women free of history of SPTB and had CL > 25 mm at the beginning of 2nd trimester as Control group which must include women of cross-matched age, gestational age (GA) and body mass index (
BMI
) in number equal to women of study group. The study protocol was approved by the Local Ethical Committee and all enrolled women signed written fully informed consent. All women of study and control groups were maintained on vaginal toilet to guard against development of infection and were asked to attend the outpatient clinic biweekly for evaluation for development of PTB, which was defined as any birth before 37 weeks completed weeks of gestation [4] [13]. At time of labor or removal of the CC suture, another CVF samples were obtained (Sample II).
2.1. Methods
2.1.1. CVF Sample Obtaining and Processing
Vaginal speculum was applied and to obtain high vaginal smear of CVF a Dacron swab was placed in posterior vaginal fornix, maintained in situ for 10 seconds to achieve saturation, and then was transferred into 750 ml of standard phosphate-buffered saline solution mixed with freshly prepared protease inhibitor solution. The swab was then removed, placed in a clean tube, vortexed for 10 sec and centrifuged at 2500 g for 10 minutes, at 4˚C and the resulting fluid was collected and added to the fluid in the original tube, well-mixed and centrifuged for a further 10 minutes to remove cell debris. Cell-free supernatants were collected and divided into aliquots and stored at −80˚C until being ELISA assayed.
2.1.2. Investigations
CVF levels of tumor necrosis factor-α (TNF-α), IL-6 and IL-10 were measured using ELISA kits according to the manufacturer’s instructions and were read using a 96 well microplate ELISA reader (Dynatech, MR 7000).
1) Human TNF-α was measured with the enzyme linked immunoassay (ELISA) kit (catalogue no. ab179886, abcam Inc., Cambridge, USA) by quantitative sandwich enzyme immunoassay technique [14].
2) IL-6 with the enzyme linked immunoassay (ELISA) kit (catalogue no. ab46042, abcam Inc., Cambridge, USA) by quantitative sandwich enzyme immunoassay technique [15].
3) Serum IL-10 was measured with the enzyme linked immunoassay (ELISA) kit (catalogue no. ab46034, abcam Inc., Cambridge, USA) by quantitative sandwich enzyme immunoassay technique [16] (Poll, 1996).
2.1.3. Cervical Cerclage
McDonald cervical cerclage (CC) was performed using within 4 days after obtaining CVF sample in women with CL < 25 mm. CC was performed using the McDonald procedure with a non-absorbable suture. After performing CC, women were asked to avoid any sexual activity, use of tampons or douching, prolonged standing for >4 h, heavy physical work, lifting heavy weights, straining or any activity that brings on symptoms of pelvic pressure or discomfort.
2.2. Study Outcomes
1) Primary outcome: the difference in cytokines’ levels between the two obtained samples.
2) Secondary outcomes included:
a) The difference in cytokines’ levels between study and control women;
b) The frequency of PTB among women of both groups;
c) The value of Sample I cytokines’ levels for prediction of PTB.
2.3. Statistical Analysis
Obtained data were presented as mean ± SD, numbers and percentages. Results were analyzed using paired t-test, One-way ANOVA Test and Chi-square test (χ2 test). Possible relationships were investigated using Pearson linear regression analysis. Regression analysis (Stepwise method) was used for stratification of studied parameters as specific predictors. Statistical analysis was conducted using the IBM SPSS (Version 23, 2015; IBM, South Wacker Drive, Chicago, USA) for Windows statistical package. P value < 0.05 was considered statistically significant.
3. Results
During the duration of study since June 2018, 67 pregnant women with history of SPTB were evaluated; 25 women were excluded and 42 women were included as study group (Figure 1). Another 42 normally pregnant women free of history
of SPTB and had CL > 25 mm was enrolled as control group. Enrollment data of women of both groups showed non-significant (P > 0.05) differences except for the inter-pregnancy interval that was significantly (P = 0.005) longer in control women than study women (Table 1).
Throughout the study duration, 4 study women (4.8%) had labor at GA < 34 GW, 12 women (14.3%) had labor at GA in range of >34 and <37 GW and the remaining 68 women (80.9%) had labor at GA > 37 GW with significantly higher frequency of women had labor at GA < 37 GW among study women. Mean duration of pregnancy of control women (38.8 ± 1.3 wk) was significantly longer than that of study women (37.2 ± 1.8 wk) (Table 2).
Estimated cytokines’ levels in CVF samples obtained from control women were significantly higher in Sample II than in Sample I as it was elevated up to 146%, 120% and 132% for IL-6, TNF-α and IL-10, respectively of its levels in Sample I. On the other hand, estimated IL-6, TNF-α and IL-10 in Sample II of CVF samples obtained from study women were significantly decreased in Sample II than in Sample I down to 82%, 80% and 76%, respectively of its levels estimated in Sample I with subsequent significant difference between the extent of change between both groups. Cytokines’ levels estimated in Sample I were significantly higher in women of study group compared to women of control group. On contrary, estimated levels of IL-6 and IL-10 estimated in Sample II were non-significantly higher, while estimated levels of TNF-α were non-significantly lower in control versus study women.
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Table 1. Enrolment data of women of both groups.
Data are presented as presented as mean ± SD; P indicates significance of difference between both groups; P > 0.05 indicates non-significant difference; P < 0.05 indicates significant difference.
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Table 2. Distribution according to duration of pregnancy of women of both groups.
Data are presented as presented as numbers, percentages, mean ± SD; *indicates significance of difference between both groups at 0.05 level.
IL-10 and TNF-α levels estimated in CVF of Sample I and II were significantly higher in samples obtained from women had pregnancy duration < 37 wk compared to that obtained from women had pregnancy duration > 37 wk. Regarding IL-6, levels estimated in CVF of Sample I were non-significantly higher, while that of Sample II were significantly higher in samples obtained from women had pregnancy duration < 37 wk compared to that obtained from women had pregnancy duration > 37 wk. The percentage of change in cytokines’ levels was significantly higher in women had pregnancy duration > 37 wk compared to women had pregnancy duration < 37 wk (Table 3).
Pregnancy duration was negatively correlated with maternal BMI and CVF cytokines’ levels, while was positively correlated with gravidity, parity and IPI. CVF cytokines’ levels were positively correlated with maternal BMI and negatively correlated with IPI that was negatively correlated with BMI (Table 4). Regression analysis defined short IPI (β = −0.179, P = 0.048) and high CVF levels of TNF-α (β = −0.554, P = 0.0007) as negative predictors for short pregnancy duration.
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Table 3. Cytokines’ levels estimated in CVF samples from women of both groups.
Data are presented as presented as mean ± SD; P1 indicates significance of difference between both groups; P2 indicates significance of difference Samples I & II of the same group; P > 0.05 indicates non-significant difference; P < 0.05 indicates significant difference.
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Table 4. Pearson’s correlation between pregnancy duration and other variables collected in women of both groups.
BMI: Body mass index; r = Pearson’s correlation coefficient; IL-: Interleukin; TNF-α: Tumor necrosis factor-α; P > 0.05 indicates non-significant value; P < 0.05 indicates significant value.
4. Discussion
The obtained results showed a negative impact of high maternal body mass index (BMI) and short inter-pregnancy interval (IPI) on duration of pregnancy and this impact was more manifest in women at high risk of preterm birth. In support of these findings, statistical analyses showed a negative significant correlation between BMI and both of IPI and pregnancy duration. Similarly, Tsur et al. [17], (2017) reported that adjusted relative risks for PTB for pregnant obese of grades I, II and III are 1.1, 1.15 and 1.26, respectively. Recently, Ratnasiri et al. [18] found women with class III obesity have a higher risk for PTB by 33% than women with a normal BMI and Liu et al. [19] found maternal obesity is significantly associated with the risk of PTB and this risk increases with maternal aging.
Moreover, in line with the impact of IPI on duration of pregnancy, Zhang et al. [20] reported that adjusted relative risk for PTB was 2.04 for IPI < 6 m and Palmsten et al. [21] reported a higher risk of PTB after IVF and non-IVF deliveries of 22.2% and 6.4%, respectively, after IPI of 12 - <24 m with increasing risk with decreasing IPI. Thereafter, Haight et al. [22] reported that in comparison to IPI of 18 - 23 months, adjusted risk ratios for PTB for IPI < 6, 6 - 11, and 12 - 17 months were 1.62, 1.16 and 1.03, respectively. Also, Ahrens et al. [23] documented that IPI < 6 m since last live birth is associated with increased risks for PTB.
For evaluation of pregnancy-associated inflammatory response, the current study estimated cytokines’ levels in CVF samples for being simple to obtain, can provide largest sample volume and having the modest concentrations of cytokines that are independent on sample weight and protein concentration [24]. In support of the use of CVF samples to reflect the intra-amniotic status and progress of pregnancy, multiple previous studies assured its advantages and higher concentration of cytokines [24] and correlation with intra-amniotic cytokines than serum [25].
Levels of cytokines estimated in CVF sample obtained at time of labor (Sample II) of control women were significantly higher than that estimated in Sample I (at the beginning of the 2nd trimester). These findings indicated a possible role for inflammatory cytokines for initiation and progression of the process of labor. In line with these findings, Ashford et al. [26] detected variations of cytokine profiles across trimesters in women delivering term and Buxton et al. [27] reported high concentrations of CVF cytokines among term pregnancies. Also, Buxton et al. [27] and Stokkeland et al. [28] suggested that these high concentrations indicated that normal pregnancy is associated with an active inflammatory state.
Women at high risk of PTB (Study group) had significantly higher cytokines levels in their Sample I compared to women of control group. Moreover, high CVF cytokines’ levels in Sample I showed negative significant correlation with pregnancy duration; a finding that suggests a role of disturbed local inflammatory cytokines in initiation of PTB. In support of this assumption, Regression analyses defined high Sample I cytokines’ levels as positive significant predictor for short pregnancy duration and this risk was magnified in women had short IPI especially in women with high BMI. In line with the diagnostic value of estimation of CVF cytokines’ levels, Jung et al. [29] reported that in women with PTB, CVF IL-6 and IL-8 and cervical length are the most important parameters that could predict spontaneous PTB within 7 days of sampling. Also, Yavari et al. [30] found no relationship between serum IL-6 and both of CVF IL-6 and preterm labor, while CVF values of IL-6 were correlated with PTB and concluded that maternal CVF not serum IL-6 is suitable as biomarker for predicting PTB. Thereafter, Yoo et al. [7] documented that estimated values of biomarkers in CVF could be non-invasive predictors of spontaneous PTB at <32 weeks in women with cervical insufficiency or short cervix and this may be improved by the combined use of clinical and laboratory data. Moreover, Ashford et al. [26] detected significantly higher values of CVF values of IL-6, IL-8, IL-10, TNF-α, and CRP in women who delivered preterm than the full-term women. Recently, Ronzoni et al. [31] documented that higher maternal anti-inflammatory cytokines increased latency until delivery in women had premature preterm rupture of membranes which causes one-third of PTB and this effect is likely due to counterbalancing of proinflammatory load.
Cervical cerclage provided both mechanical and immunological protection against PTB as evidenced by the reduction of the incidence of labor before 37 GW despite the fact that all women of study group were at high risk and had cervical length of <25 mm at the beginning of the 2nd trimester of pregnancy and by the significantly lower CVF cytokines’ levels in Sample II compared to Sample I of women of Study group.
In line with these findings, Wang et al. [32] documented that CC for women with an asymptomatic short cervix and prior PTB history had more benefits in maternal and neonatal outcomes than vaginal progesterone therapy and Kiefer et al. [33] detected beneficial effect of CC for mid-trimester short cervix for prevention of PTB especially in patients with high inflammation. Thereafter, Monsanto et al. [34] found CC led to a significant decline in CVF pro-inflammatory cytokines levels and suggested that cerclage may help reduce local inflammation. Enakpene et al. [35] found that in comparison to vaginal progesterone alone for treatment of women with extremely shortened cervix, addition of CC significantly decreased overall spontaneous PTB rates, prolonged pregnancy latency by 2-fold, and decreased the overall neonatal complications. Also, Conde-Agudelo et al. [36] reported that cerclage significantly decreased the risk of PTB, perinatal morbidity/mortality and birth weight < 1500 g in comparison to no cerclage.
5. Conclusion
High body mass index, short inter-pregnancy interval and high CVF inflammatory cytokines’ levels affect the duration of pregnancy. Pregnant women with history of recurrent SPTB are vulnerable to be seriously affected by this triad. Early prophylactic cervical cerclage for women at high-risk of SPTB can modulate local immune disturbance, so in addition to its mechanical impact can reduce the incidence of SPTB and prolong the duration of pregnancy.