Diagnosis of intraamniotic inflammation by measuring vaginal interleukin-6 in patients with cervical insufficiency: could amniocentesis be avoided?

BACKGROUND: Cervical insufficiency is a recurrent, passive, and painless dilation of the cervix in the second trimester. The etiology is unclear, but there may be an association with subclinical intraamniotic infection. Interleukin-6 (IL-6) production in the amniotic cavity is induced by bacterial invasion, it is the major proinflammatory cytokine released in response to infection. Although the gold standard method to measure it is through an amniocentesis, the procedure constitutes an invasive technique with several associated risks. The objective of this study is to determine if there is a correlation between intraamniotic and vaginal IL-6 in patients with cervical insufficiency and bulging membranes during the second trimester of pregnancy, in order to avoid an amniocentesis before the rescue cerclage. METHODS: A cohort study was performed in which all patients with cervical insufficiency and bulging membranes admitted into our tertiary hospital between 2019 and 2020 were included, and a control group of asymptomatic women in the second trimester of gestation where studied at the same time. Patients with bulging membranes underwent an amniocentesis to quantify amniotic IL-6, and a sample of vaginal fluid for vaginal IL-6 determination was obtained from both the study and the control group. RESULTS: A total of 20 women were included in each group. Median gestational age at diagnosis was 22 weeks in patients with bulging membranes, and 21 weeks in the control group. Vaginal IL-6 in control group (10.875 pg/mL) is much lower than the study group one (1308.77 pg/ml). In patients with bulging membranes, vaginal IL-6 expression was lower in the vagina than in the amniotic cavity [average IL-6 in the amniotic cavity 26890.07 pg/mL, vs 1308.77 pg/mL in the vagina (p < .01)]. Through a Spearman coefficient correlation rank [rho = 0.709 (p < .001)], there is a positive correlation between amniotic and vaginal IL-6 values. The best value of this correlation was calculated with the ROC curve, being the area under the curve 0.929 (CI 95% 0.721-0.995), and the cutoff of point less than 61.4 pg/ml (sensitivity 83.33%; specificity 92.86%). Patients with vaginal IL-6 < 61.4 pg/ml associated a longer latency time between diagnosis and delivery, a higher neonatal weight and a lower perinatal mortality. Rescue cerclage in vaginal IL-6 < 61.4 pg/ml was the best predictor of good pregnancy outcome. CONCLUSION: There is a correlation between intraamniotic and vaginal IL-6 in patients with cervical insufficiency and bulging membranes during the second trimester of pregnancy. However, further studies are needed in order to considerate the avoidance of an amniocentesis before an emergency cerclage.

Amniotic fluid HIF1α and exosomal HIF1α in cervical insufficiency patients with physical examination-indicated cerclage.

OBJECTIVE: Hypoxia inducible factor 1α (HIF1α) has been reported to activate inflammatory cascade. Recently, exosomes have been known to have pivotal roles in intercellular communication. The aim of this study was to compare the concentration of amniotic fluid (AF) HIF1α, exosomal HIF1α, and inflammatory cytokines such as interleukin 1α (IL1α), interleukin 1ß (IL1ß), interleukin 6 (IL6), and tumor necrosis factor α (TNFα) between physical examination-indicated cerclage (PEIC) and control group. We also investigated the associations between biomarkers and amniocentesis-to-delivery interval and the correlations of inflammatory cytokines, HIF1α, and exosomal HIF1α. METHODS: Case-control study was performed. Cases are defined as 16 patients who underwent PEIC and controls are 19 women who underwent amniocentesis for confirming chromosomal abnormalities. The concentration of IL1α, IL1ß, IL6, TNFα, HIF1α, and exosomal HIF1α were measured using enzyme-linked immunosorbent assay (ELISA). Exosomes were confirmed by tumor susceptibility Gene 101 (TSG 101) and transmission electron microscopy (TEM). RESULTS: The mean HIF1α in PEIC group was higher than control group (PEIC, 15.03 ± 9.60-pg/mL versus control, 2.96 ± 1.99 pg/mL; p < .01). There were significant differences in inflammatory cytokines between two groups. A significant difference in exosomal HIF1α was shown between two groups (PEIC, 27.97 ± 28.61-µg/mL versus control, 12.42 ± 8.20 µg/mL; p < .01). HIF1α, IL1α, IL6, TNFα, and exosomal HIF1α showed significantly negative association with cerclage-to-delivery interval. However, IL1ß was not associated with cerclage-to-delivery interval. HIF1α was positively correlated with exosomal HIF1α (rho = 0.93, p < .01). Both HIF1α and exosomal HIF1α were significantly associated with TNFα (rho = 0.94, p < .01; rho = 0.97, p < .01). Both HIF-1α and exosomal HIF1α had positive correlation with IL1α (rho = 0.96, p < .01; rho = 0.91, p < .01). However, IL1ß showed no correlations with HIF1α and exosomal HIF1α. A positive correlation between HIF-1α and IL6 was observed (rho = 0.58, p = .01.) Exosomal HIF1α also had correlation with IL6 (rho = 0.52, p = .03). CONCLUSIONS: This study demonstrated that amniotic fluid (AF) HIF1α and AF exosomal HIF1α were higher in physical examination-indicated cerclage (PEIC) group than control group. AF HIF1α and AF exosomal HIF1α were associated with shorter amniocentesis-to-delivery interval. More importantly, they had positive correlations with AF inflammatory cytokines such as IL1α, IL6, and TNFα. Our results may indicate that AF HIF1α and AF exosomes interact with AF inflammatory cytokines and contribute inflammatory cascade in complicated pregnancies.

Cervical cerclage placement decreases local levels of proinflammatory cytokines in patients with cervical insufficiency.

BACKGROUND: Cervical insufficiency is characterized by premature, progressive dilation and shortening of the cervix during pregnancy. If left unattended, this can lead to the prolapse and rupture of the amniotic membrane, which usually results in midtrimester pregnancy loss or preterm birth. Previous studies have shown that proinflammatory cytokines such as interleukin-1ß, interleukin-6, interleukin-8, and tumor necrosis factor alpha are up-regulated in normal parturition but are also associated with preterm birth. Studies evaluating such markers in patients with cervical insufficiency have evaluated only their diagnostic potential. Even fewer studies have studied them within the context of cerclage surgery. OBJECTIVES(S): The objective of the study was to evaluate the impact of local and systemic inflammatory markers on the pathogenesis of cervical insufficiency and the effect of cerclage surgery on the local immune microenvironment of women with cervical insufficiency. STUDY DESIGN: We recruited 28 pregnant women (12-20 weeks' gestation) diagnosed with insufficiency and referred for cerclage surgery and 19 gestational age-matched normal pregnant women as controls. Serum and cervicovaginal fluid samples were collected before and after cerclage surgery and during a routine checkup for normal women and analyzed using a targeted 13-plex proinflammatory cytokine assay. RESULTS: Before surgery, patients with cervical insufficiency had higher levels of interleukin-1ß, interleukin-6, interleukin-12, monocyte chemoattractant protein-1 and tumor necrosis factor alpha in cervicovaginal fluid compared to controls, but after surgery, these differences disappeared. No differences were found in serum of insufficiency versus control women. In patients with insufficiency, the levels of interleukin-1ß, interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and interferon gamma in cervicovaginal fluid declined significantly after cerclage compared with before intervention, but these changes were not detected in serum. CONCLUSION: Compared with normal women, patients with cervical insufficiency have elevated levels of proinflammatory cytokines in cervicovaginal fluid but not in serum, suggesting a dysregulation of the local immune environment. Cerclage intervention led to a significant decline in these proinflammatory cytokines, suggesting that cerclage may help reduce local inflammation in cervical insufficiency.

Dynamic collagen changes in cervix during the first trimester and decreased collagen content in cervical insufficiency.

OBJECTIVE: To determine the changes in cervical collagen during the first trimester of pregnancy and to evaluate the collagen deficit in cases with a previous diagnosis of cervical insufficiency (CI). MATERIALS AND METHODS: Cervical punch biopsies were obtained from 66 patients divided into three groups: patients with recurrent abortions due to CI (CI group; n = 8); first-trimester abortion group (study group; n = 37), subdivided into three groups according their gestational week (<7, 7-9 and 9-12 weeks), and patients with cervical biopsy due to gynecologic reasons (control group; n = 12). Collagen quantity was determined by a biochemical method that measured the levels of hydroxyproline (HOP) in dry cervix tissue. RESULTS: The HOP concentrations were significantly higher at lower gestational ages (p = 0.001). Collagen quantity was lowest in the CI group compared with other groups (p < 0.001). CONCLUSION: This study shows collagen component of cervix decreases as pregnancy advances through the first trimester. Cervical collagen concentration is lower in women with a history of CI compared to controls who has not a history of CI.

Oxidative status shifts in uterine cervical incompetence patients.

Uterine cervical incompetence (UCI) is a pregnancy complication affecting about 10% of the pregnancies in the western world. Studying the etiology of the UCI requires a specific approach adequate for this highly heterogenous syndrome. Oxidative status disorders are associated with various pathologies, including pregnancy complications. As such, general oxidative status profiling is a promising methodology to treat UCI. We aimed at assaying the closely interrelated oxidative status markers in the uterine cervical incompetence patients by means of the systems biology-oriented approach. Chemiluminescent assay, circulating thioredoxin 1 protein, uric acid, and homocysteine level measurements were used to assess the character of the oxidative status regulation in the UCI patients. We found UCI to be associated with the atypical plasma oxidative status deregulation; UCI plasma samples demonstrated lowered proneness to the pro-oxidative processes, and this was not due to the excessive antioxidant activity. There were neither signs of oxidative stress nor destructive pro-oxidant feedforward circuit locking in the UCI group. We also report increased circulating levels of uric acid in the UCI patients.

Cervical collagen and biomechanical strength in non-pregnant women with a history of cervical insufficiency.

BACKGROUND: It has been suggested that cervical insufficiency (CI) is characterized by a "muscular cervix" with low collagen and high smooth muscle concentrations also in the non-pregnant state. Therefore, the aim of this study was to investigate the biomechanical properties, collagen concentration, smooth muscle cell density, and collagen fiber orientation in cervical biopsies from non-pregnant women with a history of CI. METHODS: Cervical punch biopsies (2 x 15 mm) were obtained from 57 normal non-pregnant women and 22 women with a history of CI. Biomechanical tensile testing was performed, and collagen content was determined by hydroxyproline quantification. Histomorphometry was used to determine the volume densities of extracellular matrix and smooth muscle cells from the distal to the proximal part of each sample. Smooth muscle cells were identified using immunohistochemistry. Finally, collagen fiber orientation was investigated. Data are given as mean +/- SD. RESULTS: Collagen concentration was lower in the CI group (58.6 +/- 8.8%) compared with the control group (62.2 +/- 6.6%) (p = 0.033). However, when data were adjusted for age and parity, no difference in collagen concentration was found between the two groups. Maximum load of the specimens did not differ between the groups (p = 0.78). The tensile strength of cervical collagen, i.e. maximum load normalized per unit collagen (mg of collagen per mm of specimen length), was increased in the CI group compared with controls (p = 0.033). No differences in the volume density of extracellular matrix or smooth muscle cells were found between the two groups. Fibers not oriented in the plane of sectioning were increased in CI patients compared with controls. CONCLUSIONS: Cervical insufficiency does not appear to be associated with a constitutionally low collagen concentration or collagen of inferior mechanical quality. Furthermore, the hypothesis that a "muscular cervix" with an abundance of smooth muscle cells contributes to the development of cervical insufficiency is not supported by the present study.

Cervical collagen in non-pregnant women with previous cervical incompetence.

OBJECTIVE: To study the cervical collagen in non-pregnant women with a history of congenital cervical incompetence. SUBJECTS: Ten non-pregnant women with congenital cervical incompetence, defined as cervical incompetence in the first pregnancy in a woman without previous cervical trauma. Seventy-one normal non-pregnant women (31 nulligravidae, nine nulliparous and 31 parous women) served as controls. METHOD: Cervical biopsies were analysed for the hydroxyproline concentration and extractability to express the concentration and the stability of the collagen. RESULTS: Women with cervical incompetence had markedly lower median cervical hydroxyproline concentration when compared to normal parous women (11.1 (range 6.11-18.5) micrograms/mg wet weight vs. 16.9 (range 7.7-29.8) micrograms/mg wet weight; P = 0.003, Mann Whitney test) and the extractability was almost twice the value found in normal parous women (80.2% vs. 49.5%; P = 0.03, Mann Whitney test). Both pregnancy per ce and parturition caused a significant decrease in the hydroxyproline concentration when compared to values found in nulligravidae (P < 0.0001, Kruskall Wallis test). CONCLUSIONS: Congenital cervical incompetence seems associated with a low collagen concentration in the cervical connective tissue in the non-pregnant state.

Connective tissue changes in the cervix during normal pregnancy and pregnancy complicated by cervical incompetence.

When postpartum cervical biopsy specimens were compared with biopsy specimens from nonpregnant women, they revealed a 12-fold decrease in mechanical strength, a 50% reduction in the concentrations of collagen and sulfated glycosaminoglycans, a 35% reduction in hyaluronic acid, an increase in collagen extractability, and a fivefold increase in collagenolytic activity. Primiparas with relatively high concentrations of collagen and hyaluronic acid had relatively long cervical dilatation times during established labor, suggesting a physiologic importance to these variables. This correlation was not found in multiparas, even though the mean values of the biochemical parameters tested were similar to those in primiparas. Second-trimester biopsy specimens taken from patients with cervical incompetence contained normal collagen concentrations, but relatively high collagen extractabilities and collagenolytic activities, exceeding normal postpartum values. A biopsy specimen that was tested biomechanically had a very low strength and very high extensibility. Most likely, these data reflect a high turnover of collagen in incompetent cervices, resulting in a high proportion of newly synthesized collagen with low biomechanical strength.