Reproductive Biology and Endocrinology Non-infected Preterm Parturition Is Related to Increased Concentrations of Il-6, Il-8 and Mcp-1 in Human Cervix

Background: Human cervical ripening is an inflammatory process. In labour at term the mRNA-levels and protein concentrations for interleukin-6 (IL-6) and IL-8 in cervix significantly increase. The aim of this study was to investigate if there are differences in the inflammatory process of preterm and term cervical ripening.


Background
Preterm delivery occurs in approximately 6-15% of all pregnancies [1]. Furthermore, it accounts for 70-75% of neonatal mortality and morbidity [2]. The association between lower and upper genital tract infections and preterm delivery is well established [3]. However, in many cases preterm labour seems to be idiopathic [1]. The basic mechanisms underlying the initiation of both preterm and term cervical ripening and labour remain largely unknown. Both term and preterm labour requires cervical softening and regular myometrial contractions. Cervical softening is the result of an intensive remodelling process of the extracellular matrix (ECM). During cervical ripening the concentration of collagen decreases [4] and its physical state changes [5]. Additionally, the concentration of the main cervical proteoglycan decorin declines [6] in contrast to an increase of the mRNA expression and the protein concentration of the large proteoglycan versican [7]. Cytokines and several other mediators such as oestrogen, progesterone, nitric oxide and prostaglandins are involved in the human cervical ripening and the remodelling of extracellular matrix [8][9][10][11]. This remodelling process can therefore be regarded as an inflammatory reaction [12,13]. The density of immunoactive cells such as leucocytes and macrophages increases 6 to 10-fold at labour compared to first trimester pregnancy [14]. They are known producers of a variety of proinflammatory cytokines and matrix metalloproteinase's, and promote the cervical extracellular matrix degradation [13,[15][16][17]. Furthermore, chemokines and cytokines are capable of attracting these immunoactive cells to the site of inflammation and are involved in human pregnancy and parturition [18,19].
Earlier studies have shown an increase of IL-8, IL-6 and TNF-α in human gestational membranes during spontaneous term labour [18,20,21]. Furthermore, the amniotic fluid levels of IL-8, RANTES, IL-6 and TNF-α [20] and MCP-1 [22] increase markedly at the onset of spontaneous labour at term. Cytokine levels in preterm parturition are widely analysed in relation to infection. However, it is estimated that less than 50% of the cases of preterm labour with elevated cytokines are due to an infection [23]. In placental cell cultures from non-infected preterm labour significantly larger amounts of IL-1β, IL-6, and TNF-α were registered compared to those shown by nonlabouring women at term [24]. RANTES in non-infected amniotic fluid in preterm delivery is significantly higher compared to women with term delivery [25].
Only a few studies have investigated the role of the cytokines in human cervical tissue at term. The gene expression of IL-6 and IL-8 is significantly up regulated with a corresponding increase in the protein concentrations in patients in labour compared to not in labour [17,26,27]. Furthermore, animal studies have shown that IL-8 when applied intracervically induces cervical ripening in guinea pigs [28]. Studying the conditions at preterm parturition, Winkler et al. has shown that in the lower uterine segment, the IL-1β, IL-6 and IL-8 concentrations increased in relation to progressing cervical dilatation, whereas TNF-α remained unchanged [29]. Cytokines in the preterm cervix, to our knowledge, have not been investigated before.
We hypothesized that non-infected preterm cervical ripening and labour are associated with increased cytokine concentrations in the cervical tissue and increased inflammatory markers in the peripheral blood. The aim of this study was to investigate if there are any differences between inflammatory process during preterm and term cervical ripening. Therefore, protein concentrations of IL-6, IL-8, MCP-1, RANTES and TNF-α as well as mRNA expression of IL-8, MCP-1 and RANTES were determined in preterm and term groups. Furthermore, an analysis of inflammatory markers in peripheral circulation -White Blood Cell count (WBC) and C-reactive protein (CRP)was undertaken.

Study Participants
A total of fifty women undergoing singleton pregnancies were included in the present investigation. The two study groups included 17 women in spontaneous preterm labour (PTL) and 8 not in labour at preterm (PTnotL). Premature delivery was defined as delivery before the 37 th week of gestation (Table 1). 14 women in term labour (TL) and 11 not in labour at term (TnotL) served as controls. Characterization of all study groups is summarized in Table 1. There were no significant differences regarding maternal age, parity and previous preterm births between the four groups.
The labour groups were in active labour and demonstrated a ripe cervix dilated >4 cm. In all patients delivered by caesarean section the assessment of cervical dilatation was established immediately before surgery, by the same obstetrician (SAT) through vaginal digital examination. The women in preterm labour were either delivered vaginally, or by emergency caesarean section due to malpresentation. The women in term labour were also either delivered vaginally or by emergency caesarean due to threatening foetal asphyxia. Women not in labour had unripe cervices (Bishop score <5p) and were delivered by caesarean section before the onset of labour. The preterm indications were suspected ablatio or intra-uterine growth retardation and the term indications were breech presentation, humanitarian or disproportion.

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In all women clinical signs of infection were absent, during parturition as well as during the postpartal period.

Specimen Collection
Immediately following parturition, a biopsy from the anterior cervical lip was taken transvaginally at the 12 o'clock position with scissors and tweezers. Our group has since 25 years applied this technique to get samples including squamous and cylindrical epithelium, vessels, glands and ECM. The samples were immediately frozen in liquid nitrogen and stored thereafter at -70°C until further investigation. A venous blood sample was taken for analysis of CRP and WBC.
Due to the limited amount of tissue from each woman, all analyses could not be performed on every sample.
The Local Ethics Committee of the Karolinska Institute approved the study (Ref. No. 97-089) and all women gave their informed consent.

Tissue homogenisation
Frozen tissue was cut into small slices on a block of dry ice and transferred to a pre-chilled (liquid nitrogen) capsule containing Teflon coated tungsten ball. The capsule was kept in liquid nitrogen for two minutes and thereafter shaken in a dismembranation apparatus (Retsch KG, Haan, Germany) at full speed for two minutes. The procedure was repeated after intermediated freezing in liquid nitrogen until the tissue became powder. Hereafter followed either RNA extraction or measurement of cytokine concentrations.

Measurement of cytokine concentrations
Tissue preparation Following the tissue homogenisation, 1 ml of phosphatebuffered saline (PBS) was added. After centrifugation at 400 g for 5 min, supernatant was retrieved. Protein levels of IL-6, IL-8, TNF-α, MCP-1 and RANTES were expressed as picograms of cytokine per mg of total protein (pg/mg protein). Total protein concentration was determined by Bio-Rad's Protein Asssay, based on Bradford dye-binding procedure (Bio-Rad Laboratories Inc., Hercules, CA, USA), according to the manufacturer's instructions.
RANTES and MCP-1 concentrations in the supernatants of homogenized cervical samples were determined in duplicates using the quantitative sandwich enzyme-linked immunoassays (ELISA) by commercially available kits (Quantikine, R&D Systems, Minneapolis, MN, USA). The detailed procedures are described in the instruction booklets supplied by the manufacturers. The results were interpolated from the standard reference curve provided with each kit. The sensitivity of kits was 5 pg/ml for MCP-1 and 8 pg/ml for RANTES. The intra-assay and inter-assay coefficients of variation were respectively 7.8% and 6.7% for MCP-1; 3.6% and 10.3% for RANTES.

RT-PCR
All gene-specific primers (

Semi-quantification of IL-8, MCP-1 and RANTES mRNA
The PCR products were separated by electrophoresis on a 1.5% agarose gel (Amersham Biosciences AB, Uppsala, Sweden). Following staining with ethidium bromide, the gels were photographed and band intensity measured under UV light using Gel Doc 2000 (BioRad, Hercules, CA, USA). The specific mRNA level of every sample was expressed as the product's intensity, divided by the housekeeping gene 28S intensity (the product/28S intensity ratio).
The identity of PCR products was confirmed by sequencing them at KISeq, Center for Genomics and Bioinformatics, Karolinska Institutet.

Determination of WBC and CRP
WBC and CRP were determined in blood in the routine laboratory of Karolinska University Hospital Solna (Stockholm, Sweden).

Statistical Analysis
Comparison between two groups was performed using the Mann-Whitney U test. When all four groups were compared, the Kruskal-Wallis test was applied, followed by multiple comparison with Bonferroni correction. The level of significance was set at p < 0.05. Calculations were performed employing STATISTICA 6.0 software (StatSoft Inc, Tulsa, OK, USA).

Protein concentrations of cytokines
Every analysed sample revealed detectable levels of proteins.
There were no significant differences between preterm and term respective groups, but differences reached significance when comparing groups in labour with not in labour. The concentration of IL-6 was significantly higher in the PTL versus the PTnotL (p = 0.02) and TnotL (p = 0.002). Similarly, concentration was significantly higher in the TL group compared to groups not in labour ( Figure 1).
Concentrations of IL-8 were significantly higher in the TL group compared to the PTnotL and the TnotL (p < 0.01). Although the same tendency was noted in the preterm groups, it didn't reach statistical significance (Figure 2A).
MCP-1 protein levels showed a tendency to be higher in the PTL compared to PTnotL, but did not reach statistical significance, whereas in the PTL was significantly higher compared to TnotL (p = 0.02). The concentration of MCP-1 was significantly higher in the TL group than in groups not in labour ( Figure 3A).
Even more significant differences (p < 0.0001) between labouring and not labouring groups were achieved when it was looked upon the data irrespective gestational age (Table 3).
Interestingly, there were no significant changes between preterm or term groups as regards the protein concentra-tions of TNF-α or RANTES (data not shown, combined data - table 3).

IL-8, MCP-1 and RANTES mRNA expression
In line with protein data, there were no differences registered in the representative cytokine mRNA expression IL-6 concentration in the cervical tissue in all study groups between preterm and term respective groups. On the other hand, mRNA expression of IL-8 and MCP-1 was significantly higher in labour compared to not in labour groups.
IL-8 mRNA levels were significantly higher in the PTL group compared to the PTnotL (p = 0.01) and to the TnotL group (p = 0.03). Furthermore, there were significant differences comparing TL with groups not in labour ( Figure  2B).
In line with the IL-8 expression, MCP-1 mRNA levels were significantly higher in labouring groups compared to not labouring groups ( Figure 3B).
In line with protein data, differences were even more significant (p < 0.0001) comparing labouring (including PTL and TL) and non-labouring groups (including PTnotL and TnotL) ( Table 3).
Similarly to the protein levels, no significant changes were found in RANTES mRNA expression (data not shown, combined data -table 3).

WBC and CRP levels
In line with cytokine levels, there were no differences in WBC (10 9 /l) and CRP (mg/l) levels between preterm and term respective groups. However, higher levels of inflammatory markers were found in the labouring groups compared to the nonlabouring groups. WBC was significantly (p = 0.017) higher in the preterm labour group with a median value of 15.9 (range 9.2-24.3) compared to the preterm group not in labour, where the median value was 8.6 (6.9-13.6). The same tendency was seen in the term groups, where median and range in the TL group was 15.65 (7.3-28.4) and in the not labour group 10.0 (7.4-14.0) respectively, however without statistical significance.
WBC and CRP were significantly higher when compared the labouring groups (PTL and TL) to the groups not in labour (PTnotL and TnotL) (p < 0.001 and p = 0.02 respectively) irrespective to gestational age ( Figure 4, Table 3).

Discussion
To our knowledge, this is the first investigation on cytokines in preterm cervical tissues in non-infected subjects.
The hypothesis that clinically non-infectious preterm cervical ripening and labour is associated with increased cytokine levels was tested and verified for IL-8, IL-6 and MCP-1 protein concentrations. This was not valid for RANTES or TNF-α protein concentrations which all remained unchanged.
Protein concentration and mRNA expression of MCP-1 in the cervical tissue Figure 3 Protein concentration and mRNA expression of MCP-1 in the cervical tissue. Box and whisker plots represent (A) the protein concentration of MCP-1 (picograms/ mg of total protein) and (B) expression of the MCP-1 mRNA (expressed as the product/28S intensity ratio) in the cervical tissue of the study groups. The groups are: preterm labour (PTL), term labour (TL), preterm not in labour (PTnotL), term not in labour (TnotL). The number of patients analysed in each group is marked in each bar in the bar chart. The box represents median value with 25%-75% of all data falling within the box. The whiskers extend to the non-outlier range. Outliers are marked as circles. Significant differences between the groups are shown above the box plots.
Interestingly, no significant differences were revealed between the preterm and term labour samples (ripe cervices) or between preterm and term not in labour (unripe cervices) samples. In other words, this study could not identify any differences related to the gestational age, while differences were found related to the cervical state.
Our findings concerning term cervical ripening agree with those in previous studies, where increase in IL-8 and IL-6 concentrations and mRNA expression was found in cervical tissue in labouring compared to not labouring subjects [17,26,27,30]. Analogous changes were registered in the lower uterine segment [31][32][33].
Looking at the changes in preterm labour, Winkler et al has shown similarities in IL-1β, IL-6 and IL-8 increase in the lower uterine segment during preterm and term parturition [29]. The only observed difference was that this increase at preterm starts at earlier stages of cervical dilatation [29,34]. Even though the lower uterine segment contains lower amounts of extracellular matrix, undergoes less intense changes and has another function than the cervix during pregnancy and parturition, the remodelling process there may still somewhat reflect events in the cervix [35,36]. Our findings in the preterm cervical tissue agree with the study on the lower uterine segment, but we are not able to judge at which stage of cervical dilatation the increase in cytokine levels begins.
In our earlier study, we have shown a decrease in 15hydroxyprostaglandin dehydrogenase expression related to the cervical state irrespective of gestational age [37]. All these findings suggest that cervical ripening at preterm is a similar process as at term. This similarity can be further confirmed, looking upon our results allocated into two groups: in labour (containing preterm and term labour) and not in labour (containing preterm and term not in labour), where even more significant differences between labouring and not labouring groups are achieved. Analogous results are seen in WBC count and CRP levels, where these markers seem to rise significantly in the labouring groups compared to the groups not in labour. This suggests that local inflammatory process in the cervix can be reflected in the peripheral blood. IL-6 is a prominent stimulator of the acute phase response in inflammatory reactions and stimulates CRP. Taking that into account, it is logical that when we identify a significant rise in IL-6 in the labour groups we can expect a concomitant rise in the CRP level. The WBC count and the CRP level are used routinely as markers of infection in daily clinical use, but our findings may suggest that WBC and CRP could probably be markers of active labour without any infection. Further studies with larger number of patients are needed to confirm this.
All these findings cannot answer the question which signals cause the cytokine increase in the labouring cervix. Preterm labour in correlation with infection is extensively studied. A significant cytokine increase is seen in gestational membranes, amniotic and cervicovaginal fluid in infected preterm labour [3,21,[38][39][40]. In our study, none of the women showed clinical or laboratory signs of infection before or after labour. This could also account for the unchanged levels of TNF-α in our and Winkler et al study [29], as TNF-α is shown to be related to infection in the amniotic fluid [41]. However, no correlation was found between chorioamnionitis and elevated IL-6 and IL-8 levels of amniotic fluid in term pregnant women [42]. Our findings are in line with earlier studies, where cytokine increase was seen in non infected preterm partu- Table 3: In labour group compared with not in labour group. In labour group includes preterm labour (PTL) and term labour (TL) groups, not in labour group includes preterm not in labour (PTnotL) and term not in labour (TnotL) groups. Protein concentrations are expressed in pg/mg of total protein. Expression of mRNA is the product/28S intensity ratio. Significant differences between the groups were determined using Mann-Whitney U Test. NS-statistically not significant difference.

Measured factor
In rition [23,24,29] suggest that some other signals than infection could be responsible for starting preterm cervical ripening and labour process. The down-regulation of progesterone receptors and oestrogen receptor α with significant up-regulation of oestrogen receptor β (ERβ) at term pregnant cervix [8,43] may be involved. Further-more, there is a possibility of direct effect of estrogens via ERβ on cervical leukocytes [44], which are a major source of proinflammatory cytokines in the cervix during labour [45]. Foetal fibronectine could be also involved in foetalmaternal signalling, as its level in the cervicovaginal fluid rises in line with the ripening of cervix and is elevated at preterm birth [46][47][48]. It can also be localised in the cervical epithelium as well as IL-8 [26,47]. Corticotropine releasing hormone (CRH) is elevated in maternal serum in preterm labour [49] and there is possible cross-regulation between CRH and cytokines, but earlier studies show controversial results [50][51][52]. Further investigations are required to clarify the involvement of these factors in the preterm cervical ripening and labour and their relation with the cytokine increase.

Conclusion
In conclusion, preterm cervical ripening can be likened to an inflammatory process with cytokines as important mediators, corresponding to the process at term cervical ripening. The local changes in the cervix may be reflected in the peripheral blood as an increase in WBC count and CRP.
WBC and CRP levels in labour compared to not in labour group Figure 4 WBC and CRP levels in labour compared to not in labour group. Box and whisker plots represent (A) white blood cell count (10 9 /l) (B) C-reactive protein (mg/l) in the blood of women in the study groups. The two groups are: In labour (including preterm labour (PTL) and term labour (TL) and not in labour (including preterm not in labour (PTnotL) and term not in labour (TnotLabour). The number of patients analysed in each group is marked in each bar in the bar chart. The box represents median value with 25%-75% of all data falling within the box. The whiskers extend to the non-outlier range. Outliers are marked as circles. Significant differences between the groups are shown above the box plots.
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