Despite intense research over the years, the complex, but well coordinated mechanisms that control the onset and maintenance of labour remains unrevealed [1
]. Until now the research on preterm parturition has mainly been focused upon the myometrial activities and on potential inhibitors of preterm labour.
To our knowledge, this is the first study investigating NOS isomers expression in preterm cervix.
Our hypothesis is that a cervical remodelling occurs at preterm delivery as well as at term. It seems relevant to believe that a woman going into preterm labour starts from a relatively more unripe cervical status than a woman going into labour near or at term [26
]. To achieve this more extensive remodelling from a very unripe cervical state, higher level of iNOS, eNOS and bNOS mRNA at preterm would be relevant. Here we analyse biopsies from women with preterm parturition without any signs of infection. We identified the presence of all three NOS isomers mRNA in preterm and term cervix before and after onset of labour. The most prominent finding was higher mRNA levels in preterm labour compared to term labour.
The eNOS mRNA level was significantly higher for the preterm group in labour compared to all other groups. The eNOS mRNA levels in the women not in labour, thus with unripe cervices, were significantly lower compared to those in labour irrespective of gestational age. These results may indicate a role for eNOS in the very final cervical ripening both preterm and term.
The iNOS mRNA levels were generally low in all groups, exhibiting the lowest value in the term labour group and the highest value in the preterm labour group. We have only found two earlier studies analysing iNOS and eNOS mRNA levels in human pregnant cervix [11
]. Tschugguel et al. noted higher iNOS mRNA levels in the postpartum group compared to non-pregnant controls while Yoshida et al [27
] identified both iNOS and eNOS mRNA in the first trimester cervix. In the study by Ledingham et al. labouring and non labouring patients were compared at term pregnancy and the protein expression was examined by Western blot and immunohistochemistry without any differences of the three synthases, iNOS, eNOS and bNOS, being found [13
]. These different results reveal the complex story of human labour as compared to the more uniform process described in other species [7
Our results showed bNOS mRNA levels to be higher in the preterm compared to the term groups. Women in preterm labour had significantly higher bNOS mRNA levels compared to the same group at term. Interestingly, when comparing labour and not in labour groups in preterm and term patients respectively, we found that the groups not in labour had higher bNOS mRNA expression levels. This is in contrast to the study of Bao et al. where an upward trend of bNOS mRNA by RT-PCR during labour compared to not in labour was noticed.
The immunoreactivity of the NO synthases had different localization. iNOS was identified in the stroma and in the epithelium. This is in line with earlier findings [11
]. On the other hand we only found iNOS localised to the vascular endothelium in very few sections whereas Ledingham et al. showed iNOS protein localized to the vascular endothelium more generally [13
]. The differences observed may be due to the fact that Ledingham et al. used paraffin-embedded sections of cervical tissue while we in our study, just as Tschugguel et el. and Yoshida et al. used cryosections. The eNOS specific staining was localized to the endothelium in all groups, which is supported by earlier investigations [11
bNOS showed the most impressive immunoreactivity in our study. It was localised to the stroma, the glandular epithelium and to the basal membrane of the squamous epithelium. These observations agree with earlier findings by Bao et al. namely studies on cervical tissue from nonpregnant and pregnant women [12
]. Our data differs from that of Ledingham et al. who could not identify bNOS in the cervical glands [13
]. Tschugguel et al could not identify any bNOS at all in the cervical tissue, but this may be due to the fact that they used a polyclonal antibody, while we used a monoclonal antibody [11
A possible concern regarding the immunohistochemistry analysis is that the interpretation of the localization of the different NO synthases might have been more reliable if a greater number of biopsies had been studied. Due to a shortage of material only two biopsies from each of the four groups were analysed in this study.
In this study we endeavoured to include women without any clinical signs of infection, neither during labour/delivery nor during the postpartum period. However, it is well known that a systemic or intrauterine infection can cause a preterm delivery and labour [29
]. In those cases studies often shows increased cytokine response and up-regulation of the synthesis of cytokines compared to term non infected labour. In Sweden only around 25 % – at a maximum – of the preterm births seems to have an infectious genesis, but in other parts of the world, infection is estimated to be associated with preterm labour/delivery in as much as 40% of the cases [30
]. Infection as causal to preterm labour and delivery is more frequent the lower the gestational age as well as in preterm premature rupture of membranes [31
A new enigma based on earlier findings by our group and others is that a significant up-regulation of inflammatory parameters, cytokines, chemokines and MMPs are seen also in normal vaginal labour at term where no infection is present [1
It is well known that cytokines, chemokines, different MMPs, prostaglandins, Cyclooxygenas (COX) and NO are inflammatory mediators. The extensive infiltration of immune cells as leukocytes, neutrophils and macrophages into the cervical stroma produce pro-inflammatory cytokines and collagenases that promote and accelerate the degradation of the ECM. The actions of prostaglandins and nitric oxide are activated by these cascade reactions and leads to the fully ripened cervix and induction of labour [5
Nitric oxide donors are potent cervical ripeners and the up-regulation of NO by the proinflammatory cytokines is proposed to represent the final common pathway of cervical ripening [7
]. NO directly stimulates COX-II and together with the increase of the proinflammatory cytokines IL-1, TNF-alpha and IL-8 results is increased prostaglandin production.
The increased amount of macrophages is associated with enhanced iNOS activity [13
]. A strong expression of bNOS protein and mRNA in the cervical stroma at term compared to that of non-labouring or non-pregnant state is also seen [12
This study revealed a frequent occurrence of bNOS in the cervix during pregnancy and labour, which is in agreement with our earlier findings that the cervix, in contrast to the uterus, is well innervated during pregnancy and labour. The immune-related cervical ripening process may be regulated by neural signalling by different neurotransmitter [40
]. Yellon et al. also raised the question why antibiotic treatment has failed to either prolong labour or reduce the rate of preterm birth. Taking into account the theory that the cervical remodelling consists of inflammatory immune cell activation it is logical that antibiotics have no effect, as they do not affect immune cell trafficking. It thus seems plausible that the rapid final cervical ripening is not related to an exogenous infection but is instead a part of a physiological inflammatory reaction. Such a process has been registered in the endometrium during menstruation as well as in the uterus at involution postpartum [25