mRNA expression and localization of bNOS, eNOS and iNOS in human cervix at preterm and term labour

Abstract

Background: Preterm birth is the primary cause of the neonatal mortality and morbidity. There will be no preterm birth without a cervical softening. Nitric oxide (NO) is shown to be a mediator of term cervical ripening. The aim of this study was to investigate mRNA expression of the three isomers of NO synthases (NOS) and to identify them by immunohistochemistry in the human cervix at preterm birth compared to term.

Methods: The three isomers of NOS--inducible (iNOS), endothelial (eNOS) and neuronal (bNOS)--were investigated in the human cervix. The expression of mRNA was determined using Real-Time Multiplex RT-PCR. The localisation of synthases in the cervical tissue was analysed using immunohistochemistry. Cervical biopsies were obtained from 4 groups of women without clinical signs of infection: preterm (PTL), term labour (TL), preterm not in labour (PTnotL) and term not in labour (TnotL) patients. One-Way ANOVA, Kruskal-Wallis, Student t-test or Mann-Whitney test were applied as appropriate to determine statistically significant differences among the groups.

Results: Patients in preterm labour had significantly (p < 0.01) higher mRNA levels of all the three NOS isomers compared to those in term labour. Women not in labour, irrespective of gestational age, thus with unripe cervices, had significantly lower eNOS mRNA levels compared to those in labour (p < 0.01). Immunoreactivity for all three NO synthases was observed in each examined sample in all groups. The bNOS staining was the most prominent.

Conclusion: The mRNA levels were higher in the preterm labour group compared to the women at term labour. The significant increase of the eNOS mRNA expression, from the unripe to the favourable cervical state during labour, may indicate a role of eNOS and supports the role of NO in the cervical ripening process. All the three synthases were identified by immunohistochemistry in all the groups of study.

Figure 1

Expression of mRNA of NOS isomers in the cervical tissue. Expression of NOS mRNA normalized to the 'Preterm labour' group. Common superscripts indicate no significant differences. The number of patients analysed in each group are marked in each bar in the bar chart. The groups are: Preterm labour (PTL), Term labour (TL), Preterm not in labour, (PTnotL), Term not in labour, (TnotL). 1A:Expression of iNOS mRNA: Patients who delivered preterm had higher iNOS mRNA levels compared to those who delivered at term. This relationship reached significance for those who were in labour (p < 0,002). 1B: Expression of eNOS mRNA: Significantly higher levels of eNOS mRNA were registered in women with preterm labour compared to term labour (p = 0,009). Women not in labour at preterm and at term had significantly lower eNOS mRNA levels compared to preterm labour (p < 0,001) or term labor (p = 0,048) respectively. 1C: Expression of bNOS mRNA : Women who delivered preterm had generally higher bNOS mRNA levels compared to those who delivered at term, reaching significance in the labour group (p = 0,006). The lowest values were seen in those who were in labour at term. Women who were delivered by caesarean section appeared to have higher bNOS mRNA levels than those who were in labour, reaching significance in the term groups (p = 0.007).

Figure 2

Immunohistochemical localization of nitric oxide synthases in human cervix. (a) inducible nitric oxide (iNOS) localized to the stroma in preterm labour (b) iNOS localized to the squamous epithelium in preterm not in labour patient. In each of the biopsies iNOS was localized in the stroma and the epithelium. (c) Endothelial nitric oxide (eNOS) localized to the vascular endothelium in all biopsies. This is collected from a woman in preterm labour. Neuronal nitric oxide (bNOS) had a distinct staining and was generally localized to the: (d) basal membrane of the squamous epithelium, picture from a women in preterm labour, (e) the stroma, biopsy from at term in labour patient, (f) the cervical glands, this sample from at term in labour patient. Original magnification × 200, scale bar 50 μm.