Accurate dating of pregnancy during routine ultrasonographic examination in early pregnancy has resulted in a decrease in the occurrence of postterm pregnancy, as defined as pregnancy duration of 294 days (42 weeks) or longer, from approximately 8–10% down to 2%.1,2 However, postterm pregnancy does occur; repeatedly in 4–16% of cases.3,4 Moreover, postterm pregnancy tends to occur in families,2,4 which may imply a common genetic background for this pregnancy complication. Sometimes postterm pregnancy can be a result of fetal anomaly, such as fetal anencephaly for example, but in the vast majority of cases, no definitive cause of postterm pregnancy can be found.2 It is possible that in these women some aberrations exist in local bioregulators taking part in cervical ripening,5 because an unripe cervix is a characteristic feature in these women. Cervical ripening is known to resemble biochemically an inflammatory process in which prostaglandins, cytokines, and rearrangement of collagen fibers are involved.5–9 However, at present, no definitive explanation exists for failure of the cervix to ripen on time.
Nitric oxide, a free-radical gas with a large spectrum of different biologic effects, is synthesized by cervical cells through inducible or constitutive nitric oxide synthases.10–14 Cervical ripening may be promoted by nitric oxide, as suggested by data from studies on animals15,16 and humans.13 Nitric oxide could exert this effect directly, or through stimulating the cervical inflammatory cascade (Qing SS, Beier HM, Garfield RE, Chwalisz K. Local application of a nitric oxide donor induces cervical ripening [abstract]. J Soc Gynecol Invest 1996;3:A462),5 through matrix metalloproteases,6,17 or through suppressing proteoglycan synthesis.18 Moreover, nitric oxide may operate jointly with prostaglandins and cytokines in cervical ripening.5,13
Nitric oxide is rapidly converted to stable nitrate and nitrite (nitric oxide metabolites), which can be measured in cervical fluid and which is higher in concentration in late pregnancy and during labor than in early pregnancy.19 Thus, it is possible that cervical release of nitric oxide is deficient in women going beyond term. Therefore, we compared cervical fluid nitric oxide metabolite concentrations in women going beyond term versus those delivering at term.
MATERIALS AND METHODS
We studied, with the permission of the local committee on ethics, 208 healthy women with singleton pregnancies between 37 and 43 weeks of gestation (Table 1). The duration of gestation was assessed by means of menstrual history and by ultrasonographic examinations at 12–14 weeks and 17–19 weeks of pregnancy, and the date of confinement was corrected on the basis of ultrasonographic dating if a discrepancy of 2 or more days existed. The material was collected between June 1, 2002, and December 30, 2002. Each volunteer received written and oral information as to the purpose and conduct of the study, and written consent was obtained. Women going beyond term (n = 108) were included in the study when the expected time of confinement had been exceeded by a mean of 11 days (10–14 days; in 12 women by 14 days); these women had been admitted to a routine consultation at our outpatient department. All these women finally carried beyond term (postterm group), defined as pregnancy duration of 294 days (42 weeks) or longer. We included in the term control group 100 women between weeks 37 and 41 of gestation (mean of 271 days) who paid a routine prenatal visit to the outpatient department and who went into spontaneous labor at a mean of 281 days (Table 1). No woman in either group reported uterine contractions or any other signs of initiation of labor preceding inclusion in the study. The postterm and term pregnancies were otherwise normal, but 8 women (7 in the postterm group and 1 in the term group) had previously experienced a cesarean delivery as a result of breech presentation (n = 4) or failure to progress during labor (n = 4). Nineteen women of 65 parous women in the postterm group (29%) had previously delivered beyond term, compared with none in the term group.
Excluded from the study were women who had any evidence of labor, effusion of amniotic fluid, or symptoms of cervical or vaginal infection or bleeding.
At inclusion into the study, cervical fluid samples were collected by introducing a Dacron swab (DuPont, Wilmington, DE) into the cervix under visual control. The swab, kept in the cervical canal for precisely 20 seconds, was then flushed in 1.5 mL of physiologic saline for 2 minutes as described previously.19 The saline samples were kept frozen at −21°C and assayed for nitric oxide metabolites by means of the Griess reaction.19 All macroscopically bloody cervical fluid samples were discarded. The detection limit of the assay was 3.8 μmol/L, and the intraassay and interassay coefficients of variation were 1.6 and 2.4%, respectively. To reduce the impact of interassay variation, the samples were assayed in only 5 batches.
After sampling, the women in the postterm group visited the clinic every third day, whereas the term control group was seen next time during term labor. All women in the postterm group delivered beyond term spontaneously (n = 66), after labor induction (n = 40), or at elective cesarean delivery (n = 2) (Table 1). Labor was induced with vaginal administration of misoprostol (n = 25) or by amniotomy (n = 15), and it was classified as successful (n = 34) if regular uterine contractions and clinical signs of labor progression were seen. The women in the term group all went into spontaneous labor (Table 1). Emergency cesarean was performed in 36 cases (22 in the postterm group and 14 in the term group), primarily as a result of failure to progress in labor (18 women in the postterm group and 10 women in the term group). Twelve infants in the postterm group and 6 infants in the term group were delivered by vacuum extraction. The postpartum period and neonatal outcome were uneventful.
Categorical data were analyzed by the χ2 test or Fisher exact probability test. Medians with their 95% confidence intervals were used to describe nitric oxide metabolite levels.20 The nitric oxide metabolite values were analyzed by nonparametric tests, such as the Mann-Whitney test, the Kruskal-Wallis one-way analysis of variance, and Spearman rank correlation test. All tests were 2-sided and processed by using NCSS 2000 software (NCSS Inc, Kaysville, UT). Probability values of P < 0.05 were considered statistically significant.
The women who went beyond term were slightly older than the women in the term group (Table 1). Previous postterm pregnancy had occurred more often in the postterm group than in the term group (Table 1). In addition, the mean sample-to-delivery interval was shorter, and the median Bishop score was lower in the postterm group than in the term group (Table 1). No other significant differences were seen in clinical variables between the study groups (Table 1).
Nitric oxide metabolites in cervical fluid were less often detectable (P = .001) in women going beyond term (60%) than in those delivering at term (87%) (Table 2). The median cervical fluid nitric oxide metabolite level in the postterm group was 4.5 times lower (P < .001) than in the term group (Table 2). This difference was also seen when analysis was carried out separately among women with a gestational age of less than 294 days versus 294 days or more at sampling (Table 2). Sixty-six women in the postterm group went into spontaneous labor, and their cervical fluid nitric oxide metabolite levels were 7.6 times lower than those in women going spontaneously into labor at term (Table 2). However, in the postterm group, women with spontaneous initiation of labor had lower (P = .01) levels of nitric oxide metabolites in their cervical fluid than did women who delivered after induced labor or elective cesarean (Table 2), even although the median Bishop score (3 and 2, respectively) did not differ in these subgroups. Nulliparity was associated with lower levels of cervical fluid nitric oxide metabolites in both groups (Table 2).
Cervical fluid nitric oxide metabolite levels were significantly and similarly related to Bishop score both in all women going beyond term (r = 0.45; P < .001) and in those delivering at term (r = 0.41; P < .001). However, women who went beyond term exhibited a lower median cervical fluid nitric oxide metabolite concentration against one Bishop score; this ratio was 7.8 in the postterm group compared with 17.7 in the term group.
The cervical fluid nitric oxide metabolite level was inversely related to the time elapsed from sample collection to spontaneous initiation of labor in women delivering beyond term (r = −0.56, P = .02; n = 66) (Figure 1), but not in women delivering at term (r = −0.12, P = .35; n = 100). Cervical fluid nitric oxide metabolite levels were inversely related to duration of delivery in women delivering vaginally beyond term (r = −0.37, P = .005; n = 84), but not in those delivering at term (r = −0.12, P = .34; n = 86).
Nineteen women in the postterm group with a history of previous postterm pregnancy had nitric oxide metabolite levels similar to those in 46 parous women without such a history (medians 55.1 and 24.1 μmol/L). In women going beyond term, a “low” level of cervical fluid nitric oxide metabolites was associated with a less ripe cervix, lower inducibility of labor and a longer duration of labor than a “high” cervical fluid nitric oxide metabolite level (Table 3). Women with failed progression of labor were 8.1 times more likely (P < .001) to belong to the low than high cervical fluid nitric oxide metabolite group (Table 3).
The cervical fluid nitric oxide metabolite level bore no association to neonatal outcome in either group, although infants born beyond term were slightly heavier than infants born at term, and 15 infants (14%) born beyond term had 1-minute Apgar scores of 7 or lower compared with 4 infants (4%) born at term (P = .003) (Table 1). Apgar scores at 5 minutes did not differ.
Cervical nitric oxide appears to have a role in cervical ripening.5,13,15,16,20 To study the release of cervical nitric oxide in women destined to go beyond term, we assessed cervical fluid nitric oxide metabolite levels in women who had carried to a mean of 11 days after the expected time of confinement and who also finally went beyond term. It was conspicuous that 29% of the parous women in the postterm group had previously carried beyond term, suggesting that at least these women had an inherent tendency for postterm pregnancy. Our study groups were similarly assessed with 2 early ultrasonographic screenings, and we are confident that the durations of pregnancies in both the postterm group and the term group were accurate. In fact, our postterm group constituted 1.9% of all parturients (n = 5,680) in our unit during the study period (7 months), which is in line with previously reported incidences.1–3 The main clinical variables were comparable between study groups except for slightly higher age in the postterm group; however, we consider that this bias does not affect cervical nitric oxide release. Cervical nitric oxide release was assessed by means of the Griess reaction as the combined accumulation of nitrate and nitrite, as described previously.19 This reflects the total formation of nitric oxide metabolites, probably in the cervix itself,10–14 but we cannot differentiate the proportion of nitric oxide metabolites that came by way of inducible or constitutive nitric oxide synthase action. Cervical fluid nitric oxide metabolite concentrations are not related to plasma nitric oxide metabolite levels or to the dietary nitric oxide metabolite content,19 and therefore no dietary restrictions were involved in the present study.
There are several issues to be considered as possible explanations for the nitric oxide deficiency in women going beyond term. First, we must take into account the fact that the cervical samples in the postterm group were collected at a mean of 20 days later in gestation than the samples in the term group. In view of the higher levels of cervical fluid nitric oxide metabolites in late versus early pregnancy,19,21 and abundant evidence of the possible involvement of nitric oxide in cervical ripening,10–14 the more advanced gestational age at sampling in the postterm group should have resulted in higher nitric oxide metabolite levels than in the term group,19,21 and not in reduced ones, as was the case in the present study. Hence, the bias in gestational age at the time of collection of cervical fluid samples cannot explain our main result, and it may have blunted the impact of the nitric oxide deficiency in women going beyond term in our study. Second, we must acknowledge the fact that sampling took place closer to labor in the postterm group than in the term group. If cervical nitric oxide is a factor in cervical ripening,5,10–14 this bias can hardly explain reduced levels of cervical fluid nitric oxide metabolites in the postterm group, but it may explain, at least in part, the closer relationships between cervical fluid nitric oxide metabolite concentrations and some labor variables in the postterm group. Thus, we believe that the release of cervical fluid nitric oxide metabolites is specifically reduced in women destined to go beyond term.
Our data do not allow us to deduce if a deficiency of cervical nitric oxide in women going beyond term is a primary phenomenon, and thus a true contributing factor to postterm pregnancy, or whether it is a reflection of relative insufficiency of prostaglandins, cytokines, matrix metalloproteases, or some other agents that may be primarily involved in cervical ripening5,6–9,18,19,22 and that may stimulate nitric oxide release. Our finding that the need for induction of postterm labor was associated with increased cervical nitric oxide levels was surprising and it may imply that cervical nitric oxide is not primarily involved in the onset of postterm labor. However, our subjects going beyond term were not randomly assigned to induction of labor or expectant management, and therefore, a difference in cervical fluid nitric oxide metabolite levels between women with induced versus spontaneous onset of postterm labor may not necessarily exist in randomized studies.
Postterm pregnancy has been reported to occur repeatedly at a rate of 4–16%,3,4 and it occurred in 29% of our subjects. Thus, some women may have an inherent cervical-ripening incapacity, either primary or secondary. This incapacity may involve nitric oxide release, although nitric oxide metabolite levels showed no difference in women with and without a history of prolonged pregnancy in our study. Further studies are needed to assess if cervical fluid nitric oxide metabolite levels could perhaps serve as a marker for the inducibility of labor in women going beyond term or in those who need labor induction at term.
1. Saari-Kemppainen A, Karjalainen O, Ylöstalo P, Heinonen OP. Ultrasound screening and perinatal mortality: controlled trial of systematic one-stage screening in pregnancy. The Helsinki Ultrasound Trial. Lancet 1990;336:387–91.
2. Crowley P. Interventions for preventing or improving the outcome of delivery at or beyond term. Cochrane Database Syst Rev 2000;(2):CD000170.
3. Campbell MK, Ostbye T, Irgens LM. Post-term birth: risk factors and outcomes in a 10-year cohort of Norwegian births. Obstet Gynecol 1997;89:543–8.
4. Mogren I, Stenlund H, Högberg U. Recurrence of prolonged pregnancy. Int J Epidemiol 1999;28:253–7.
5. Kelly RW. Inflammatory mediators and cervical ripening. J Reprod Immunol 2002;57:217–24.
6. Stygar D, Wang H, Vladic YS, Ekman G, Eriksson H, Sahlin L. Increased level of matrix metalloproteinases 2 and 9 in the ripening process of the human cervix. Biol Reprod 2002;67:889–94.
7. Sennström MB, Brauner A, Lu Y, Granström LMM, Malmström AL, Ekman G. Interleukin-8 is a mediator of the final cervical ripening in humans. Eur J Obstet Gynecol Reprod Biol 1997;74:89–92.
8. Sennström MB, Ekman G, Westergren-Thorsson G, Malmström A, Byström B, Endresen U, et al. Human cervical ripening, an inflammatory process mediated by cytokines. Mol Hum Reprod 2000;6:375–81.
9. Osman I, Young A, Ledingham MA, Thomson AJ, Jordan F, Greer IA, et al. Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term. Mol Hum Reprod 2003;9:41–5.
10. Tschugguel W, Schneeberger C, Lass H, Stonek F, Zaghlula MB, Czerwenka K, et al. Human cervical ripening is associated with an increase in cervical inducible nitric oxide synthase expression. Biol Reprod 1999;60:1367–72.
11. Ledingham MA, Thomson AJ, Young A, Macara LM, Greer IA, Norman JE. Changes in the expression of nitric oxide synthase in the human uterine cervix during pregnancy and parturition. Mol Hum Reprod 2000;6:1041–8.
12. Ekerhovd E, Brännström M, Weijdegard B, Norström A. Nitric oxide synthases in the human cervix at term pregnancy and effects of nitric oxide on cervical smooth muscle contractility. Am J Obstet Gynecol 2000;183:610–6.
13. Ekerhovd E, Weijdegard B, Brännström M, Mattsby-Baltzer I, Norström A. Nitric oxide induced cervical ripening in the human: involvement of cyclic guanosine monophosphate, prostaglandin F2[alpha], and prostaglandin E2. Am J Obstet Gynecol 2002;186:745–50.
14. Bao S, Rai J, Schreiber J. Brain nitric oxide synthase expression is enhanced in the human cervix in labor. J Soc Gynecol Invest 2001;8:158–64.
15. Chwalisz K, Shao-Qing S, Garfield RE, Beier HM. Cervical ripening in guinea-pigs after local application of nitric oxide. Hum Reprod 1997;12:2093–101.
16. Chwalisz K, Garfield RE. Regulation of the uterus and cervix during pregnancy and labor: role of progesterone and nitric oxide. Ann N Y Acad Sci 1997;828:238–53.
17. Yoshida M, Sagawa N, Itoh H, Yura S, Korita D, Kakui K, et al. Nitric oxide increases matrix metalloproteinase-1 production in human uterine cervical fibroblast cells. Mol Hum Reprod 2001;7:979–85.
18. Liu GZ, Ishihara H, Osada R, Kimura T, Tsuji H. Nitric oxide mediates the change of proteoglycan synthesis in the human lumbar intervertebral disc in response to hydrostatic pressure. Spine 2001;26:134–41.
19. Väisänen-Tommiska M, Nuutila M, Aittomäki K, Hiilesmaa V, Ylikorkala O. Nitric oxide metabolites in cervical fluid during pregnancy: further evidence for the role of cervical nitric oxide in cervical ripening. Am J Obstet Gynecol 2003;188:779–85.
20. Newcombe RG. Two-sided confidence intervals for the single proportion: comparison of seven methods. Stat Med 1998;17:857–72.
21. Maul H, Longo M, Saade GR, Garfield RE. Nitric oxide and its role during pregnancy: from ovulation to delivery. Curr Pharm Design 2003;9:359–80.
22. Brune B, von Knethen A, Sandau KB. Nitric oxide and its role in apoptosis. Eur J Pharmacol 1998;351:261–72.