Changes in Cervical Nitric Oxide Concentration

Correlate With Bishop Score and Cervical

Length Modifications in Prostaglandin
E2-Mediated Induction of Labor
Giuseppe Chiossi, MD, Gianluca Verocchi, MD, Paolo Venturini, MD, and
Fabjo Facchinetti, MD

OBJECTIVE: Nitric oxide (NO) plays afundamental role in cervical ripening and it is synthesized in the
human cervix. We studied the effect 9/f the dinoprostone on cervical NO release in pregnant women, and we
investigated the relationship between cervical NO metabolites, cervical length, and Bishop score.
METHODS: Seventy-seven women underwent induction of labor at .37 weeks of gestation, due to
post-terin pregnancy (23. 8%o), oligohydramnios (36.3) or preeclampsia (29.9%). Cervicalfluid samples for
NO metabolites (NOx), Bishop score, and cervical length were assessed immediately before (time 0 LTOJ)

and 6 hours qfter (T6) the local application qf dinoprostone, a commercially available prostaglandin E2
(PGE2) analog.
RESULTS: Thie mean patients' age was 34 3.2 years, mean gestational age at enrollment uwas 284
+ 9.2 days, and nulliparous represented 31.2% of the study population. At time 0, Bishop score was less
than 4 iu 74% (57/77) of the subjects, mean cervical length 28.6 5.8 mm, mean NOx
was

concentration was 208.6 + 103.8 pRiV/mL; 6 hours later, at T6, tile mean cervical length decreased to
19.5 + 8.8 mnm, and the mean NOx concentration increased up to 3 16. 7 240.9 [L.7M/mL. Data were
uniafected by parity or by regular uterine contraction patterns. A statistically significant positive correlation
wasfound bcetveen changes in cervical NOX levels and Bishop score modifiaction (P <.0 1; r = .494), as
well as between the modification of NO metabolites concentration and cervical shortening (P <.01; r
= .3 07).

CONCLUSIONS: Prostaglandin (PG)-induced cervical ripening is associated with local NO release. NO

plays an active role in cervical remodeling since it positively correlates with both cervical shortening and Bishop
score increase. NO oxide and PG are the two pathways that, cross activating each other, trigger the cascade
of events responsible of cervical ripening. UJ Soc Gynecol Investig 2006; 13:203- 8) Copyright (© 2006
by the Societyfor Gynecologic Investigation.

KEY WORDS: Induction of labor, dinoprostone, prostaglandin E2,
metabolites, cervical ripening.
ervical ripening is the process that allows the cervix to
change from a tightly closed structure designed to
maintain an intrauterine pregnancy, to a compliant
organ that dilates accommodating the passage ofthe fetus. Such
changes have been likened to an apyretic inflammatory reac-
tion1 and are accompanied by extensive neutrophilic granulo-
cytes invasion in the cervical tissue.'-1 Cervical remodeling is
a multifactorial process involving prostaglandins (PGs), in par-
ticular Prostaglandin E-2 (PGE2)," inflammatory cytokines
such as interleukin (IL)-1, IL-8, tumor necrosis factor-ot
(TNF-u),6 and steroid hormones.7'8
Fronm the Mother-Infant Department, Section of Obstetrics, Modena Policlinico
Hospital, University of Modena and Reggio Emilia, Modena, Italy.
Address correspondence and reprint requests to: Professor Facchinetti Fabio, MD,
Mother-Infant Department, Section of Obstetnics, Modena Polichnico Hospital, Univer-
sity of Modena and Reggio Emilia, via del Pozzo 71, 41100, Modena, Italy. E-mail:
facchinetti.fabiogunitnore.it
Copyright ©O 2006 by the Society for Gynecologic Investigation.
Published by Elsevier Inc.
nitric oxide, nitric oxide
Recently, nitric oxide (NO), a free radical gas, was ascribed
a role as a mediator of cervical remodeling.9 The local appli-
cation of the NO donor sodium nitroprusside produced effec-
tive maturation of the guinea pig cervix.10 An endogenous
NO system is present within the human uterine cervix, and
human cervical ripening at term is associated with an increased
expression of the inducible form of NO synthase (iNOS)." 13
NO donors have been proved to be fundamental in cervical
remodeling also in humans. Ultrastructural analysis revealed
that both isosorbide mononitrate (IMN)14 and sodium nitro-
prusside (SNP) when applied locally" destroy the organization
of cervical connective tissue. Moreover, IMN and SNP have
been demonstrated to be effective and safe to facilitate first
trimester termination of pregnancy when administered before
dilatation and curettage.14 16-19 In humans, cervical fluid NO
metabolite level nises after NO donor admiinistration, produc-
1071 -5576/0)6/$32.(0)0
doi:10. 1016/j jsgi.2006.01 .001

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204 J Soc Gynecol Investig Vol. 13, No. 3, April 2006
ing cervical maturation.2-) Furthermore, reduced NO release in
the uterine cervix is associated with prolonged pregnancy1
while the opposite findings have been found in case ofpreterm
labor.22
The highly complex process of cervical ripening involves
considerable interactions between NO, PGs, and inflammatory
cytokines, 23,24 suggesting that self-regulatory paracrine
mechanisms are important in inducing cervical modifications.
As NO plays a pivotal role in the process of cervical remod-
eling, we investigated the possibility that PGE2-operated cer-
vical ripening could be associated with a local activation of NO
release.
MATERIALS AND METHODS
Subjects
This prospective observational study, which was approved by
the Institutional Review Board of the University of Modena
and Reggio Emilia, was performed between May 2004 and
April 2005 on women undergoing induction of labor due to
prolonged pregnancy (ie, greater than or equal to 291 days of
gestation), oligohydramnios (amniotic fluid index less than 5),
or preeclampsia. Inclusion criteria consisted in singleton preg-
nancy, gestational age greater than 259 days (ie, 37 gestational
weeks) previously ascertained through a first trimester ultra-
sound examination, and a Bishop score less than 6. Exclusion
criteria were rupture of membranes, previous uterine surgery,
presence of uterine activity (more than one contraction in 30
minutes), known hypersensitivity to PGs, any contraindication
to vaginal delivery, and any drug assumption except folic acid.
Furthermore, oxytocin was not to be required for induction of
labor. Seventy-seven women met the inclusion criteria and
were enrolled in the study population.
STUDY PROTOCOL
Women were admitted to the Labor and Delivery ward,
Modena Policlinico Hospital, early in the morning, and un-
derwent a nonstress test (NST) for 60 minutes. Patients were
subsequently enrolled in the study as they signed the informed
consent form. After cervical fluid collection for NO metabo-
lites (NOx) measurement, subjects underwent Bishop score
evaluation and cervical length assessment by ultrasound, ac-
cording to Berghella et al.i' Induction of labor was performed
using dinoprostone, a commercially available PGE2 analog. Six
hours after induction of labor was started (T6), cervical fluid
was recollected for NOx determination, and Bishop score and
cervical length were reassessed.
If the Bishop score was less than 4 at enrollment, cervical
ripening was operated by either a single application of slow-
release dinoprostone vaginal insert (Propess, Ferring, Den-
mark) or two applications of 0.5 mg dinoprostone intracervical
gel (Prepidil 0.5 mg, Upjohn, Kalamazoo, MI), 6 hours apart.
In case the Bishop score was 4 to 5, dinoprostone 2 mg vaginal
gel (Prepidil 2 mng) was used, and a second dose was adminis-
tered after 6 hours ifBishop score remained less than 6. On the
following morning, 24 hours apart, intracervical or vaginal
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Chiossi et al
Chiossi et al
dinoprostone was applied according to the Bishop score. In
clinical practice, oxytocin infusion is administered indepen-
dently of the Bishop score if active labor has not been reached
after three dinoprostone applications, or when the Bishop
score is greater than 7. According to the study protocol,
women requiring oxytocin for induction were not included in
the study population. For study purposes, amniotomy was
performed only in laboring women with at least 5 cm dilata-
tion. As previously described, the beginning of active labor was
considered when the cervix reached 3 to 4 cm ofdilatation due
to effective uterine contractions, corresponding to an abrupt
change in the slope of the curve that results when cervical
dilatation is plotted against time.26
Nitric Oxide Metabolites Assessment
Under speculum visualization, a preweighted Dacron swab
(DuPont, Wilmington, DE) was introduced under visual con-
trol into the distal portion ofthe cervix, at the external cervical
os. As previously described, the swab was kept in place for
precisely 20 seconds avoiding shear, and then flushed in 1.5 mL
of saline solution for 2 minutes. The saline samples were
immediately centrifuged at 800 X g for 15 minutes, kept frozen
at -20C, and assayed for nitrites/nitrates (NOx).20 Each swab
was weighted before and after sample collection to assess the
volume of cervical fluid that had been soaked up by the
Dacron swab. The weight increase represented the volume of
cervical fluid that was obtained. The mean raise in weight after
cervical sampling at time 0 (TO) was 91 + 7 gug, corresponding
to 91 ± 7 pL; similarly, the mean weight increment at T6 was
85 ± 8 fig, equal to 85 ± 8 4L. Samples weighting +3 SD
were discarded, as were all of the macroscopically bloody
cervical fluid samples. NO was evaluated spectrophotometri-
cally using the Griess reagent, as the accumulation of its
metabolites (NOx). ?0
Statistical Analysis
Continuous variables were evaluated by Student t test, both for
paired and unpaired observations. Categorical variables were
evaluated by x2 analysis and Fisher exact test where appropri-
ate. Pearson correlation was also used. Nonparametric Wil-
coxon pairs t test was used for values with non normal
distributions. All statistical analysis were performed with SPSS
for Windows Release 11.0 (SPSS Inc, Cary, NC).
RESULTS
Clinical features of the population are reported in Table 1.
Induction of labor was due to prolonged pregnancy in 23.8%
of the cases, oligohydramnios in 36.3%, and preeclampsia in
29.9%. Neither cervical length nor cervical NOx differed
according to the indication of labor induction (Table 1) or
parity (Table 2).
At baseline (TO), Bishop score was less tharf 4 in 74%
(57/77) of the study population, mean cervical length assessed
by ultrasound was 28.6 + 5.8 mm, and mean concentration of
cervical NO metabolites was 208.6 + 103.8 [iM/mL. Six
hours after dinoprostone was applied (T6), Bishop score was
Cervical Nitric Oxide in PGE2-Mediated Induction of Labor J Soc Gynecol Investig Vol. 13, No. 3, April 2006 205

Table 1. Clinical Features of the Study Population According to Reason for Induction of Labor
Prolonged
Pregnancy Oligo Preeclampsia P Total
Gestational age (d) 294 + 1.5* 280.5 + 12 277.5 + 14.1 <.01 284 + 9.2
Nulliparity 8/26 9/28 7/23 NS 24/77
CL TO (mm) 28.7 6 29.1 6.1 28 + 5.3 NS 28.6 + 5.8
CL T6 (mm) 18.9 + 8.6 19.7 8.7 19.9 + 9.1 NS 19.5 + 8.8
NOx TO (p.M/mL) 199.8 + 96.2 215.6 + 115.6 210.4 + 99.6 NS 208.6 + 103.8
NOx T6 (p.M/mL) 310.2 + 232.5t 318.7 + 239.1t 321.2 ± 251.1t NS 316.7 + 240.9
CL = cervical length; NOx NO netabolites; Oligo = oligohydramnios; NS = not significant.
* Mean + SD.
P < 0.05 vs TO.

less than 4 in only 31.2% (24/77) of the subjects enrolled,

still
mean cervical length was significantly reduced to 19.5 ± 8.8
mm (P <.01), and mean concentration of cervical NOx sig-
nificantly increased to 316.7 ± 240.9 ,uM/mL (P <.01). Both
cervical shortening and increased cervical NOx were observed
irrespective of parity (Table 2).
Time from induction to active labor was 20.8 ± 10.3 hours
for nulliparous, and 18.5 ± 8.9 hours for multiparous women.
No further PG analog was administered to eight women
(10.4%) presenting with regular uterine contractions 6 hours
from the initial dinoprostone application. In this subgroup of
patients, both cervical shortening and cervical NOx increase
did not differ from the remaining study population; further-
more, the diagnostic criteria for active labor were not met at
T6.
A statistically significant positive correlation was found be-
tween Bishop score modification (A Bishop TO Bishop -
T6 Bishop) and changes in cervical NOx (A NOx = T6 Nox
- TO NOx) (P <.01; r = .494). A direct association was also
detected between A NOx and cervical shortening (A cervical
length [CL] = TO CL - T6 CL) (P <.01; r = .307) (Figure
1). The correlations among A Bishop, A CL, and A NOx were
not affected by parity (P = not significant).
Finally, in nulliparous women, cervical NOx values reached
at T6 were inversely correlated with the time interval from PG
of the cervical modifications induced by the PGE2 analog
dinoprostone. Moreover, among nulliparous women, the
higher was the cervical NOx increase, the shorter was the time
interval from PG administration to labor.
Cervical ripening is an active biochemical process, similar to
an apyretic inflammatory reaction6 controlled by multiple fac-
tors: progesterone,27 PGs,5 62829 NO,629 and inflammat
cytokines, such as IL-1, IL-8, and TNF-ot.24,30,31 In inflam-
matory processes, PGE2 is thought to act principally as a
vasoactive agent that facilitates inflammatory cell infiltration,
promotes secretion of inflammatory cytokines such as IL-8,32
increases neutrophil chemotaxis,33 and stimulates the release of
metalloproteinases (MMP) by monocytes.34 NO induces local
vasodilatation, leukocytes infiltration, extracellular matrix re-
modeling, and MMP activation.6 8 9'3 36 This metabolite also
stimulates apoptosis of smooth muscle cells and fibroblast, an
=
important process in cervical remodeling,3738 promotes IL-8
and elastase secretion from cervical explants,34 and has the
capacity of altering the composition of proteoglycas and gly-
coproteins in the cervix.9'39'4t)
A considerable "cross talk" exists between NO and PG
biosynthetic pathways, as these molecules act in concert. Mul-
tiple experimental studies demonstrated both in vitro and in
vivo that NO directly stimulates cyclooxygenase (COX) II,
increasing PG production during inflammation. Sim

application to delivery (r = .421, P <.05).
DISCUSSION
This study demonstrates that PGE2 application produces an
increase in local NO release along with cervical ripening in
term pregnant women. The positive correlation between de-
crease in cervical length, augmentation in Bishop score, and
cervical NO secretion suggests that NO is a fundamental index
ilarly, abundant evidence also showed that PGs can stimulate
NO synthesis.4346 Vaisanen-Tommiska et al studied the effect
of misoprostol on cervical NO release in non-pregnant and
pregnant women.47 Fifteen non-pregnant women, 26 women
in early pregnancy, and 31 in late pregnancy were administered
either vaginal misoprostol or vaginal placebo. Cervical fluid
samples were collected up to 3 hours after misoprostol/placebo
administration and were assessed for NOx by means of the

Table 2. Cervical Features of the Study Population According to Parity

Nulliparous Multiparous P Total
Gestational age (d) 287 + 6.3* 281 + 12.1 NS 284 + 9.2
CL TO (mm) 29.4 + 7.6 27.8 + 4 NS 28.6 + 5.8
CL T6 (mm) 20 68 19 9.6 NS 19.5 8.8
NOx TO (p.M/mL) 201 + 94 215.2 + 113.6 NS 208.6 + 103.8
NOx T6 (p.M/mL) 311 + 230t 322.4 + 251.8t NS 316.7 + 240.9
CL = cernical length; NOx
*
= NO netabolites, NS = not significant.
Mean ± SD.
t p < .05 vs TO.

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_
206~~~ ~ ~Investig
206 J SocJSoGynecol
Gynco Inesi Vol. 13, No. 3, April 2006
Vol 13 No 3, Api 200 Chiossi et al

Griess reaction. Placebo had no effect on cervical NOx level

and misoprostol did not affect cervical fluid metabolites in
non-pregnant women. Instead, misoprostol induced 4.3- to
5.20-fold elevations in cervical fluid NOx concentration in
early pregnancy and 4.4- to 18.2-fold elevations in late preg- IL-1 IL-8 TNFa i
nancy (P <.01). In the late pregnancy, group elevation of Chemotaxis
NOx level following mi'soprostol was not related to changes ill
Bishop score, although the median cervical fluid NOx con-
centration per 1 Bishop score rose fourfold after misoprostol. iNOS COX II
Our data confirm that PGE analogs stimulate cervical NO
release in pregnancy, as previously proved by Vaisanen-Tom-
miska et a.47 Furthermore, a statistically significant correlation
1
NO Amplification
I
GE
was demonstrated between decrease in cervical length, aug-
mentation of Bishop score, and NOx levels. Thus, dinopros-
tone could possibly activate a chain reaction in the cervix of
pregnant women, as the initial NO stimulation caused by the
PGE2 analog is followed by a local secretion of PGs triggered Glycosaminoglycan Apoptosis MMPs Vascular
by NO. Both responses account for cervical ripening in the synthesis Permeability
pregnant patient. Cervical remodeling seems to be sustained by
two parallel systems that can cross activate each other and
trigger the cascade of events that ultimately modifies the bio-
chemical structure of the cervical tissue. ECM degradation
Multiple factors are involved in the process of cervical Figure 2. Proposed model of cervical ripening also according to the
ripening (Figure 2). Evidence exists that progesterone acts as an data of the actual study. ECM = extracellular rnatnx.
immunosuppressor in the cervix blocking that inflammatory
reaction that causes cervical softening, effacement, and dilata-
tion. In fact, progesterone stops IL-8 release from cervical
explants and stimulates the production of a potent neutrophil proteins, and they contain COX II and iNOS, capable of
inhibitor (secretory leukocyte protease inhibitor [SLPI]).4 producing substantial amounts of PGE2 and NO.48
Furthermore, such hormone inhibits cytokine release, sup- Moreover, iNOS has been demonstrated to be expressed
presses iNOS and the inducible form of COX II, and blocks after stimulation with lipopolysaccharide, IL-1, and TNF-
some MMPs.9'29 A local decrease in progesterone availability 09,5°;
the same inflammatory stimuli are also capable of pro-
moting the transcription of COX II.1(.29 31 This is the reason
or interference with its action would stimulate IL-8 release and
inhibit SLPI production, triggering neutrophil and macro- why IL-1, IL-8, IL-6, and TNF-ox have been shown to be
phage chemotaxis, immigration, and activation, as previously released in the cervix and to induce cervical remodeling in
suggested by Denison et al.34 These cells are well-known vivo.23'24'52 Therefore, inflammatory cytokines contribute to
sources of MMPs capable of digesting extracellular matrix promote both NO and PG synthesis, the effectors of cervical
ripening. As demonstrated by the present study, local applica-
tion of a PG analog represents one of the different ways to
35
activate a self-maintaining process that is ultimately responsible
30 of cervical maturation.
a_,
-I 25
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