Association of genitourinary infections and cervical length with preterm childbirth

A prospective cohort study was conducted on a convenience sample of 1370 pregnant women with a gestational age of 20 to 25 weeks in the city of Ribeirão Preto. Data on obstetrical history, maternal age, parity, smoking habit, and a history of preterm delivery was collected with the application of a sociodemographic questionnaire. Cervical length was determined by endovaginal ultrasound, and urine and vaginal content samples were obtained to determine urinary tract infection (UTI) and bacterial vaginosis (BV), respectively. The aim of this study was to verify the association of cervical length and genitourinary infections with preterm birth (PTB). Ultrasound showed no association of UTI or BV with short cervical length. PTB rate was 9.63%. Among the women with PTB, 15 showed UTI (RR: 1.55, 95%CI: 0.93–2.58), 19 had BV (RR: 1.22, 95%CI: 0.77–1.94), and one had both UTI and BV (RR: 0.85, 95%CI: 0.13–5.62). Nineteen (14.4%) PTB occurred in women with a cervical length ≤2.5 cm (RR: 2.89, 95%CI: 1.89–4.43). Among the 75 patients with PTB stratified as spontaneous, 10 showed UTI (RR: 2.02, 95%CI: 1.05–3.86) and 14 had a diagnosis of BV (RR: 1.72, 95%CI: 0.97–3.04). A short cervical length between 20 and 25 weeks of pregnancy was associated with PTB, whereas UTI and BV determined at this age were not associated with short cervical length or with PTB, although UTI, even if asymptomatic, was related to spontaneous PTB.


Introduction
Preterm birth (PTB) is an important public health problem due to its high incidence and perinatal morbiditymortality (1). Brazilian studies have reported a prevalence of PTB ranging from 11.7 to 12.3% (2). PTB and its complications are responsible for 78% of all neonatal deaths and are associated with high morbidity, with serious short-and long-term consequences involving physical, psychological, and economic costs (3,4).
Studies have demonstrated that infections such as bacterial vaginosis (BV) and urinary tract infections (UTI) may be associated with a higher risk of PTB and low birth weight (5,6), since these processes are related to the inflammatory response present in infections. These conditions are fundamentally involved in the physiopathology of PTB since cytokines (TNF-a, interleukins, and prostaglandins -PGE2, PGD2, and PGF2a, in particular) resulting from the cascade of inflammatory events that directly participate in the triggering of uterine contractions (7,8).
Cervical effacement causing cervical shortening precedes by five to six weeks the clinically determined labor (9,10). Thus, the identification of cervical shortening at early gestational ages represents an important risk factor for PTB (11)(12)(13) and its evaluation by transvaginal ultrasound is one of the parameters showing a good correlation with the risk for PTB (14).
On this basis, our objective was to assess the association of genitourinary infections with cervical shortening and their relationship with previous PTB (PPTB). This case-control study was nested in a prospective convenience cohort investigated in 2010 and 2011. The cohort has been described in the thematic project entitled "Ecological factors of preterm birth and consequences of perinatal factors on children's health: birth cohorts in two Brazilian cities -BRISA" (https://nesca.fmrp.usp.br/brisabotoes) conducted in Ribeirão Preto (SP) and São Luís (MA). The Ribeirão Preto data were used in the present study.
In this study, we used a convenience sample from the BRISA thematic project whose size was based on the reported prevalence of the explanatory variables studied in the initial project, which varied from 10 to 50%. Thus, considering a 12% prematurity rate, we started from the initial sample of 1500 women and their respective newborns. Considering losses and lack of information, the sample effectively consisted on 1370 mother-child pairs and a total of 132 PTB. Of all PTB, we were able to obtain information about the characteristics of childbirth, spontaneous or not, for 102 participants.
We included pregnant women residing in Ribeirão Preto, seen in public and private services, carrying a single fetus, and evaluated during prenatal care at a gestational age of 20 to 25 weeks. The criteria for inclusion in the prenatal BRISA cohort were: having done an obstetrical ultrasound exam before the 20th week of gestation, having a gestational age of 20 to 25 weeks at the time of data collection, and having a single fetus.
Women who were lost to follow-up between the prenatal and birth evaluations and women with incomplete information were excluded from the study. Gestational age was estimated from the date of last menstrual period (DLM) and based on the gestational ultrasound performed before 20 weeks of pregnancy, thus minimizing determination bias in the estimate of outcome (15).
After responding to a standardized questionnaire in a face-to-face interview, the women were submitted to gynecological examination and collection of vaginal material for fresh examination and for the preparation of Gramstained slides. The criterion used for the diagnosis of BV was the presence of clue cells and/or a Nugent score with counts of existing Lactobacillus, Gardnerella/Bacteroides, and Mobiluncus morphotypes. Morphological types where scored as 1+ to 4+ according to the number of microorganisms present. A score of seven or more was considered to indicate positivity for BV (16)(17)(18). Urine was collected for culture, considered the gold standard method validated for the investigation of asymptomatic bacteriuria and the detection of UTI (19).
Ultrasound exams were performed at the Clinical Research Unit of FMRP-USP by trained observers calibrated for the method, using an HDI instrument, model 11 (USA), and a technique systematized and validated by the Fetal Medicine Foundation (20). Controls were obtained from the cases that did not progress to PTB, at a 2:1 proportion (21). In view of the well-established association of short cervix with prematurity, all patients with a cervical length of 2.5 cm or less were referred to a center specialized in high-risk pregnancies.
After birth, newborn data regarding hospital, weight, and gestational age were obtained and a standardized puerperium questionnaire was applied in order to obtain childbirth information about the 95 participants, with the analysis of replies such as presence of painful contractions preceding the outcome in order to determine whether labor was spontaneous. Participants whose questionnaires did not contain sufficient information were contacted by telephone and 7 of them were located. The difference between groups was analyzed by the Fisher exact test, and the relative risk of the various variables was calculated by the adjustment of log-binomial models and confidence intervals (95%CI) using the SAS 9.2 software (USA).

Results
The general characteristics of the study population are shown in Table 1. The study was conducted on 1370 pregnant women, 132 (9.63%) of whom had PTB. Among these 132 patients with PTB, information about childbirth was available for 102, with a total of 74 spontaneous PTB The Fisher exact test was applied to determine the association between the presence of genitourinary infections and a short cervix in an ultrasound exam. A cervical length of 2.5 cm or less was observed in 7 (5.79%) of 121 patients with a positive UTI exam and in 68 (5.46%) of the 1245 patients with no UTI (P=0.83). A cervical length of 2.5 cm or less was observed in 13 (7.07%) of 184 patients with a positive BV exam and in 61 (5.21%) of those with no BV, again without statistical significance (P=0.30). Among the 13 patients with a positive exam for the two infections, no cervical length p2.5 was observed.
Regarding  Table 3 and  Table 4. Data are reported as number and percentage. The difference between groups was analyzed by the Fisher exact test and the relative risk of the various variables was calculated by the adjustment of log-binomial models and confidence intervals (95%CI). UTI: urinary tract infection.

Discussion
The present study revealed a 9.63% incidence of PTB, a value below that reported in the literature. In Brazil, a 2010 study coordinated by the Postgraduate Program of Epidemiology of the Federal University of Pelotas with the participation of 12 universities, reported that 11.7% of the births occurring in Brazil are preterm (26). The 2014 Brazilian Multicenter Study on Preterm Birth (EMIP), involving 20 reference centers distributed throughout Brazil and data obtained from April 2011 to July 2012, pointed out that 12.3% of the births were preterm (27). The lower rate of PTB in our study could be due to the referral of patients diagnosed with a short cervix to a prematurity center, reducing the rate of prematurity.
Data analysis revealed that cervical shortening was not associated with the occurrence of infections (UTI and BV) between 20 and 25 weeks of pregnancy. The hypothesis of such association was raised because infections, as well cervical shortening, are associated with  PTB. We considered the immuno-inflammatory theory of PTB and attempted to determine whether the cytokines and prostaglandins produced in inflammatory and infectious processes, which trigger uterine contractions and depolymerization of the cervix, were linked to cervical shortening, as proposed by Romero et al. (5), Hanna et al. (7), and Chalis et al. (8). We wish to point out that the infections of the women studied here were asymptomatic and might have released lower amounts of inflammatory mediators, thus having a lower influence on cervical size. A cervical length p2.5 cm determined between 20 and 25 weeks of gestation was associated with PTB, supporting data obtained by others. In 1966, Schaffner and Schanzer (9) were the first to report the association of cervical shortening with PTB and many important studies have been conducted since then showing this association. An example is a study by Iams et al. (12)  Of 199,093 patients studied by Scheiner et al. (28), 4890 (2.5%) showed asymptomatic bacteriuria, 13.3% of whom had PTB, as opposed to 7.6% of patients with a negative exam (OR 1.9, 95%CI: 1.7-2.0), demonstrating the increased risk of pregnant women with asymptomatic bacteriuria.
The determination of BV between 20 and 25 weeks of gestation was not associated with PTB in our study. pregnant women with BV. Screening for BV using clinical criteria or Gram and treatment of the condition eradicated the bacteria from the genital tract within 20 weeks (RR 0.20; 95%CI: 0.05-0.76) but did not reduce significantly the occurrence of childbirth at a gestational age of less than 37 weeks (RR 0.88; 95%CI: 0.71-1.09). Analysis of evidence from the literature and from the present study may suggest that asymptomatic BV detected between 20 and 25 weeks is not associated with PTB.
The present data demonstrated an association of other risk factors with spontaneous PTB: a history of PTB is a strong predictor of a new PTB in subsequent pregnancies, as previously reported by Esplin et al. (23). The crude and adjusted RR values were very high both among patients with 1 PPTB and those with 2 or more PPTB. This information was obtained with the prenatal questionnaire, which was applied by lay persons, and was only used by us during data processing. For this reason, even though this is a well-defined risk factor, these patients were not identified at the proper time for referral to reference centers specialized in prematurity. There was no association between maternal age and prematurity although literature data indicate a higher risk of PTB among women younger than 19 years and older than 35 years (24). The reference used here regarding parity was having 2 or 3 children. There was no increase in the risk of prematurity among primiparous patients and only an increase in crude RR was detected among them. This result supports data obtained by Bezerra et al. (22) in a study in which no association was detected between number of gestations and prematurity. Regarding the smoking variable, the crude risk of women that were smokers was similar to that reported in the literature by Grantz et al. (25).
In the present study, objective techniques were used for the measurement of the major exposures, with previous calibration of the instruments. The design of the study permitted the assessment of biological material of the pregnant women for the detection of infections. Although a specific sample calculation was not performed, with the total number of subjects in the initial thematic project (BRISA) being used to assess the association of factors related to PTB, it was observed that the sample size used had enough power for the estimate of associations of interest.
A limitation of the present study was that the patients were not monitored during the prenatal period, being evaluated only once prenatally and once after childbirth. Thus, there was no control of the number of pregnant women who had symptomatic UTI and BV throughout gestation and it was not known whether or not they had been treated. An additional limitation was that we did not know how the patients with a short cervical length had been followed up.

Conclusions
In the present study, there was no association of asymptomatic UTI or BV with cervical shortening determined between 20 and 25 weeks of pregnancy. UTI, but not BV, was associated with spontaneous PTB. There was a relationship between early cervical shortening and prematurity, with a lower incidence than that reported in the literature.