Ureaplasma parvum detected in umbilical cord tissues diagnosed with funisitis associated with adverse pregnancy outcomes and neonatal pneumonia

Introduction

Genital colonization, a unique group of microorganisms that reside primarily in the genital mucosa, have garnered significant attention in recent years due to their association with numerous of adverse outcomes of pregnancy [1]. Among various genital microorganisms, Ureaplasma spp. – particularly the two clinically significant species Ureaplasma parvum (UP) and Ureaplasma urealyticum (UU) – along with Mycoplasma hominis (MH) demonstrate particularly high prevalence [2], 3]. These pathogens have been clinically associated with multiple obstetric and gynecological complications, including spontaneous abortion, stillbirth, low birth weight, preterm delivery, chorioamnionitis, intra-amniotic infection, premature rupture of membranes (PROM), postpartum endometritis, pyelonephritis, pelvic inflammatory disease, and sepsis [4], [5], [6], [7], [8].

Intrauterine inflammation, with or without infection, is commonly associated with various adverse pregnancy outcomes and neonatal complications [9], 10]. Inflammation can lead to pathological changes in the placenta, umbilical cord, and even various fetal organs. Clinically common chorioamnionitis is defined by neutrophilic infiltration within the chorioamniotic membranes (fetal membranes) or chorionic plate, while funisitis refers to inflammation of the umbilical cord [11]. Chorioamnionitis represents as evidence of a maternal (host) inflammatory response, whereas funisitis indicates a fetal inflammatory reaction [12]. The placenta and umbilical cord serve as critical physiological structures bridging the maternal and fetal systems, facilitating the transport and exchange of nutrients and blood while enabling the elimination of metabolic waste products. Intrauterine infection can transmit pathogens to the fetus via the placental or umbilical cord route, potentially leading to adverse clinical outcomes [13]. Funisitis is a recognized higher risk factor for adverse neonatal outcomes than chorioamnionitis [14].

Chorioamnionitis and funisitis may occur either due to microbial infection or in the absence of detectable microorganisms (i.e., “sterile inflammation”) [15], 16]. The predominant bacterial pathogens associated with intrauterine inflammation are primarily Ureaplasma species, followed by Fusobacterium species, Streptococcus species, Gardnerella species, and Mycoplasma species [17], [18], [19]. Clinical studies demonstrate that Ureaplasma species (U. parvum and U. urealyticum) emerge as the most common microbial isolated from amniotic fluid, umbilical cord and placental tissues [2], 20]. How do genital microorganisms traverse the chorioamniotic barrier to infect the placenta, umbilical cord and fetus? The most widely accepted route is that lower genital tract microorganisms migrate through the cervix into choriodecidual space, and then reaching the amnoitic fluid and fetus [21], 22]. Infected fetus may face a series of health problems, including pneumonia, enteritis, encephalitis, feeding difficulties and sepsis [23]. While other authors have reported that genital tract Ureaplasma colonization has no statistically significant differences with the chorioamnionitis and funisitis [24], [25], [26].

Still, the impact of genital microorganisms on funisitis and chorioamniotic is conflicting. Understanding the relationship among them is crucial for preventing and treating related diseases and ensuring the health of mothers and infants. This study investigated seven genital tract microorganisms in FFPE cord tissue samples from cases of funisitis using qPCR. Combining this result with maternal prenatal examination data, pregnancy outcomes, and neonatal clinical symptoms, it analyzed the associated risks between these microorganisms and these clinical outcomes.

Materials and methods

Results

Study population

A total of 120 umbilical cord samples from women diagnosed with funisitis and chorioamnionitis were analyzed in the present study and 22 cases were excluded for the following reasons: induced abortion (n=16), in vitro fertilization (IVF) (n=6) (Figure 1). The remaining 98 subjects were categorized into two groups according to perinatal outcomes. Fifty one (52.0 %) pregnant women were included in the adverse pregnancy outcomes group, comprising five fetal deaths, 19 miscarriages, and 27 preterm births. Forty seven (48.0 %) pregnant women who gave birth at or beyond 37 weeks of gestation served as a term birth group (Figure 1).

Figure 1:

Flowchart of this study.

Genital microorganism present in the umbilical cord associated with adverse clinical phenotypes

Figure 2 shows the association between four genital tract microorganisms detected in the umbilical cord and adverse clinical phenotypes. Here, preterm birth and pneumonia correlated with infection by the four positive microorganisms (p < 0.05).U. parvumwas identified by qPCR in 19 specimens, while GBS, U. urealyticum and M. hominis were detected in 8, 4, and 3 samples, respectively. Three umbilical cord samples with U. urealyticum positive were co-infected with M. hominisor or GBS (Supplementary Table S1). In 19 cases with U. parvum positive, the corresponding pregnant women exhibited clinical symptoms of varying severity in their pregnancy outcomes, including premature rupture of membranes (PROM), miscarriage, and fetal death (Supplementary Table S1). We compared the associated risks of different pregnancy outcomes and fetal symptoms with positive vs. negative detection of U. parvum in umbilical cord specimens. In the fetal death and miscarriage group (p = 0.4011) and the premature rupture of membranes (PROM) group (p = 0.3891), there was no significant difference in U. parvum detection (positive vs. negative). However, in the preterm birth group (p < 0.0001) and the neonatal pneumonia group (p = 0.0021), positive detection of U. parvum showed a significant association with these two outcomes (Figure 2).

Figure 2:

Correlation between adverse clinical phenotypes and microorganisms identified positive in umbilical cord samples. FD&M, fetal death and miscarriage; PROM, premature rupture of the membranes; PD, preterm birth;***p < 0.0005;**p < 0.005.

Discussion

Among 98 umbilical cord tissue samples, 30 (30.6 %) tested positive for seven microorganisms via qPCR, with U. parvum (UP) identified as the predominant species. This finding aligns with the dominant microbial profile detected in clinical vaginal swabs from pregnant women.

The results of this retrospective study inferred that pregnant women with genital tract microorganisms infection, especially U. parvum, were at increased risk for adverse clinical outcomes, including fetal death, miscarriage, preterm birth and neonatal pneumonia. This finding matches with a recent case-control study by Maria et al. In that study, U. parvum was significantly associated with histological acute chorioamnionitis but not with pneumonia [28]. While our research findings were partly contrary to that conclusion. The significant relation between the detection of Ureaplasma spp. and pneumonia is also supported by previous studies. Sobouti et al. reported that U. parvum can be detected in the genital tract of pregnant women and can be vertically transmitted to the fetus, potentially causing infections in the newborn [29]. These findings suggest a significant transmission of U. parvum from mothers to newborns. Abe et al. collected gastric fluid samples from 47 newborns in the NICU immediately after birth and tested using PCR assays targeting Ureaplasma spp.The subspecies of nine positive samples were U. parvum [30]. In a similar study of ours, U. parvum was detected with the highest frequency in the gastric fluid of newborns, with an odds ratio (OR) of up to six for bronchopulmonary dysplasia (BPD) [31]. Previous studies have shown that the detection of U. urealyticum in the umbilical cord is associated with funisitis [2], 32], while reports linking U. parvum to funisitis remain scarce. Our findings revealed a 19.4 % detection rate of U. parvum in umbilical cord tissues with confirmed funisitis, suggesting its potential role as the predominant pathogenic bacterium underlying the inflammatory process.

The incidence rates of funisitis and chorioamnionitis are not consistent, and they may occur independently [12], 33]. In the cases examined in this study, those diagnosed with funisitis also had a concurrent of chorioamnionitis. These discrepant findings may be attributed to variations in the primary disease focus across different research studies. Chorioamnionitis and funisitis can be caused by non-infectious factors or infectious pathogens. Common pathogens such as U. parvum and marginally U. urealyticum, GBS and other Gram-negative bacteria have been implicated in the development of funisitis and chorioamnionitis [13]. These organisms can gain access to the amniotic cavity by ascending infections from the lower genital tract through the cervix into the intraamninotic space [13], 21]. Our study demonstrated a significant association between genital tract microorganisms and chorioamnionitis severity (p = 0.0254), showing higher prevalence in stages II/III compared to stage I.

Traditionally Ureaplasma spp. classified as biovar 1 and biovar 2, these microorganisms underwent taxonomic reclassification in 2002 based on genomic evidence. Current nomenclature designates the former biovar 2 as Ureaplasma urealyticum and biovar 1 as Ureaplasma parvum [8], 34], 35]. Although previous studies have linked U. urealyticum to adverse pregnancy outcomes including stillbirth, spontaneous abortion, chorioamnionitis, and preterm labor, most investigations neither differentiated between the two clinically relevant species (U. urealyticum and U. parvum) nor employed sufficiently sensitive detection methodologies [36]. In Kataoka et al. study shown that vaginal colonization with U. parvum, but not U. urealyticum, is associated with late abortion or early preterm birth [37]. This result was also confirmed in present study. According the phenotype and genotype, serovars 1, 3, 6, and 14 subspecies are classified as U. parvum, while 2, 4, 5, 7–13 subspecies are classified as U. urealyticum [38], 39]. Rittenschober-Bohm et al. serotyped 1316 samples that colonization with U. parvum serovar 3, but not serovar 1 or serovar 6, in early pregnancy is associated with preterm delivery at very and extremely low gestational age [40]. In a cohort of Australian pregnant women, Payne et al. identified U. parvum serovar 6 (SV6) as a genotype demonstrating particular clinical significance in preterm birth outcomes, according to their molecular epidemiological study [41]. But our result, the serovar sequencing of U. parvum showed that the detection rates of the three main serotypes were close, which inconsistent with above conclusion.

Although numerous clinical observational studies have been conducted over a period of over 30 years, the clinical significance of Ureaplasma spp. infection is still under debate [42]. There is a viewpoint that the Ureaplasma spp. is a commensal in the female genital tract and considered to have of low virulence, because Ureaplasma spp. lacks a cell wall, resulting in weak immunogenicity [43]. Pavlidis et al. have shown that cervical epithelial damage facilitates ascending UP infection into the uteri of pregnant mice, with accompanying PTB and elevation of pro-inflammatory cytokines in the myometrium, fetal membranes and placenta [44]. Previous studies have demonstrated that Ureaplasma spp. are strongly associated with funisitis and chorioamnionitis. Dose a positive vaginal swab test require antibiotic treatment? Studies have shown that there is no difference in the colonization rate of Ureaplasma spp. in the endocervix between fertile women with and without symptoms of genital tract infection [45], 46]. Additionally, the pathogenic role of Ureaplasma spp. is often unclear because most of these infections are clinically asymptomatic. U. parvum infections can persist asymptomatically in humans for up to 2 months [47]. Ureaplasma spp. are not always suspected as causative agents of funisitis and chorioamnionitis. Coinfection with multiple microorganisms may require more than one antibiotic to successfully eradicate the infection [48]. Additionally, pregnant women face limited treatment options due to their concerns about the teratogenic and harmful effects of antibiotic use on the fetus. Antibiotic therapy is not recommended on a broad scale unless there is evidence of intra-amniotic infection.

Although it is currently unclear why some women infected with U. parvum experience adverse pregnancy outcomes while others do not, some researchers attribute these differences sequelae into the toxicity of the infecting serotype and bacterial load [49], [50], [51]. In our study, the Ct values, which serve as an indicator of bacterial load, has no correlation with adverse perinatal outcomes. We further recommend that molecular pathology testing be performed to identify the pathogen when pathological tissue definitively confirms funisitis or chorioamnionitis but the etiology remains undetermined. Compared to traditional cultivation methods, molecular detection, especially multiplex qPCR, can detect multiple pathogens simultaneously.

There are several limitations in our study. Firstly, the relatively small sample size due to the retrospective inclusion of FFPE tissue samples diagnosed with funisitis from archival collections within the past five years. Secondly, the lack of consistent detection time points and methods for the clinical test results of pregnant women may cause certain biases in the conclusions. Thirdly, as this is a retrospective study, vaginal swabs and neonatal oral swabs were not preserved, which hindered the verification of the correlation between vaginal colonization infection and the occurrence of preterm birth and pneumonia. Fourthly, this study only involved seven colonizing bacterias, and the impact of other bacteria on adverse pregnancy outcomes was not covered. Fifthly, the unavailability of data about other well-known factors associated with preterm birth, pneumonia, funisitis, and chorioamnionitis.

Conclusions

Genital mycoplasmas was associated increased inflammation of the chorioamnionitic membranes. The detection of U. parvum in umbilical cord tissue was significantly associated with pretem labor and neonatal pneumonia. Larger prospective studies with adjusted analyses are needed to confirm these findings and clarify mechanistic roles. By understanding and addressing this relationship, healthcare providers can improve the health outcomes for both mothers and newborns. Regular vaginal swab screening for pathogens in early pregnancy may helps in formulating treatment plans. However, further prospective studies are needed to confirm this hypothesis.