As the incidence of preterm birth continues to rise each year, the management of preterm infants has become an increasingly important research focus in perinatal medicine. Preterm premature rupture of membranes (PPROM) is one of the leading causes of preterm birth. PPROM is often closely associated with intrauterine infection: occult intrauterine infections can trigger PPROM, while prolonged PPROM facilitates easier and faster retrograde pathogen invasion. The severity of infection correlates with the duration of PPROM [1][2]. Fetal exposure to pathogens such as bacteria, viruses, and fungi in utero can induce an inflammatory response known as fetal inflammatory response syndrome (FIRS). FIRS resembles adult systemic inflammatory response syndrome (SIRS) and may lead to multi-system involvement in the fetus or neonate. It can initiate labor, increase neonatal morbidity, and cause multi-organ dysfunction. Both mother and fetus produce pro-inflammatory cytokines in response to intra-amniotic infection, resulting in a high risk of adverse outcomes—particularly preterm birth, early-onset sepsis, bronchopulmonary dysplasia, intraventricular hemorrhage, periventricular leukomalacia, neonatal respiratory distress syndrome, and even death [3]-[5].

In utero, the fetus may already be exposed to bacteria in the amniotic fluid, with both fetus and mother encountering microorganisms and their metabolites. When PPROM occurs, it remains unclear whether and how the composition of the intrauterine microbiota changes, and which specific alterations are linked to the onset and progression of FIRS. We hypothesize that PPROM leads to dysbiosis of the intrauterine microbiome, and that these changes are closely associated with intrauterine inflammation and directly influence the prognosis of preterm infants. This study aims to use metagenomic detection to identify early abnormal shifts in the intrauterine microbiota and to screen for specific pathogens. Such an approach would be valuable for clarifying the mechanisms of intrauterine inflammation, predicting its occurrence, and improving the diagnosis, treatment, and outcomes of preterm infants.

It has traditionally been assumed that the blood-placental barrier and fetal membranes protect the amniotic fluid from microbial invasion, allowing the fetus to develop in a sterile environment [6]. However, with advances in detection technologies, recent studies have identified microbial DNA colonizing both amniotic fluid and the placenta. These microorganisms are thought to originate from the birth canal and the maternal gastrointestinal tract. Through fetal breathing and swallowing movements, the fetal respiratory and gastrointestinal tracts are exposed to microorganisms and their components. Related studies have shown that meconium contains viable microorganisms and their DNA, and that the microbial composition of amniotic fluid, meconium, and placenta varies with gestational age. Importantly, these microorganisms do not appear to trigger fetal inflammatory response syndrome (FIRS) [7]-[9].

In this study, among cases without preterm premature rupture of membranes (PPROM) or other evidence of maternal infection, none developed chorioamnionitis. However, metagenomic analysis of the amniotic fluid still revealed the presence of a microbial ecosystem, with detectable gene copies of bacteria, fungi, and viruses. Among the bacteria, Cutibacterium acnes, Staphylococcus epidermidis, and Staphylococcus hominis were the most frequently detected species. The most common fungi and virus were Lasiodiplodia, Aspergillus, and human endogenous retrovirus K, respectively, although their copy numbers were very low. These findings only indicate that bacterial, fungal, and viral genetic material can be detected in amniotic fluid, which may constitute a unique microbial genetic environment within the amniotic cavity. However, as previous studies have pointed out [7][9], distinguishing true colonizing microbiota from background contamination or transient microbial DNA remains highly challenging in low-biomass samples. Therefore, whether the microbial DNA detected in this study represents an active, colonizing “normal amniotic fluid microbiota” requires further investigation, and its physiological significance remains to be clarified.

When the fetus is exposed in utero to pathogenic microorganisms such as bacteria, viruses, or fungi, these pathogens must first cross the placenta via the umbilical vessels to enter the fetal circulation, triggering chorioamnionitis [10][11]. The fetus then mounts an inflammatory response by producing pro-inflammatory cytokines against the intra-amniotic infection, which can induce fetal inflammatory response syndrome (FIRS). The pathophysiological process that initiates FIRS is histologically characterized by umbilical vasculitis. FIRS can trigger the onset of labor, leading to preterm birth. If pregnancy is not terminated, FIRS may cause persistent multi-system damage to the fetus that continues after delivery, markedly increasing the risk of neonatal complications and adverse outcomes [12]-[15].

PPROM may be a consequence of intrauterine infection, or it may induce intrauterine infection due to prolonged membrane rupture. However, PPROM does not necessarily indicate the presence of inflammatory pathology in the placenta or umbilical cord [16]-[18]. In this study, placental and umbilical cord pathology revealed chorioamnionitis and/or funisitis in fewer than half of the PPROM cases. This suggests that PPROM does not inevitably trigger inflammatory responses in the placenta or fetus. Furthermore, there were no significant differences in inflammatory markers between the PPROM and non-PPROM groups among the preterm infants. Nevertheless, the copy numbers of bacteria and fungi in the amniotic fluid were significantly higher in the PPROM group, indicating that PPROM leads to alterations in the amniotic fluid microbiome, characterized primarily by an increase in bacterial and fungal abundance. This change may be one of the reasons why PPROM is a high-risk factor for neonatal infection. Among the PPROM cases, no significant differences were observed in the composition or quantity of amniotic fluid microorganisms between the subgroups with and without placental/umbilical cord inflammatory changes. However, due to the small sample size in this subgroup analysis, the statistical power was limited. Therefore, this result should be considered inconclusive rather than evidence of no association. For example, the median Ureaplasma copy number was higher in the inflammation group, but the difference did not reach statistical significance. Future studies with larger sample sizes are needed to further clarify this relationship.

Currently, when PPROM occurs, antibiotics are routinely administered to both pregnant women and preterm infants [19]-[21]. In the present study, mothers in the PPROM group received prenatal antibiotics, whereas those in the non-PPROM group did not. However, there was no significant difference between the two groups in the duration of early postnatal antibiotic use among preterm infants. Notably, the non-PPROM group showed no evidence of intrauterine inflammation, and PPROM itself does not necessarily lead to intrauterine inflammatory responses. These findings suggest that empirical antibiotic use should be approached with caution in the absence of PPROM and other evidence of intrauterine infection. Even in cases of PPROM, antibiotics should be discontinued promptly once intrauterine bacterial infection has been excluded. Otherwise, unnecessary antibiotic exposure may disrupt the normal intrauterine microbiome, with potential but still unknown consequences for preterm infants.

This study has an important limitation: all mothers in the PPROM group received antenatal antibiotic treatment, whereas those in the control group did not. This represents an unavoidable confounding factor. Therefore, the observed differences in microbial load in the amniotic fluid between the PPROM and control groups may be attributable to PPROM itself, antibiotic exposure, or their combined effects. Antibiotic use may have suppressed the growth of certain susceptible microorganisms, thereby altering the composition and abundance of the microbial community. Accordingly, the conclusions of this study should be interpreted as reflecting an association between PPROM status combined with antibiotic exposure and the microbial findings, rather than being attributed solely to PPROM. Future studies with more refined designs are needed to distinguish the independent effects of PPROM and antibiotics on the amniotic fluid microbiome.

In conclusion, a unique microecological environment exists in the amniotic fluid, which may play an important role in fetal growth, development, and maturation. The occurrence of PPROM is associated with changes in the composition of the amniotic fluid microbiome, and these alterations may be related to the development of intrauterine inflammatory responses.

Natural Science Fundation of Changsha City (kq 2208439); Provincial Natural Science Foundation of Hunan (2024JJ9501).

Wenqi Zhou performed the ELISA and pathological analyses and contributed to manuscript writing. Liping Zhu and Ke Huang collected the specimens. Lu Bai conducted the metagenomic analysis. Qin Yang provided guidance on manuscript writing.