Analysis of the microbiome in maternal, intrauterine and fetal environments based on 16S rRNA genes following different durations of membrane rupture

The incidence of chorioamnionitis and neonatal sepsis increases with the increasing time of rupture of membranes. Changes in the amount and categories of microbiomes in maternal and fetal environments after membrane rupture have yet to be discussed. In order to determine the microbiome diversity and signature in the maternal, intrauterine, and fetal environments of different durations following membrane rupture, we collected samples of fetal membrane, amniotic fluid, cord blood and maternal peripheral blood from singleton pregnant women and divided them into five groups according to the duration of membrane rupture. DNA was isolated from the samples, and the V3V4 region of bacterial 16S rRNA genes was sequenced. We found that the alpha diversity of the fetal membrane microbiome increased significantly 12 h after membrane rupture, while the beta diversity of the amniotic fluid microbiome increased 24 h after membrane rupture. In cord blood, the mean proportion of Methylobacterium and Halomonadaceae reached the highest 12 h after membrane rupture, and the mean proportion of Prevotella reached the highest 24 h after membrane rupture. The LEfSe algorithm showed that Ruminococcus, Paludibaculum, Lachnospiraceae, and Prevotella were detected earlier in cord blood or maternal blood and then detected in fetal membranes or amniotic fluid, which may suggest a reverse infection model. In conclusion, the microbes may invade the placenta 12 h after membrane rupture and invaded the amniotic cavity 24 h after membrane rupture. In addition to the common ascending pattern of infection, the hematogenous pathway of intrauterine infection should also be considered among people with rupture of membranes.

Survey of reproductive microbiome content.Alpha diversity was calculated to reflect the "within" community richness of the microbial community for each sample.In the fetal membrane, the microbiotas of the 12 to < 24 h group were found to be more diverse than the bacterial communities found in the < 6, 6 to < 12 and 24 to < 48 h groups when compared in Wilcoxon's test (Fig. 1A).There was no significant difference in the microbiome of amniotic fluid, cord blood or maternal peripheral blood (Fig. 1B-D).
Beta diversity was calculated to reflect the overall microbiota dissimilarities in samples of different groups.In amniotic fluid, the microbiome of the 24 to < 48 and ≥ 48 h groups were more diverse than that of the < 6 and 12 to < 24 h groups (Fig. 2B).There was no statistically significant difference in the microbiome of the fetal membrane, cord blood or maternal peripheral blood between the different groups (Fig. 2A,C,D).
Microbial features associated with the duration of membrane rupture.We compared the bacteria (mean proportion > 1%) at the genus level among different durations of membrane rupture using the Kruskal-Wallis test and found diversity.The results showed that the mean proportion of Prevotella and Halomonadaceae in cord blood, Methylobacterium in both fetal membranes and cord blood, was different between the five groups (Fig. 3A,B).There was no significant difference between different durations of membrane rupture in amniotic fluid and maternal peripheral blood samples.
To identify the specific taxonomic biomarkers in each tissue of different membrane rupture durations, we additionally applied the linear discriminant analysis effect size (LEfSe) algorithm to identify microbial species that were significantly more prevalent in each period.The specific microbes are shown in Table 3. Ruminococcus, Paludibaculum, Lachnospiraceae, and Prevotella were detected earlier in the fetal membrane and amniotic fluid and then detected in cord blood, while Rhodobium, Christensenellaceae, Nonomuraea, Thauera, and Pseudonocardia were detected earlier in cord blood or maternal blood than in the fetal membrane and amniotic fluid.

Discussion
Our 16S-based operational taxonomic unit (OUT) analysis of samples found that the alpha diversity of the fetal membrane microorganisms increased significantly 12 h after membrane rupture, while the beta diversity of the amniotic fluid microbiome significantly increased 24 h after membrane rupture.According to the analysis of the specific bacteria shared among different groups in different tissues, it was found that some bacteria may appear in maternal blood or cord blood earlier, suggesting the possibility of a hematogenous pathway of intrauterine infection.However, further studies should focus on the morbific abundance or duration of these microorganisms to have a better understanding of their association with fetal or neonatal diseases and to minimize the bias of potential contamination.
Our study indicated that the diversity and abundance of microorganisms in amniotic fluid increased 24 h after the rupture of membranes, which was consistent with Seaward's finding that the risk of chorioamnionitis and neonatal infection significantly increased if membrane rupture was over 24 h 11 .Our study also showed that compared with women of membrane rupture time less than 6 h, women of membrane rupture time over 24 h had a higher rate of maternal infection, Although most guidelines recommended expectant management for 24 h after PROM of term births in the absence of signs of infection 12 , a large systemic review found that immediate intervention or intervention within 24 h reduced the risk of maternal infectious morbidity compared with expectant management for PROM at 37 weeks' gestation or later 13 .Our result may provide evidence for induction of labor earlier after membrane rupture of term or near term births if spontaneous labor does not occur, considering that at least 12-18 h of effective contractions should be allowed for induced labor 14 .Our study also showed that in cord blood, the mean proportion of Methylobacterium and Halomonadaceae reached the highest 12 h after membrane rupture, and the mean proportion of Prevotella reached the highest 24 h after membrane rupture.Both Methylobacterium and Prevotella are gram-negative bacilli and have been reported to contribute to some clinical infections 15,16 .Besides, of all the newborn diagnosed of neonatal infection, only one baby's blood  www.nature.com/scientificreports/culture was positive of known common pathogen.Whether the elevated WBC or CRP of other newborn were caused by components of the microbiota need further investigation by 16S rRNA genes.Future studies could focus on their association with fetal or neonatal infections.The LEfSe algorithm showed that Ruminococcus, Paludibaculum, Lachnospiraceae, and Prevotella were detected earlier in the fetal membrane or amniotic fluid and then detected in cord blood or maternal peripheral blood, which was consistent with the ascending infection model.Fetal membranes constitute a barrier against bacterial invasion of the amniotic cavity.Following premature rupture of membranes, which was often preceded by cervical dilation, loss of integrity of the membranes directly exposed the amnionic epithelia surface to the endogenous bacterial microbiome of the cervix.Factors including loss of amniotic fluid with its antibacterial properties, increased physical proximity between the cervix and the product of gestation due to loss of amniotic fluid and negative pressure following uterine contractions all contributed to the spread of the microbiome as a "biofilm" along the amnionic epithelia surface 17 .All four bacteria we listed were anaerobes, and their effect on neonates has yet to be explored.
However, it was also found that Rhodobium, Christensenellaceae, Nonomuraea, Thauera, and Pseudonocardia were detected in cord blood or maternal blood earlier than in fetal membrane and amniotic fluid, which was different from the ascending pattern of microbiomes.The reason might be that these microorganisms reached the fetus through the hematogenous pathway and reversely seeded into the amniotic fluid from the fetus.There have been an increasing number of reports supporting hematogenous invasion of microbes in intrauterine infection in recent years.Riggs et al. found that the hematogenous spread of Mycoplasma pulmonis to the rat fetus can occur without amniotic fluid infection and indicated that the fetus itself can spread the amniotic fluid with microbes 18 .Mendes et al. found that Streptococcus mutans could migrate from saliva to maternal peripheral blood and cord blood and that tooth brushing increased S. mutans detection in blood samples 19 .However, the mechanism by which oral microbial species move from gravidae to the fetus is not yet fully understood.In addition, Fardini et al. found that after intravenous injection of gingival plaque samples, some species selectively had a higher prevalence in the placenta than in the oral cavity, which indicated that sometimes the translocation was species specific 20 .Among the bacteria discovered in our study, Pseudonocardia carboxydivorans was isolated from human cerebrospinal fluid (CSF) from individuals suffering from meningitis, indicating its relationship with infectious Figure 1.Alpha diversity analysis of bacterial 16S rRNA genes was calculated at the OTU level to reflect the differentiation among the duration following rupture of membranes in the fetal membranes, amniotic fluid, cord blood and maternal peripheral blood.The Sobs index of OTU-level abundance (y-axis) is represented by a vertical bar, where each bar indicates a group by the duration following rupture of membranes.In the fetal membrane, the 12 to < 24 h group was more diverse than the < 6, 6 to < 12 and 24 to < 48 h groups according to Wilcoxon's test.
diseases, which should be further studied 21 .Ascending infections should be alerted among women with PROM, which is commonly recognized.However, any systemic maternal infection before term could trigger the onset of labor, and rupture of membranes may be a result of infection instead of the causes 3 .Our study demonstrated that the hematogenous pathway of infection should also be considered in PROM patients.However, the specific microorganisms involved and their morbific abundance or duration are poorly understood.Future studies will focus on the associations between these microorganisms and fetal or neonatal diseases.If such a link is revealed, it may allow targeting of neonatal investigation and treatment.
Changes in the microbiomes of maternal, intrauterine, and fetal environments after membrane rupture have rarely been reported in previous studies.Further investigation to identify how each type of bacteria may be clinically relevant with maternal or fetal infection could provide evidence for clinical interventions for PROM patients, including antibiotic use or earlier induction of labor after membrane rupture for term or near term patients.Methylobacterium, Halomonadaceae and Prevotella could be potential targets for the study of fetal or neonatal infection.In addition, microbiomes may spread in the hematogenous pathway, suggesting the importance of recessive systemic maternal infections in PROM patients.
In our study, 16S rRNA gene amplicon high-throughput sequencing was used to compare the microbiome diversity and signature in maternal, intrauterine, and fetal environments of different durations following membrane rupture, which enabled researchers to further identify potential intrauterine-related bacteria and their possible origins.However, our study has several limitations.The primary limitation was that the blood and intrauterine samples might be more heavily influenced by contaminants produced during the experiment 22,23 .
Figure 2. Beta diversity analysis of bacterial 16S rRNA genes was calculated at the OTU level to reflect the differentiation among the duration following rupture of membranes in the fetal membranes, amniotic fluid, cord blood and maternal peripheral blood.The distance calculated on the OTU level in Bray-Curtis (y-axis) is represented by a vertical bar, where each bar indicates a group by the duration following rupture of membranes.In amniotic fluid, the microbiome of the 24 to < 48 h and ≥ 48 h groups were more diverse than that of the < 6 and 12 to < 24 h groups.
Vol:.( 1234567890 To reduce the impact of this problem, we used the LEfSe algorithm and Kruskal-Wallis test to minimize its impact.During comparisons, the LEfSe algorithm was set to identify microbes that were significantly enriched in the period than in all other periods of membrane rupture, which may result in the absence of some microorganisms due to the algorithm.Besides, information of the pathological examination of placenta, the number of digital examinations were lost, and amniotic fluid has been sampled only in case of c-section, which may cause bias.What's more, part of samples was collected from preterm patients, the difference of gestational age between the group of PROM < 6 h and the group of PROM > 48 h could also cause bias.Last one, although the baseline characteristics of women showed that the rate of antibiotic administration was the same in different groups, however, type of antibiotic administered was not totally the same, which could also cause bias.Further large-scale prospective studies were needed to better investigate these problems.In summary, we found that the diversity of the fetal membrane microorganisms increased significantly after 12 h of membrane rupture, while the diversity of the amniotic fluid microbiome changed after 24 h.Methylobacterium, Halomonadaceae and Prevotella in cord blood changed significantly between the five groups.Source tracking of microorganisms showed that in addition to the common ascending pattern of infection, the hematogenous pathway of intrauterine infection should also be considered in PROM patients.

Methods
Study population.This was a secondary analysis of a case-control study to compare the microbiome signature in the maternal, intrauterine, and fetal environment in women who experienced preterm birth and term birth of the Obstetrics and Gynecology Hospital of Fudan University between March 2014 and December 2014.The eligibility criteria were as follows: (1) singleton birth; (2) gestational week of birth ≥ 28 weeks; (3) the successful acquisition of cord blood for test.The exclusion criteria were as follows: (1) women with demonstrated infection or fever before labor; (2) women with a history of antibiotic use one month before PROM; (3) twin Table 3.The differential bacteria among the duration following rupture of membranes in the fetal membranes, amniotic fluid, cord blood and maternal peripheral blood.Bacteria superscripted with * were detected in fetal membrane or amniotic fluid earlier than in cord blood or maternal peripheral blood.Bacteria superscripted with + were detected in cord blood or maternal peripheral blood earlier than they were detected in fetal membrane or amniotic fluid.After consent was obtained, clinical information was extracted directly from the medical records.Maternal clinical metadata included maternal age, times of gestation and parity, gestational weeks at delivery, maternal comorbidities, temperature during labor, the results of blood count assay (including white blood cell [WBC] and neutrophilic granulocyte [N] percent), duration of membrane rupture, the presence of maternal infection during delivery, the presence of meconium stained amniotic, intrapartum antibiotic administration and mode of delivery.The neonatal outcomes we collected included Apgar scores at 1 and 5 min, admission to the neonatal ward, neonatal infection, and perinatal mortality.Neonatal infection was defined as follows: the blood test of newborn showed that WBC ≥ 50 × 10 9 /L and/or CRP (C-reactive protein) ≥ 20 mg/L within 12-24 h after delivery; WBC ≥ 30 × 10 9 /L and/or CRP ≥ 8 mg/L within 24-48 h after delivery; or the blood culture (before the administration of antibiotics) of newborn was positive.Gestational age was confirmed by first trimester sonogram.The duration of membrane rupture was recorded as the time from the beginning of membrane rupture to the delivery of the newborn.Maternal infection was considered when maternal temperature is greater than 38 °C in addition to two other signs (uterine tenderness, maternal or fetal tachycardia and foul/purulent amniotic fluid) 24 .Screening test of Group B streptococcus was carried out for women at 35 gestational weeks and screening tests of mycoplasma, chlamydia, gonococcus and other common bacteria of cervical secretions were carried out for PROM women at the administration of the labor room.Antibiotics such as second-generation cephalosporin or clindamycin (for patients allergic to penicillin) were given at the presence of suspected maternal infection, preterm birth or group B streptococcus positive.Admission to the neonatal ward was at the discretion of experienced neonatologists.According to the duration of membrane rupture, five groups were established (< 6 h, 6 to < 12 h, 12 to < 24 h, 24 to < 48 h and ≥ 48 h).

Figure 3 .
Figure 3. Differentiation of microbe abundance (mean proportion > 1%) at the genus level among the different durations following rupture of the membrane in the Kruskal-Wallis test.The y-axis represents bacteria at the genus level, and the x-axis represents their mean proportion.(A) Comparison in fetal membranes, (B) comparison in cord blood. https://doi.org/10.1038/s41598-023-41777-z

Table 1 .
Demographic characteristics of women in different groups.*Compared with < 6 h group, p ≤ 0.05.WBC white blood cells, N neutrophilic granulocyte, GDM gestational diabetes mellitus, HDP hypertensive disorders of pregnancy, FGR fetal growth restriction.

Table 2 .
Numbers of samples in each specimen of different durations of membrane rupture.