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No microorganism was detected in amniotic fluid of healthy pregnancies from the second trimester to the delivery

Microbiome volume 13, Article number: 20 (2025) Cite this article

Abstract

Background

The early colonization and establishment of the microbiome in newborns is a crucial step in the development of the immune system and host metabolism. However, the exact timing of initial microbial colonization remains a subject of ongoing debate. While numerous studies have attempted to determine the presence or absence of intrauterine bacteria, the majority of them have drawn conclusions based on sequencing data from maternal or infant samples taken at a single time point. In this study, we aimed to investigate the microbial population in amniotic fluid (AF) from the second trimester until the time of delivery using multiple microbiological methods.

Methods

AF samples were collected during the second trimester (19–21 gestational weeks) and at the time of delivery. Cohort 1 included 51 women who underwent the term and elective cesarean section, with both their second trimester and delivery AF samples (n = 55, respectively) analyzed. Cohort 2 contained 22 women who experienced infection-related adverse pregnancy outcomes (including preterm birth, histological chorioamnionitis, and stillbirth), with only their second trimester AF samples (n = 24) examined. Additionally, multiple procedural negative controls and technical positive controls were applied to this study to remove potential contamination. Microbial profiles were assessed through cultivation, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and cytokine analysis.

Results

In cohort 1, the bacterial load and community structure in the second trimester AF samples were indistinguishable from negative controls. Although marginally higher bacterial loads and different bacterial communities were observed in the delivery AF samples compared to negative controls, these bacterial DNA were not considered biologically functional due to the absence of maternal inflammatory responses. In cohort 2, the bacterial load and community structure of the second trimester AF samples differed significantly from those of negative controls, with Ureaplasma and Lactobacillus identified as the most prevalent genera against negative controls.

Conclusions

Our study demonstrates that no microorganisms were detected in the AF of healthy pregnancies from the second trimester to the delivery. The presence of Ureaplasma and Lactobacillus in the second trimester AF may be associated with infection-related adverse pregnancy outcomes.

Video Abstract

Background

The initial colonization and establishment of microbiota play a fundamental role in early life development and immune, metabolic, and physiological processes, ultimately impacting long-term health [1]. Despite its importance, the origin and exact time of the first microbial colonizers in humans remain controversial and have significant implications for short and long-term health trajectories. The traditional “sterile womb paradigm” has been challenged by recent studies that have detected unique bacterial communities in the amniotic fluid [2,3,4], placenta [5,6,7], meconium, and uterus [3, 8]. However, contamination from sampling procedures and clinical or laboratory environments are widely recognized as sources of false positive bacterial signals, particularly for low-biomass sample types [9]. Our previous pilot study found that the presence of microorganisms in mid-trimester amniotic fluid samples from healthy pregnancies was indistinguishable from negative control samples [10], which aligns with previous studies that reported the microbiota of amniotic fluid [11, 12] and placenta [13, 14] were indistinguishable from technical controls, putting the topic of “in utero colonization hypothesis” into the controversy [15].

By the end of the first trimester, mucosal immunity is evident, and memory T cells are generated in the human fetal intestine [16], suggesting that the potential presence of intrauterine microbes may influence prenatal immune development. Rackaityte et al. identified bacteria-like morphology in human fetal meconium at mid-gestation by scanning electron microscopy [17], while Mishra et al. found that healthy human fetal tissues in the second trimester contained sparse bacterial biomass and an active memory T cell response toward fetal bacteria. These findings indicate that antigenic priming of the fetal immune system begins during gestation. Complementing these observations, mouse studies revealed viable and cultivatable bacteria in the fetal intestine during mid-gestation but not late gestation, prompting speculation that immune regulation of the maternal–fetal barrier during gestation may influence the presence and biomass of selected microbiota [18]. As pregnancy progresses, the uterine environment undergoes various changes, transitioning from an early pro-inflammatory condition to an anti-inflammatory condition in the second trimester and returning to a pro-inflammatory state before the onset of labor [19]. The majority of studies have attempted to identify the intrauterine microbiota based on samples collected at the time of delivery, and the research of bacterial prevalence and dynamics profile across gestational timelines is warranted. Amniotic fluid (AF) samples could be obtained sequentially during the second trimester when amniocentesis is performed and at the time of delivery, making them an ideal sample type for reflecting intrauterine microbial profiles during pregnancy.

Infectious diseases are one of the common causes of adverse pregnancy outcomes, especially in preterm birth, spontaneous miscarriage, and histological chorioamnionitis. Preterm birth, occurring in 9–12% of pregnancies worldwide [20], is closely linked to spontaneous intra-amniotic infection [21], which is typically caused by the microbial invasion of the amniotic cavity (MIAC). The ascending pathway is considered the most common route of intra-amniotic infection [22]. The Human Microbiome Project recently reported that women who experience preterm birth are less likely to harbor a vaginal microbiota dominated by Lactobacillus crispatus and have a higher concentration of vaginal pro-inflammatory cytokines [23], possibly through mechanisms involving microbiota traveling from the vagina to the uterus [19]. However, studies examining the relationship between intrauterine bacterial profiles in the second trimester and preterm birth are lacking. Besides that, due to the limitations of clinical examination techniques, even if no clinical signs of infection are found in cases of stillbirth or spontaneous miscarriage, potential intrauterine microbial invasion cannot be excluded.

In this study, we applied multiple complementary methods, including bacterial cultivation, quantitative real-time polymerase chain reaction (qPCR), 16S ribosomal RNA (rRNA) gene sequencing, and cytokines detection, to conduct a prospective study (cohort 1) investigating bacterial presence and dynamic profiles in AF samples obtained at the second trimester and the time of delivery from the term, non-labored, and elective cesarean deliveries. In parallel, we retrospectively analyzed the relationship between the microbial profiles in the second trimester AF and pregnancy outcomes in women who underwent infection-related adverse pregnancy outcomes (cohort 2).

Methods

Study design and participants

Women who underwent amniocentesis for prenatal diagnosis were enrolled at 19 ~ 21 weeks of gestation in Peking University First Hospital from May 2018 to January 2020 and followed up until delivery. Exclusion criteria included fetal malformation, clinical infection, or antibiotics use within 2 weeks. This study was reviewed and approved by the institutional ethics committee of Peking University First Hospital (2015[886]), and all the participants provided written informed consent. AF samples were collected during the second trimester when amniocentesis was conducted and the time of cesarean delivery, respectively. Ultimately, 201 women were recruited, and their AF samples were collected in the second trimester, and ultimately 55 AF samples from term, non-labored, and elective cesarean section deliveries were collected at the time of delivery. The subsequent pregnancy outcomes of all the pregnancies were followed, and clinical information was obtained through electronic medical records. In this study, infection-related adverse pregnancy outcomes were defined as spontaneous preterm birth, histological chorioamnionitis, late miscarriage, and stillbirth.

Several types of negative controls were designed to reflect potential contamination, including sterile saline solution collected at the same time as amniocentesis or cesarean section (sampling control, SC, n = 8), DNA extraction kit buffers obtained in the laboratory (extraction control, EC, n = 4), and PCR amplification reagents and DNA-free water (amplification controls, AC, n = 4). To ensure the reliability of the experiment, positive controls were also included, consisting of stool samples (n = 4), vaginal swabs (VS, n = 4), and artificial bacterial communities (ABC, n = 7). Artificial bacterial communities 1–7 contained various Gram-positive and Gram-negative bacteria with known numbers of colony-forming units.

Sample collection and preparation

AF samples were obtained via direct syringe aspiration through intact amniotic membranes at the time of amniocentesis, as well as the time of cesarean section through the surgical incision. Both amniocentesis and cesarean section were conducted in sterile operating rooms, and sample preparation was carried out by trained researchers wearing sterile surgical gowns and gloves. After centrifugation at 1300 × g for 10 min, supernatants were collected and divided into 5 aliquots in a biosafety cabinet for further analysis. Negative controls were collected in the same operating room, following the same experimental procedures during amniocentesis and cesarean section, respectively.

Bacterial culture

Each aliquot of AF samples and negative controls (1–3 ml) was injected into anaerobic and aerobic culture vials (BD BACTET™, Mississauga, Canada), respectively. The whole operation was performed in a biological safety cabinet, and the vials were incubated for 72 h at 35 °C under the manufacturer’s protocol (BD BACTET™). Simultaneously, a genital mycoplasma cultivation assay was also performed according to the manufacturer’s instructions (Autobio Diagnostics Co., Ltd., Zhengzhou, China). The AF samples were transferred into a reconstituted medium and incubated for 24 h at 35–37 °C.

DNA extraction

Following the protocol of the QIAamp DNA Stool Mini kit (Qiagen, Hilden, Germany), DNA extraction was performed in a sterile biological safety cabinet by trained researchers wearing sterile laboratory coats, masks, and sleeves. During the entire experimental process, the extraction of negative controls and AF samples was performed using the same procedure. DNA concentrations were measured with a Qubit 3.0 Fluorometer (Q32866, Life Technologies, Carlsbad, CA) and stored at − 20 ℃.

16S rRNA quantitative PCR

To investigate the bacterial DNA abundance in AF samples, bacterial DNA copy number was detected using TaqMan quantitative PCR of the V3–V4 regions of the 16S rRNA gene. Considering that the concentration of amniotic fluid and negative control samples were extremely low, with some qubit readings below 5 ng/μl, the samples were diluted into 5 ng/μl if their concentrations were over 5 ng/μl, otherwise, the samples were used directly without dilution if their concentrations were below 5 ng/ul. Each reaction contained 10 µl of Premix Taq (2 ×) Mix (Takara, Shiga, Japan), 5 µl of DNA, and 0.0125 nmol of primer (primer-F, 5′-ACTCCTAYGGGRBGCASCAGT-3′; primer-R, 5′-CCTAGCTATTACCGCGGCTGCT-3′;) and probe (probe, 5′−6FAMCGGCTAACTMCGTGCCAGB HQX-3′) [24]. The reaction was performed as previously described [10] with Roche 480 Real-Time PCR instrument and supporting software (Roche, Basel, Switzerland). A plasmid containing the 16S PCR amplicon from E. coli was serially diluted from 107 to 10 copies to generate a standard curve with a slope of − 4.4936 (Fig. 1a), which indicated a qPCR efficiency of 66.93% [25], falling below the ideal range of 80–110%. We mainly attributed the reduced PCR amplification efficiency to the use of primers and probes containing multiple degenerate bases, which aimed to broaden the detection coverage of multitudinous bacterial species. Each sample was amplified in duplicate.

Fig. 1
figure 1

The comparison of 16S rRNA gene copy number among various sample types. a Standard curves for tenfold diluting series (107 copies to 10 copies) of Escherichia coli 16S ribosomal DNA. b Comparison of 16S rRNA gene copy numbers among various sample types. Statistical significance was assessed by the Mann–Whitney U test.  Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Del AF, delivery amniotic fluid in cohort 1; Sec AF in cohort 2, second trimester amniotic fluid in cohort 2; SC, sampling controls; EC, extraction controls; AC, amplification controls; VS, vaginal swab

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16S rRNA gene sequencing and analysis

The 16S rRNA gene V3–V4 region was chosen for Illumina sequencing (Illumina Inc, San Diego, CA, USA) to identify the bacterial taxonomic composition by a 2-step PCR. Extracted DNA was first amplified using digital droplet PCR. Droplet generation, droplet transfer, and plate sealing were performed according to the protocol (Supplementary material 1: Supplementary methods). DNA was amplified with 1 × KAPA HiFi Master Mix (16SAFP02; Coyote, Beijing, China), 0.01 nmol of each primer (primer-F: 5′-CCTAYGGGRBGCA SCAG-3′; primer-R: 5′-GGACTACNNGGGTATCTAAT-3′), and 9 μL of DNA. The reaction conditions were performed with the use of a 96-well PCR instrument (Coyote) as described previously [10], which included 1 cycle at 95 °C for 3 min, followed by 30 cycles of denaturation at 98 °C for 15 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, as well as 1 cycle at 72 °C for 10 min. Then amplification products were purified with VAHTS clean beads (Na44-02; Coyote). After the attachment of barcode adapters (16SAFP03; Coyote), the second PCR was performed under the same conditions, with 8 cycles and an increased annealing temperature of 58 °C for 30 s. Amplicon libraries were purified with VAHTS clean beads (Na44-02) and quantified with a Qubit dsDNA HS Assay Kit (Q32851; Life Technologies). The final library was sequenced with the use of the Illumina HiSeq 2500 platform (Illumina Inc.).

Multiplex bead array assay for cytokines

AF concentrations of the following 6 cytokines were measured with an EMD Millipore Milliplex Kit (LXSAHM-06; Merck Millipore, Burlington, MA, USA) following the manufacturer’s instructions. The cytokines measured in AF samples included interleukin (IL)−1β, IL-2, IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α) to reflect the potential inflammatory response in the amniotic cavity. Standard curves were generated as previously reported [10], and the values of samples were calculated from the curve.

Data analysis

The cycle of quantification (Cq) of the qPCR assay, defined as the number of thermal cycles required for the detection threshold [25], was converted to copy numbers according to the standard curve. All profiles were subsampled to 25,000 reads, under which the minimum value of good coverage was 0.9918, ensuring the coverage of sequencing data. Using the Greengenes database, sequences of the 16S rRNA gene were clustered using QIIME with 97% nucleotide similarity and taxonomic classification. Bacterial compositions were visualized with a heat map, which was generated via Seaborn, a Python data visualization library. Alpha diversity was evaluated with the Chao1, Shannon, and Simpson indexes. Beta diversity was assessed by unweighted UniFrac distance matrices and visualized by principal coordinates analysis, with 1000 permutations, and statistically calculated by the nonparametric multivariate analysis of variance methods with the use of the Adonis function [26], included in the R package vegan (http://CRAN.R-project.org/package=vegan). The metric variable was shown as the mean ± standard deviation or median (interquartile range) and compared by Student’s t test or Mann–Whitney U test according to the normality of the data distribution. Chi-square and Fisher’s exact tests were used to compare of proportions of analytes. A probability value of P < 0.05 was considered significant. GraphPad Prism (version 7.0; GraphPad Software, San Diego, CA, USA) was used for the statistical and graphic analyses.

Results

Participant characteristics

A total of 194 AF samples were obtained in the second trimester. At the time of delivery, 55 AF samples from 51 pregnancies (including 47 single pregnancies and 4 twin pregnancies) of term, non-labored, and elective cesarean section deliveries were collected, along with 55 AF counterparts from the second trimester, forming Cohort 1. Twenty-two women (including 20 single pregnancies and 2 twin pregnancies) experienced infection-related adverse pregnancy outcomes—1 with twin was stillborn at 22 gestational weeks, three were preterm birth (one with twin was extremely preterm birth at 27 gestational weeks, while 2 were late preterm birth at 35 and 36 gestational weeks respectively), 13 were histological chorioamnionitis, and 5 women suffered both histological chorioamnionitis and preterm birth (2 of them were moderate preterm, while 3 of them were late preterm). However, the AF samples were lost at the time of delivery, and their 24 AF samples from the second trimester were included in cohort 2 (Fig. S1).

The demographic and clinical characteristics of the two cohorts are shown in Table 1. Gravidity, gestational age at amniocentesis, the ratio of twins, and infant gender were comparable between the two cohorts, while maternal age, parity, gestational age at delivery, birth weight, and cesarean section rate were significantly different between cohort 1 and cohort 2, which were intrinsically related to pregnancy outcomes.

Table 1 Participant characteristics
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No bacteria found by bacterial culture

All AF samples and sampling negative controls were performed in aerobic and anaerobic cultures, as well as genital mycoplasma cultivation. Among the 134 AF samples and 8 samplings of negative control, only one second trimester AF sample (subject 45) yielded Paenibacillus lactis in aerobic conditions. However, no bacteria was found in its delivery AF counterpart, and the subject 45 did not suffer any adverse pregnancy outcome. As Paenibacillus lactis is a common environmental contaminant in clinical culture, we are inclined to confirm positive results in the sequencing data.

Amniotic fluid has low bacterial DNA abundance

To quantify the microbial DNA abundance in AF samples, the qPCR assay was used to measure the copy numbers of the 16S rRNA gene. A standard curve over a range of 10–107 gene copies was generated by the linear regression analysis of an E coli plasmid (Fig. 1a, slope =  − 4.4936; R2 = 0.99), and the copy number of each sample was calculated based on the mean Cq value and the regression equation of standard curve (y =  − 44936x + 43.414). We also converted the calculated results according to the amplification efficiency. Compared with stool samples (med = 1.5 × 107 copies/μL, min = 1 × 107 copies/μL, max = 5.1 × 107 copies/μL) and vaginal swabs (med = 3.7 × 105 copies/μL, min = 1.2 × 105 copies/μL, max = 6.2 × 105 copies/μL), AF samples contained extremely low bacterial DNA abundance (Fig. 1b, med = 67 copies/μL, min = 21 copies/μL, max = 1401 copies/μL for the second trimester AF in cohort 1, med = 207 copies/μL, min = 67 copies/μL, max = 4950 copies/μL for the delivery AF, med = 180 copies/μL, min = 64 copies/μL, max = 92,400 copies/μL for the second trimester AF in cohort 2). Furthermore, the 16S rRNA gene copy number was assessed in negative control samples. There was no significant difference between the second trimester AF in cohort 1 and the sampling controls (med = 52 copies/μL, min = 30 copies/μL, max = 125 copies/μL; Mann–Whitney U test; U = 81; P = 0.4038), while the second trimester AF in cohort 1 contained higher numbers of 16S gene copies than the extraction controls (med = 25 copies/μL, min = 7 copies/μL, max = 80 copies/μL, U = 56; P < 0.01) and amplification controls (med = 17 copies/μL, min = 5 copies/μL, max = 50 copies/μL; U = 24; P < 0.0001), indicating that the potential contamination may occur during the sampling procedure. The delivery AF showed a significant increase in bacterial DNA copies in comparison to the second trimester AF counterparts (U = 389; P < 0.001), which suggested the varying bacterial DNA abundance as the pregnancy progressed, but still with extremely low DNA abundance. Interestingly, the numbers of 16S gene copies of the second trimester AF in cohort 1 were significantly lower than that of cohort 2 (U = 226; P < 0.0001). Since the copy number of the 16S rRNA gene was considerably low in negative control and AF samples, we attempted to confirm these results in the sequencing data.

The bacterial community structure of the second trimester AF in cohort 1 is indistinguishable from negative controls

The bacterial DNA of all the AF samples and control samples were assessed by 16S ribosomal RNA gene sequencing. As the positive control samples, the taxonomic composition and relative abundance of each artificial bacterial community were consistent with expectation (Fig. S2), which guaranteed the reliability and stability of sequence data in this study.

At first, we compared the read counts of second trimester AF and delivery AF in cohort 1, as well as negative and positive control samples. The second trimester AF had a median read count of 10 (Fig. 2a, min = 0, max = 32), which was much lower than that of stool (med = 68,799, min = 53,033, max = 97,564, P < 0.0001) and vaginal swab samples (med = 26,914, min = 7658, max = 70,343, P < 0.0001). No difference was found between the second trimester AF and sampling controls (med = 7, min = 3, max = 10, P = 0.29), extraction control (med = 2.5, min = 1, max = 37, P = 0.28), or amplification control (med = 2, min = 0, max = 30, P = 0.06). The read counts of the second trimester AF were slightly lower than those of the delivery AF counterparts (med = 10, min = 2, max = 55, P = 0.04), and the latter was higher than that of the amplification control (P < 0.01).

Fig. 2
figure 2

The analysis of 16S rRNA gene sequencing. a The comparison of 16S rRNA read counts between sample types. b The comparison of 16S rRNA OTU numbers between sample types. c The comparison of alpha diversity (Chao1, Shannon, and Simpson indices) between sample types. d Principal coordinates analysis based on unweighted UniFrac distance is shown along the first two principal coordinate (PC) axes, and percentages are the percent variation explained by each PC axis. e The comparison of between-group UniFrac distance of second trimester AF and delivery AF in cohort 1. Statistical significance was assessed by the Mann–Whitney U test. Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Del AF, delivery amniotic fluid in cohort 1; Sec AF in cohort 2, second trimester amniotic fluid in cohort 2; SC, sampling controls; EC, extraction controls; AC, amplification controls; VS, vaginal swab

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After annotation to bacterial operational taxonomic units (OTUs), we found similar results, showing that the OTU numbers of the second trimester AF (Fig. 2b, med = 7, min = 0, max = 16) were much lower than those of stool (med = 1011, min = 952, max = 1107, P < 0.0001) and vaginal swab samples (med = 56.5, min = 29, max = 68, P < 0.0001). However, they did not differ significantly from any negative controls (P > 0.05 for all comparisons). The OTU numbers of the delivery AF (med = 8, min = 2, max = 23) were significantly higher than those of the second trimester AF (P < 0.01), sampling control (P = 0.01), and amplification control (P = 0.01). Furthermore, we analyzed within-sample diversity and found that the alpha diversity (Fig. 2c; Chao1, Shannon, and Simpson indices) of the second trimester AF were comparable to that of sampling control (P = 0.25, P = 0.09, and P = 0.11, respectively), extraction control (P = 0.41, P = 0.40, and P = 0.41, respectively), and amplification control (P = 0.26, P = 0.19, and P = 0.21, respectively), and were significantly lower than that of stool samples (P < 0.0001 for all). Also, we found that the Chao1, Shannon, and Simpson indices of the delivery AF samples were significantly higher than those of the second trimester AF counterparts (P = 0.02, P < 0.01, and P = 0.01, respectively).

To investigate the bacterial community composition, the beta diversity of all samples was investigated by performing principal coordinate analysis (PCoA) based on unweighted Unifrac distance. As shown, the second trimester AF clustered with all negative controls (Fig. 2d, P = 0.12 for sampling control, P = 0.08 for extraction control, and P = 0.06 for amplification control), and were distinct from stool and vaginal swab samples (P = 0.01 and P < 0.01, respectively). The delivery AF samples were different from negative controls (P = 0.002 for sampling control, P = 0.027 for extraction control, and P = 0.002 for amplification control), and distinct from stool (P = 0.001) and vaginal swab (P = 0.025). Furthermore, we found the delivery AF samples appeared to cluster separately from the second trimester AF counterparts (P < 0.01), indicating varying bacterial communities from the second trimester to the time of delivery. In addition, we calculated the unweighted UniFrac distances to quantify the community similarity between sample types (Fig. 2e). Compared with the second trimester AF, the community distance between delivery AF and vaginal swab were significantly smaller (P = 0.001), suggesting a gradually increased similarity to vaginal microbiota as the cervix shortens and the cervix mucus plug is lost near delivery.

Dynamic changes of predominant bacterial OTU across gestational timeline

Given the differences in microbial community structure between the second trimester AF samples and the delivery AF counterparts, we identified the specific OTU associated with bacterial genera based on the average abundance and distribution in each group, respectively. As shown, we highlighted the enrichment of 8 bacterial OTU genera specifically predominant in the second trimester AF, including Listeria, Enterococcus, Streptococcus, Bacteroides, Propionibacterium, Megasphaera, Prevotella, and Sphingomonas (Fig. 3a). Additionally, we identified 8 bacterial OTU genera specifically predominant in delivery AF, including Lactobacillus, Ureaplasm, Bacteroides, Streptococcus, Listeria, Enterococcus, Erwinia, and Gardnerella (Fig. 3b).

Fig. 3
figure 3

The distribution of specific bacterial genera in second trimester AF and delivery AF in cohort 1. a Bacterial genera predominant and high in second trimester AF samples are depicted as “second trimester AF-specific genera”. b Bacterial genera predominant and high in delivery AF samples are depicted as “delivery AF-specific genera”. Dot-plot showing the distribution and abundance of each specific genera in both second trimester and delivery AF samples. Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Del AF in cohort 1, delivery amniotic fluid in cohort 1

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The prevalence and abundance of the second trimester AF-specific OTU genera were significantly decreased in the corresponding delivery AF (Fig. 3a), and the second trimester AF-specific OTU genera showed a homogeneous distribution among samples, suggesting systemic contamination. On the other hand, the delivery of AF-specific OTU genera was rare in the corresponding second trimester AF, and some of them were common in the vagina, prompting the hypothesis that microbiota might travel from the vaginal to the amniotic cavity through the ascending pathway as the cervix shortens and cervix mucus plug is lost near the delivery.

The specific bacterial OTUs of the second trimester AF in cohort 2 resemble vaginal microbiota

Microbial invasion of the amniotic cavity (MIAC) has been closely linked to obstetrical complications, including spontaneous preterm birth, late miscarriage, preterm premature rupture of membranes (PPROM), histological and clinical chorioamnionitis, and stillbirth [27]. In this study, we further retrospectively analyzed the second trimester AF from pregnancies who suffered infection-related adverse pregnancy outcomes. Firstly, the read counts of the second trimester AF in cohort 2 were much more variable, with a median read count of 12 (Fig. 2a, min = 2, max = 63,759), which were higher than those of the second trimester AF in cohort 1 (P = 0.02), and much lower than those of stool (P < 0.001) and vaginal swab samples (P < 0.001). Similarly, the OTU numbers of the second trimester AF in cohort 2 (Fig. 2b, med = 9, min = 2, max = 117) were higher than those of the second trimester AF in cohort 1 (P < 0.01), sampling control (P = 0.03), and amplification control (P = 0.01), and also lower than those of stool (P < 0.0001) and vaginal swab samples (P < 0.01), indicating significant enrichment of bacterial content in amniotic cavity of symptomatic pregnancies at the second trimester.

Regarding within-sample diversity, the second trimester AF in cohort 2 had slightly higher alpha diversity than the second trimester AF in cohort 1 (Fig. 2c, P < 0.05 for Chao1 index, P = 0.09 for Shannon index, and P = 0.33 for Simpson index, respectively), and lower than that of stool (P < 0.0001, P < 0.001, P < 0.01, respectively) and vaginal swab (P < 0.01, P < 0.01, and P < 0.01, respectively), as expected.

Overall, the microbial community structure of the second trimester AF in cohort 2 was distinguishable from those of stool (Fig. 4a, P = 0.001) and negative controls (P = 0.01 for both sampling control and amplification control). When compared with the second trimester AF in cohort 1, the second trimester AF in cohort 2 showed a more variable and dissimilar distribution (P = 0.001). Notably, no significant difference was observed between the second trimester AF in cohort 2 and vaginal swabs in the community structure (P = 0.06). These results were further consolidated by comparison of between-sample unweighted UniFrac distances. The UniFrac dissimilarity between the second trimester AF in cohort 2 and sampling control was significantly higher than that of the second trimester AF in cohort 1 and sampling control (Fig. 4b, P = 0.019), while the distance between the second trimester AF in cohort 2 and vaginal swab was significantly lower than that of the second trimester AF in cohort 1 and vaginal swab (P = 0.021), which stimulated the speculation that an ascending microbial colonization of the intrauterine cavity may have occurred at the second trimester.

Fig. 4
figure 4

The analysis of 16S rRNA gene sequencing. a Principal coordinates analysis based on unweighted UniFrac distance is shown along the first two principal coordinates (PC) axes, and percentages are the percent variation explained by each PC axis. b the comparison of between-group UniFrac distance of second trimester AF in cohort 1 and second trimester AF in cohort 2. Statistical significance was assessed by the Mann–Whitney U test. Sec AF in cohort 1, second trimester amniotic fluid in cohort 1; Sec AF in cohort 2, second trimester amniotic fluid in cohort 2; SC, sampling controls; EC, extraction controls; AC, amplification controls; VS, vaginal swab

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To identify the bacterial OTUs unique to the second trimester AF samples in cohort 2, the sequence data were filtered by the following criteria: (1) absence in negative controls and the second trimester AF in cohort 1; (2) presence in at least two AF samples. Eventually, 9 bacterial OTUs at the genus level were identified, including Ureaplasma, Lactobacillus, Turicibacter, Bradyrhizobium, Streptococcus, Gardnerella, Ruminococcus, Anaeroplasma, and Mucispirillum (Table 2). Ureaplasma was the most abundant and predominant genus, found in 13 AF samples from 12 women. Subject 94, who harbored the highest number of Ureaplasma reads (a total of 62,819), underwent spontaneous preterm birth at 34 gestational weeks with intact membrane and histologic chorioamnionitis, but cervicovaginal fluid and AF culture were negative. The pregnancy outcomes of women with these 9 bacterial OTUs are described in Table 2.

Table 2 Clinical complications of pregnancies who harbor bacterial OTU in cohort 2
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The second trimester AF in cohort 2 has moderately high concentrations of inflammatory cytokines

To determine the potential inflammatory response in the amniotic cavity, the profile of 6 cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)−6, IL-8, IL-10, IL-1β, and IL-2, were further investigated. Generally, in all AF samples, the levels of all cytokines were considerably low, and the data that did not meet the measurement threshold was removed. In cohort 1, no significant difference was found between the second trimester AF and delivery AF regarding the 6 cytokines. These results suggested that the bacterial OTU might be non-biologically functional in the absence of maternal inflammation responses. When compared with the second trimester AF in cohort 1, the levels of TNF-α, IL-10, and IL-1β were significantly higher in the second trimester AF in cohort 2, while the levels of IL-2, IL-6, and IL-8 were comparable between them (Table 3).

Table 3 Cytokines concentrations in AF samples
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Discussion

By applying complementary approaches and multiple experimental controls, we aimed to investigate bacterial presence in the intrauterine environment and the dynamics of the bacterial DNA profiles from the second trimester to the time of delivery, as well as bacterial and inflammatory profiles in AF associated with infection-related adverse pregnancy outcomes. The main findings in our study were as follows: (1) both anaerobic and aerobic cultivation of AF samples did not yield viable bacteria in any cases; (2) the microbial loads and community structure of the second trimester AF in healthy pregnancies were both indistinguishable from negative controls. Although delivery AF had marginally higher bacterial loads and different microbial communities compared to negative controls, the absence of maternal inflammation responses suggested that the bacterial OTU were non-biologically functional; (3) the bacterial loads and microbial community structure of the second trimester AF of women who suffered infection-related adverse pregnancy outcomes were different from negative controls, which were dominant by Ureaplasma and Lactobacillus.

The relationships between host-microbe range from mutualism to commensalism, to pathogenesis, and these relationships are extensively involved in host responses. However, our understanding of how and when microorganisms occur and colonize is incomplete. The long-held “sterile womb” view has been challenged by sequence-based intrauterine microbiome studies. A key report published in 2014 claimed that placenta tissue from uncomplicated pregnancy harbored a unique microbiome [28], which was similar to the microbiota of the human oral cavity through 16S rRNA gene sequencing, as well as metagenomics sequencing in a subset of samples. This publication sparked a wave of uterine microbiota research, involving the placenta [5, 7, 13, 14, 29,30,31], amniotic fluid [11, 12, 32, 33], uterus tissue [3, 34, 35], and meconium [8] samples. A considerable part of subsequent studies reported that there was detectable microbiota in the uterus based on 16S rRNA gene sequencing and/or metagenomics, while more and more scientists questioned those positive findings for the absence of adequate controls and the possibility of unavoidable background DNA contamination during the whole experimental process [36,37,38,39]. The findings were challenged by de Goffau et al., who conducted a comprehensive study containing 537 participants and found a range of species that are common in the vagina, such as Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii. After accounting for the presence of vaginal microorganisms and those in the laboratory reagents, there was no evidence to support that the placenta is colonized by microorganisms in healthy pregnancies [29], concluding that the presence of any detected bacteria was associated with contaminated DNA and/or batch effect.

It is undisputed that the placenta or AF cannot contain a concentration of microbiota as high as that found in the oral cavity, vagina, and gut. Contamination is always a potential confounder in microbial studies, especially concerning low- or no-microbial biomass samples [9, 38, 40]. It is challenging to distinguish bacterial DNA from background noise. Therefore, it is necessary to include multiple controls during the whole experimental procedure. There is no gold standard for recognizing a real bacterial DNA from contamination but removing all sequences in negative control may result in the loss of authentic signals. In this study, we designed not only negative (from sampling to extraction, to amplification) and positive (stool and vaginal swab) controls but also seven artificial bacterial communities, which consisted of both Gram-positive and Gram-negative bacteria. The Gram-positive bacteria served as quality controls, as their high mechanical strength of the cell wall is less likely to be affected by extraction methods [41, 42]. The consistency between the expected and detected abundance of Gram-positive bacteria can ensure the feasibility of the extraction method applied in this study (Fig. S2). In line with Lim et al. [12], the 16S rRNA gene copy numbers in AF were extremely low when compared with stool samples and vaginal swabs. The results of qPCR showed that the microbial abundance of the second trimester AF of healthy pregnancies was similar to that of sampling controls, but higher than extraction and amplification controls, raising the possibility that sampling contamination may be the most important confounder.

In our study, we identified a marginally higher bacterial load in the delivery AFs compared to the second trimester AF counterparts. In the late pregnancy trimester, AF contained fetal hair, meconium, and fetal sebum, leading to more efficient DNA extraction. This DNA carrier effect might offer an alternative explanation for the higher bacterial load in AF at delivery than in the second trimester in the same woman. Two of the delivery AF-enriched genera were Lactobacillus and Ureaplasm. As the predominant microorganisms in the vagina, their abundances were only slightly higher than those in the second trimester AF counterparts. It was hard to determine the authenticity of those bacteria, as the shortening of cervical length and loss of cervical mucus plug might breach the balance between long-term colonizers in the vaginal mucosal and amniotic-chorionic membranes barrier near delivery. However, it was certain that those microorganisms were of low- or non-pathogenic potential for the absence of maternal inflammatory response, which allowed healthy pregnancy outcomes.

Fetal immune maturation is a key factor in lifelong health [43]. Recent studies reported that the presence of intrauterine microbiota contributes to the maturation and development of the fetal immune system. In this study, we identified several bacterial OTU of vaginal microorganisms in the second trimester AF of women who suffered infection-related adverse pregnancy outcomes against negative controls, but no evidence supported the presence of living microorganisms by bacterial culture assay. To identify the biological function of those bacterial OTUs, we further investigated the profile of cytokines in AF samples. We found a significant increase of TGF-α, IL-1β, and IL-10 in the second trimester AF in cohort 1 compared with cohort 2, although the levels of these cytokines were far below the cut-off value of intra-amniotic inflammation. Intra-amniotic inflammation was diagnosed when the AF IL-6 concentration was ≥ 2.6 ng/ml [44, 45], and none of the AF samples met this threshold. It should be pointed out that the level of IL-10 in second trimester AF in cohort 2 far exceeded that in cohort 1. As a pleiotropic anti-inflammatory cytokine, IL-10 down-regulates immune response by inhibiting IL-6 and IL-1β, acting as a crucial contributor to the balance of anti- and pro-inflammatory mediators that results in proper pregnancy outcomes [46, 47]. Payne et al. found that elevated amniotic fluid IL-10 (defined as > 10.1 pg/mL) was significantly associated with pre-labor rupture of membranes in Chinese pregnancies [48], while Thaxton et al. showed that poor IL-10 production may be a trigger for preterm birth [49]. Despite the controversial findings regarding IL-10 concentration, the IL-10 dramatic elevation indicated disturbances of immune response coupled with adverse pregnancy outcomes in cohort 2. Unfortunately, no AF samples in cohort 2 were collected at the time of delivery, making it hard to define the pathogenicity of microbial OTU in the second trimester AF in cohort 2. Interestingly, we indeed detected the existence of Ureaplasma and Lactobacillus in subject 208, who suffered stillbirth, but no pathogenic causes or infection signs were found through routine clinical examination. Although it is hard to conclude that the Ureaplasma and Lactobacillus were the exact cause of stillbirth, our finding could motivate the investigations on the causal relationship between intrauterine microbiota and adverse pregnancy outcomes.

Conclusions

By combining the complementary results of culture, qPCR, 16S rRNA gene sequence, and cytokines analysis, we concluded that no bacteria were found to be distinguishable from negative controls in the second trimester AF in healthy pregnancy. A marginally higher bacterial load in the delivery AF counterpart was not biologically functional. The presence of Ureaplasma and Lactobacillus in the second trimester AF might be responsible for the infection-related adverse pregnancy outcomes.

Data availability

The datasets used and/or analyzed during the current study are available from the China National Center for Bioinformation (CNCB), under HRA007453 (https://ngdc.cncb.ac.cn/gsa-human/browse/HRA007453).

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Acknowledgements

We acknowledge Mr. Yichen Liu for contributing to the 16S rRNA gene sequencing data analysis and Mr. Jiming Yin (Capital Medical University Affiliated Beijing You An Hospital, Beijing, China) for technical support with the multiplex bead array assay.

Funding

The research was supported by the National Key Research and Development Program of China (No. 2021YFC2700700).

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Author notes

  1. Yu Liu and Jingmei Ma contributed equally to this work.

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Peking University First Hospital, Beijing, China

    Yu Liu, Jingmei Ma, Yulong Tong & Huixia Yang

  2. Beijing Key Laboratory of Maternal Fetal Medicine of Gestational Diabetes Mellitus, Beijing, China

    Yu Liu, Jingmei Ma, Yulong Tong & Huixia Yang

  3. Women and Children’s Hospital, Chongqing Medical University, Chongqing, 401147, China

    Yu Liu

  4. COYOTE Medical Laboratory, Beijing, China

    Xiang Li, Haijuan Zhao, Qubo Ai & Lanying Zhang

  5. Department of Laboratory, Peking University First Hospital, Beijing, China

    Lingzhen Meng

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Contributions

Y.L., J.M., and H.Y. conceived the study design. Y.L. and Y.T. were responsible for the recruitment and collection of samples. X.L., H.Z., Q.A., and L.M. were responsible for the laboratory assays. Y.L. and L.Z. performed the data analysis. Y.L. and J.M. completed the initial manuscript. H.Y. revised the manuscript. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Huixia Yang.

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This study was reviewed and approved by the institutional ethics committee of Peking University First Hospital (2015[886]), and all the participants provided written informed consent.

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Supplementary Information

Supplementary Material 1. Supplementary methods.

40168_2024_2024_MOESM2_ESM.tif

Supplementary Material 2. Figure S1. Study Design Overview. Flowchart of prospective cohort study. The “n” means the number of amniotic fluid sample.

40168_2024_2024_MOESM3_ESM.tif

Supplementary Material 3. Figure S2. The detected compositions and relative abundances of 7 artificial bacterial communities according to 16S ribosomal RNA gene sequencing is consistent with expectation. ABC, artificial bacterial community.

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Liu, Y., Ma, J., Li, X. et al. No microorganism was detected in amniotic fluid of healthy pregnancies from the second trimester to the delivery. Microbiome 13, 20 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s40168-024-02024-3

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Microbiome

ISSN: 2049-2618

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