Evidence for contamination as the origin for bacteria found in human placenta rather than a microbiota

Abstract

Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota.

Fig 1. Quantity of bacteria (A-C) and number of different species (D-F) found in fetal membranes, umbilical cords and chorionic villi collected after cesarean section using an Alexis^® (CSA group; A, D), cesarean section (CS group; B, E) or vaginal delivery (VD group; C, F).

CFU: colony forming unit; CSA: cesarean section using an Alexis^®; CS: cesarean section; VD: vaginal delivery; M: fetal membranes; U: umbilical cord; V: chorionic villi. ns: non-significant; *: P < 0.05; **: P < 0.01.

Fig 2. Prevalence (%) of each bacterial species/genus in fetal membranes (A), umbilical cords (B) and chorionic villi (C).

Samples that did not harbor any bacteria were removed. The higher diversity was observed in external samples (fetal membranes and umbilical cords) coming from vaginal delivery. CSA: cesarean section with Alexis^®; CS: cesarean section; VD: vaginal delivery; Centre 1: Port Royal maternity; Centre 2: Antoine Béclère Hospital maternity; Centre 3: Sainte Félicité maternity.

Fig 3. 16S rRNA gene copy number in fetal membranes, umbilical cords, chorionic villi and extraction blanks collected after C-section with (A) or without (B) an Alexis^® or after vaginal delivery (C).

CSA: cesarean section using an Alexis^®; CS: cesarean section; VD: vaginal delivery; M: fetal membranes; U: umbilical cord; V: chorionic villi. ns: non-significant; **: P < 0.01; ***: P < 0.0001.

Fig 4. Example of microbial composition in negative control (A, C, E) or chorionic villi (B, D, F) using different databases for analysis with KrakenUniq.

DNA was found in both negative control and chorionic villi and was highly variable depending on the database. Databases used: RefSeq v24 (A, B); RefSeq v48 (C, D); NCBi ‘nt’ (E, F).