Microbiome in Preterm Birth

A total of 100 study participants will be included, assigned to one of the following groups:

Study group:

• This group will consist of 50 pregnant women, who experienced pPROM between 22+5 and 28+0 gestational weeks, either presenting at the primary study site, or being referred from other hospitals, and delivered at preterm by cesarean section.

Control group:

• This group will consist of 50 pregnant women, who are scheduled for elective cesarean section at the outpatient department of the primary study site, between a 32+0 and 37+0 gestational weeks, and delivered at term by cesarean section.

Recruitment:

Recruitment of all patients will take place at the Medical University of Vienna, Department of Obstetrics and Gynecology. Women in the study group will be referred from outside hospitals or will present for any reason at our department. Verification of pPROM will be performed by speculum examination and detection of amniotic fluid pooling. In case of unclear findings, an enzymatic test (e.g., Amnisure®, QUIAGEN Sciences, LLC; Germantown, MD 20874, USA) will be conducted. Following pPROM verification, women who meet the inclusion criteria will be offered to participate in the study. Those who experience a spontaneous vaginal delivery instead of a cesarean section due to any reason, will be considered as drop-out. Women in the control group will be recruited during their routine presentation for elective cesarean section (for any reason that does not meet the exclusion criteria of the study) that will be scheduled at term. Those who experience preterm birth although being scheduled for elective cesarean at term, will be considered as drop-out. During their consultation at the outpatient department, these women will be offered to participate in the study.

Sampling:

All swabs will be collected from sub-investigators of this study using a standardized procedure. For anonymization, only the collection time point, location and ward will be marked on the swab tubes. After informed consent, vaginal swabs will be collected during speculum examination from the lateral vaginal wall and posterior fornix vaginae using a sterile cotton swab combined with an epithelial brush. A rectal swab will be collected by insertion of a sterile swab into the anal sphincter. Intraoperative swabs of the placenta and uterine cavity will be collected during caesarean section under sterile conditions. Neonatal swabs (buccal mucosa and skin) will be collected directly after delivery and in the neonatal period. Stool samples will be taken from the meconium, defined as first stool of the infant and the stool of the newborn in the neonatal period. All specimens will immediately be stored at -80°C after collection. The epithelial brush will be put into RNAlater® RNA stabilization solution and stored at -80°C.

Microbiome analysis:

Microbiome analysis will be performed at the Joint Microbiome Facility (JMF) of the Medical University of Vienna and the University of Vienna. Testing will be performed by sub- investigators at the JMF. The microbial community composition in collected stool swab samples will be determined by 16S rRNA gene amplicon sequencing. Briefly, DNA will be extracted with the QIAamp Microbiome Kit or QIAamp DNA Mini Kit (for swab and stool samples, respectively), followed by 16S rRNA gene amplification and barcoding as previously described. Multiplexed amplicon samples sequenced on the Illumina MiSeq platform at the JMF. Negative controls performed during DNA extraction and 16S rRNA gene amplification are routinely included in the sample processing workflow. The obtained sequence data will be quality-filtered and demultiplexed, followed by amplicon sequencing variant (ASV) inference with DADA2,16 enabling analysis at the highest possible taxonomic resolution. Resulting ASV sequences will be taxonomically classified using SINA with the newest release of the the SILVA SSU rRNA database. If necessary, contaminants will be removed in silico using the decontam software package.

Perinatal data:

In addition to swab sampling and analysis, the following perinatal parameters will be collected, using the PIA Fetal Database, version 5.6.16.917 (GE Viewpoint, Munich, Germany): Maternal age [number], parity [number], tertiary education [yes/no], ethnicity [category], relationship status [category], body mass index [number], nicotine abuse [yes/no], history of pPROM [yes/no], history of PTB [yes/no], preexisting diseases [category], vaginal infection screening [yes/no], cervical insufficiency [yes/no], preeclampsia [yes/no], bleeding [yes/no], antenatal steroid prophylaxis [yes/no], ongoing antibiotic treatment [yes/no], tocolysis [yes/no], magnesium prophylaxis [yes/no], gestational week at delivery [number], birthweight [number], Apgar score at 1/5/10 minutes [number], umbilical cord arterial pH [number], transfer to neonatal intensive care unit [yes/no].