Table 1.

Patient demographics, study design, and profile results of current literature describing next-generation sequencing studies of uterine microbiota in chronological order.

	Patient population and objective	Patient cohort (subjects and age)	Race and/or ethnicity	BMI	Sample type analyzed and pH collected	Procedures to avoid contamination from vagina or cervix?	Contamination controls and type	Sequencing platform and variable region	Top identified phyla	Summary of findings
Mitchell et al. (28)	Patient population: women undergoing hysterectomy for non-cancer indicationsObjective: to evaluate the presence of vaginal bacterial species in the uterus	58 subjects Average age: 43 No use of antibiotics within the last 30 days	White: 79% African American: 10% Hispanic: 7% Declined to answer: 3%	NR	Endometrial swabs from excised uterus Vaginal swabs collected before hysterectomy pH: NR	Specimens were collected only if the surgeon was able to complete the procedure using a noninvasive vaginal fornix delineator (Colpo-Probe; Cooper Surgical, Trumbull, CT, USA) or a vaginal sponge stick rather than an intracervical manipulator	NR	qPCR for 12 species	Firmicutes Bacteroidetes	- 95% of subjects had upper genital tract colonization The three most common species in uterus were L. iners, Prevotella spp., and L. crispatus - No significant difference in soluble markers of inflammation in endometrial swabs between women with (a) no bacteria, (b) only Lactobacillus species, or (c) detectable non-Lactobacillus species pH: NR
Franasiak et al. (48)	Patient population: women undergoing IVFa Objective: to assess the impact of microbiome obtained from IVF catheter tip at the time of embryo transfer on pregnancy outcome following IVF	33 subjects Average age: 35.9 Antibiotics: NR	Caucasian: 79% Asian: 15% African American: 3% Hispanic: 3%	NR	Distal 5 mm of IVF catheter tip pH: NR	Formable outer sheath advanced under ultrasound guidance	Positive controls utilizing Escherichia coli along with negative controls were run to detect contamination from reagents	The Ion 16S Metagenomics Kit (V2–4–8 and V3–6, 7–9)	Firmicutes Proteobacteria	- Lactobacillus was the top genus found on the IVF catheter tip in both successful and unsuccessful IVF outcomes - Flavobacterium was the second most prevalent genus found across both groups - There were no characteristic differences in microbiomes between successful and unsuccessful IVF groups pH: NR
Verstraelen et al. (50)	Patient population: women with various reproductive conditions (recurrent implantation failure, recurrent pregnancy loss, or both) but no uterine abnormalities and a diverse medical history Objective: to determine the presence of a uterine microbiome in non-pregnant women	19 subjects Median age: 32 No perioperative antibiotic treatment	White: 100%	NR	Tao BrushTM IUMC Endometrial Sampler pH: NR	Cervical surface and external os were thoroughly rinsed with an aqueous 0.5% chlorhexidine gluconate solution (antiseptic and disinfectant). Tao BrushTM IUMC Endometrial Sampler protected by a plastic covering sheath laterally and by a small plastic bead on top to protect the brush on all sites from contamination during passage through the vaginal lumen and endocervical canal	NR	Illumina (V1–V2)	Bacteroidetes	90% of the subjects had uterine microbiomes in which Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides fragilis, and an undetermined Pelomonas taxon made up over a third of the total pH: NR
Fang et al. (49)	Patient population: women with EP and “healthy” asymptomatic women with partners with MFI Objective: to determine the difference between uterine microbiota composition between EP, EP + CE, and “healthy” asymptomatic controls	30 subjects Average age: H: 30.90 EP + CE: 35.2 EP: 34.4 No antibiotic use within last 3 weeks	NR: study conducted in China	H: 21.04 ± 1.03 EP: 20.47 ± 0.67 EP + CE: 21.29 ± 0.99	Vaginal swabs and endometrial swabs collected pH: NR	Vaginal and cervical canal disinfection Endometrial swabs with sleeves	NR	Illumina (V4)	Proteobacteria Firmicutes	- Subjects with EP and EP/CE had microbiomes with much higher proportions of Firmicutes than healthy subjects - At the genus level, Lactobacillus, Gardnerella, Bifidobacterium, Streptococcus, and Alteromonas were significantly higher in the healthy group compared with either the EP or the EP/CE group - Enterobacter and Sphingomonas were found at lower proportions and Prevotella at a higher proportion in the EP/CE group pH: NR
Khan et al. (53)	Patient population: healthy asymptomatic women operated on for dermoid cyst/serous cyst adenoma/mucinous cyst adenoma or for uterine myoma and women with endometriosis. Both groups were further divided into GnRHa treated and GnRHa-untreated Objective: to assess the impact of endometriosis and/or GnRHa treatment on the intrauterine microbiome	32 subjects Average age: Control GnRHa−: 33.6 Control GnRHa+: 42.1 Endometriosis GnRHa−: 35.7 Endometriosis GnRHa+: 37.5 Antibiotics: NR	NR: study conducted in Japan	NR	Seed swabs were used to collect endometrial samplesCystic fluid was collected during laparoscopy pH: NR	Seed swab was inserted under visual control into the uterine cavity taking care to avoid any contact with vaginal walls	NR	Illumina (not specified)	Firmicutes Proteobacteria	- Lactobacillaceae were significantly decreased in women with endometriosis being treated with GnRHa compared with without endometriosis but were also treated with GnRHa - Streptococcaceae, Staphylococcaceae, and Enterobacteriaceae were significantly increased in women treated with GnRHa compared with women without endometriosis but were also treated with GnRHa pH: NR
Moreno et al. (47)	Patient population: women undergoing IVF 19–29 kg/m2 whom had at least one good-quality embryo transferred but had not used antibiotics within the last month before the study Objective: to determine the impact of the uterine microbiome obtained from IVF catheter tip at the time of embryo transfer, and its hormonal regulation by collected endometrial fluid at 2 days after luteinizing hormone surge as well as 7 days after, on reproductive out in those undergoing IVF	Subject numbers: Impact of uterine microbiome on reproductive success: 35 Impact of hormonal regulation on uterine microbiome: 22 Comparison of vaginal microbiome and uterine microbiome: 13 Average age: LD: 40.06 NLD: 39.00 No antibiotics or probiotics used within the last month	NR: study conducted in Spain as part of the ovum donation program	LD: 24.18 ± 5.18 NLD: 22.45 ± 4.02	Endometrial fluid collected with catheter inserted transcervically pH: endometrial	To prevent contamination by cervical mucus during catheter removal, suction was dropped at the entrance of internal cervical oss (ICO), and cervical mucus was also aspirated before EF aspiration	NR	454 Pyrosequencing (V3–5) Firmicutes Actinobacteria		- Uterine microbiota did not differ at two timepoints in the hormonal cycle - The presence of a non-Lactobacillus-dominated uterine microbiota in a receptive endometrium was associated with significant decreases in implantation, pregnancy, ongoing pregnancy, and live birth rates pH: endometrial pH not associated with microbiota composition or reproductive outcome
Walther-António et al. (35)	Patient population: women undergoing hysterectomy for either benign uterine conditions, endometrial hyperplasia or endometrial cancer Objective: to determine uterine microbiome in patients with and without endometrial cancer	31 subjects Median age: Benign: 44.5 Cancer: 64 Hyperplasia: 54 No antibiotic 2 weeks prior	Caucasian: 100%	Median: Benign: 26.6 Cancer: 32.1 Hyperplasia: 35.4	Uterine, fallopian tube, ovary, and peritoneal swabs following hysterectomy. The uterus, vagina and cervix also had scrapes taken. Urine and stool samples were also taken pH: vaginal		A total of 14 controls were performed, with five of them not retrieving any sequence readsA Petri dishwith Lysogeny broth was kept open on the grossing station during sample collection to detect any possible airborne contamination of the specimen (findings NR)	Illumina (V3–5) Proteobacteria Bacteriodetes		- Vaginal, cervical, fallopian tube, and ovary microbiomes are significantly correlated within an individual - Atopobium vaginae and a Porphyromonas sp. in the gynecologic tract were statistically associated with endometrial cancer pH: high vaginal pH associated with endometrial cancer
Miles et al. (51)	Patient population: women undergoing hysterectomy and salpingo-oopherectomy for a variety of conditions Objective: to determine the microbial compositions at various sites in the female reproductive tract (FRT) and to what extent it varies between patients	10 subjects Average age: 50.6 No antibiotic treatment within the last 30 days. All patients received antibiotics 30 min before surgery	NR	NR	Endometrial, vaginal, cervical, myometrial, fallopian tube, and ovarian swabs taken post-hysterectomy pH: NR		Quality assurance and control of the reactions were performed with both positive and negative control samples to ensure fidelity of the reagents and lack of contamination	454 Pyrosequencing (V1–3)	Firmicutes Proteobacteria	- At a phylum level, Firmicutes were highly abundant - At a genus level, Lactobacillus were highly abundant - Bacterial profiles were highly related across all samples and across all patients pH: NR
Tao et al. (52)	Patient population: women undergoing IVFa Objective: to determine the microbiome obtained from IVF catheter tip during embryo transfer and to assess the limit of accurate quantification of microbiota	70 subjects Average age: 36.2 Antibiotics: NR	Caucasian: 61% Asian: 17% African American: 1.4% Hispanic: 5.6% Unknown: 15%	NR	Distal 5 mm of IVF catheter tip pH: NR	Formable outer sheath advanced under ultrasound guidance	Positive controls at varying concentrations for both single species and polymicrobial samples were used to validate the detection of low abundance bacteria. A negative control was also included	Illumina (V4)	Firmicutes	- Firmicutes were highly abundant from IVF catheter tip - At a genus level, Lactobacillus were highly abundant from the IVF catheter tip - Lactobacillus were detected in all patients sampled along with other vaginal bacteria pH: NR
Chen et al. (27)	Patient population: women operated for conditions not known to involve infectionObjective: to determine the microbiota along the FRT and its association with menstrual cycle, adenomyosis and endometriosis	110 subjects Age: NR No recent use of antibiotics	Asian: 100%	NR	Nylon flocked swabs used to sample: lower third of vagina, posterior fornix, cervical mucus, endometrium, left fallopian tube, and right fallopian tube. Peritoneal fluid was sampled after sterile saline was injected into the peritoneal cavity		Negative diluent controls used: sterile PBS, sterile physiological saline, dry sterile swabs rubbed on preoperative skin, and dry sterile swabs rubbed on surgeon’s gloved fingers. The controls were then cultured on PYG agarPeritoneal fluid was collected from 15 women and were cultured on PYG agar	Ion Torrent Personal Genome Machine system (V5–V4)	Firmicutes	- Unique microbiota compositions were found to exist in cervical canal, uterus, fallopian tubes and peritoneal Fluid which differed from the vagina - Microbiota was also found to correlate with endometriosis and stage in the menstrual cycle - Uterine microbiome shown to be culturable in 5 out 15 subjects

IVF, in vitro fertilization; EP, endometrial polyps; CE, chronic endometritis; MFI, male factor infertility; NR, not reported; GnRHa, gonadotropin-releasing hormone agonist; LD, Lactobacillus dominant; NLD, non-Lactobacillus dominant.

^aNo report of any prevailing medical conditions which may modulate microbiota.