Comparison of microbial profiles and viral status along the vagina-cervix-endometrium continuum of infertile patients

Figures & data

Figure 1. Diversity of microbial communities in vaginal, cervical, and endometrial samples of infertile patients. Vaginal, cervical, and endometrial samples were obtained from each study participant (n = 100). The abundances of vaginosis-associated bacteria and fungi were analyzed using a real-time PCR kit ‘Femoflor 16’ (cat# R1-P801-S3/6) (DNA-Technology, Moscow, Russia)), which includes reagents for the detection of 24 relevant bacterial and fungal taxa. The line inside the box represents the median value; the borders of the box – are the first and third quartiles; whiskers – the maximum value before the upper limit (third quartile + 1.5 × interquartile range); circles above whiskers are outliers.

Figure 2. Comparison of detection rates of microorganisms and viruses in vaginal, cervical, and endometrial samples of infertile patients. Vaginal, cervical, and endometrial samples were obtained from each study participant (n = 100). Samples were analyzed using real-time PCR kits ‘Femoflor 16’, ‘TNC Complex’, ‘Herpes Multiplex’ (cat# R1-P801-S3/6, R1-P111-S3/9, R1-P210-S3/9, respectively, (DNA-Technology, Moscow, Russia)). The recommended manufacturer cycle threshold value for qualitative analysis was 24, whereas during quantitative analysis DNA levels of less than 10³ copies were considered negative. The detection of bacterial DNA was based on the amplification of a conservative procaryotic sequence. Some closely related taxa were analyzed collectively due to limitations of the applied real-time PCR assay. In such cases, the names of the taxa are listed together and joined by a “+” sign. P-values were calculated using Cochran’s Q test. *significant difference for all types of samples (p < 0.05). **significant difference for endometrial samples vs other types of samples. ***significant difference for endometrial samples vs vaginal samples.

Table 1. Results of comparative quantitative microbiota analysis in vaginal, cervical, and endometrial samples.

Variable	Abundance, %			p-Value
	V	C	E	E vs V	E vs C	C vs V
Lactobacillus spp.	100 (8–100)*	100 (4–100)*	100 (13–100)*	>0.05	>0.05	>0.05
Enterobacteriaceae	0.04 (0.001–63.1)	0.08 (0.001–100)	N/D	>0.05	>0.05	>0.05
Streptococcus spp.	42.87 (6.3–79.4)	28.2 (6.3–50.1)	29.9 (20.0–39.8)	>0.05	>0.05	>0.05
Staphylococcus spp.	0.016 (0.001–3.162)	0.016 (0.001–3.98)	N/D	>0.05	>0.05	>0.05
Gardnerella vaginalis + Prevotella bivia + Porphyromonas spp.	35.7 (0.0126–100)	35.7 (0.01 –100)	45 (0.04–100)	0.03**	>0.05	>0.05
Eubacterium spp.	3.98 (0.002–25.12)	6.3 (0.006–39.811)	25.11 (0.04–100)	<0.001**	0.002**	>0.05
Sneathia spp. + Leptotrichia spp. + Fusobacterium spp.	27.8 (15.8–39.8)	37.8 (12.6–63.1)	41.5 (20–63)	>0.05	>0.05	>0.05
Megasphaera spp. + Veillonella spp. + Dialister spp.	7.94 (1.26–15.85)	5.01 (1.26–12.6)	6.3 (3.2–10.0)	>0.05	>0.05	>0.05
Lachnobacterium spp. + Clostridium spp.	0.179 (0.001–1.995)	0.18 (0.001–3.981)	12.6**	>0.05	>0.05	>0.05
Mobiluncus spp. + Corynebacterium spp.	0.0199 (0.001–0.631)	0.016 (0.001–1.585)	N/D	>0.05	>0.05	>0.05
Peptostreptococcus spp.	5.01 (3.98–5.01)	5.01 (2.51–7.94)	3.98 (3.16–6.31)	>0.05	>0.05	>0.05
Atopobium vaginae	5.01 (0.1–50.12)	5.01 (0.63–39.81)	3.16 (1–50.1)	>0.05	>0.05	>0.05
Candida spp.****	0.025 (0.0003–1.9953)	0.126 (0.006–1.585)	5.15 (3.98–6.31)	>0.05	>0.05	>0.05
Mycoplasma hominis	0.001 (0.0001–0.0631)	0.026 (0.0013–0.0501)	0.251***	>0.05	>0.05	>0.05
Ureaplasma (urealyticum + parvum)	0.159 (0.0004–10)	0.2 (0.004– 25.119)	0.631 (0.002–19.95)	<0.001**	0.005**	>0.05

Abundances of analyzed microorganisms (% of total bacterial load measured by amplification of conservative prokaryotic DNA sequence) are presented as median (minimum–maximum range). *Abundance of Lactobacillus spp. of 100% in most samples is a result of analytical limitations of the implemented real-time PCR approach and should be interpreted as “approximately 100%”. P-values were calculated using Friedman’s test, whereas in cases where quantitative data was available only for two groups – using Wilcoxon signed-rank test. ** significant value. *** minimum–maximum range cannot be calculated since corresponding taxa detected only in one endometrial sample. **** Candida spp. concentrations were also normalized by a total bacterial load to compensate for sampling quality heterogeneity. (C) cervical; (E) endometrial, (V) vaginal samples; N/D, microorganism not detected; N/A, not applicable (either in cases when overall comparison p-value was not significant, or when microorganism was not detected in any of the endometrial samples). Due to low detection rates of most taxa (especially in endometrium), data of samples with an abundance of corresponding microorganisms 0% were excluded (as it would significantly impact the analysis and lead to complete duplication of the results of the qualitative analysis presented earlier.

Table 2. Clinical and demographic characteristics of the participants.

Parameter	Value
Age, yrs	36.7 (29.0–46.0)
Body mass index, kg/m^2	22.4 (16.9–31.1)
Infertility factors
Tubo-peritoneal factor	16/100
Endometriosis	38/100
Diminished ovarian reserve	40/100
Uterine factor	13/100
Endocrinologic factor	22/100
Unknown	17/100
Uterine adhesions	7/100
Uterine fibroids	15/100
Chronic endometritis	11/99*
Uterine polyps	42/99*
STIs in anamnesis	17/100
Recurrent implantation failure	9/100
Recurrent pregnancy loss	4/100

Data are mean (range) or number observed/number of subjects. *results of histological and immunohistochemical analyses for one patient were not available. STIs: sexually transmitted infections.

Data availability statement

Results of microbiological analysis and clinical data of study participants are presented in Supplemental material 2.