Microbiological and Cytokine Profiling of Menstrual Blood for the Assessment of Endometrial Receptivity: A Pilot Study

Abstract

Endometrial receptivity (ER) is a key factor required for the successful implantation of the embryo. However, the evaluation of ER is challenging, as a nondisruptive sampling of endometrial biomaterial by conventional methods is only possible outside of the embryo transfer (ET) cycle. We propose a novel approach for the assessment of ER-microbiological and cytokine profiling of menstrual blood aspirated directly from the uterine cavity at the beginning of the cryo-ET cycle. The aim of the pilot study was to evaluate its prognostic potential regarding the outcome of the in vitro fertilization procedure. Samples collected from a cohort of 42 patients undergoing cryo-ET were analyzed by a multiplex immunoassay (48 various cytokines, chemokines, and growth factors) and a real-time PCR assay (28 relevant microbial taxa and 3 members of the Herpesviridae family). Significant differences between groups of patients who achieved and did not achieve pregnancy were observed for G-CSF, GRO-α, IL-6, IL-9, MCP-1, M-CSF, SDF-1α, TNF-β, TRAIL, SCF, IP-10, and MIG (p < 0.05), whereas microbial profiles were not associated with the outcome of cryo-ET. It appeared that levels of IP-10 and SCGF-β were significantly lower (p < 0.05), in patients with endometriosis. Menstrual blood may provide great opportunities to noninvasively investigate various parameters of the endometrium.

Keywords: assisted reproduction; chemokines; cytokine profile; cytokines; endometrial microbiota; endometrial receptivity; growth factors; in vitro fertilization; menstrual blood.

Figure 1

Heat map for the cytokine analysis in menstrual supernatant. Data presented as pg per mg of total protein. The scale was capped at 100 pg/mg to preserve the color separation at lower concentrations of cytokines. Uncapped numerical values for each sample are available in Table S1.

Figure 2

Boxplots for the comparison of levels of analyzed immune mediators between groups. Data presented as pg of analyte per mg of total protein. Only statistically significantly differing cytokines are shown. p-values were calculated using the Mann–Whitney U test. *, p-values significant after the Benjamini–Hochberg adjustment for multiple comparisons.

Figure 3

Correlation matrix for 48 immune mediators analyzed in menstrual supernatant. Correlation of quantitative variables was assessed via Spearman’s rank coefficient. Data were calculated based on the results in the total cohort of patients. Cytokines that were upregulated in patients who achieved pregnancy are marked with “*”.

Figure 4

Boxplots for the comparison of levels of analyzed immune mediators based on the presence of endometriosis and diminished ovarian reserve. DOR, diminished ovarian reserve. Data presented as pg of analyte per mg of total protein. Only statistically significantly differing cytokines are shown. p-values were calculated using the Mann–Whitney U test. *, p-values significant after the Benjamini–Hochberg adjustment for multiple comparisons.

Figure 5

Heat map for the microbiological analysis in menstrual sediment. Data presented as abundance (% of total bacterial load measured by amplification of conservative prokaryotic DNA sequence). For convenience, the levels of Candida spp. DNA were added to the values of the total bacterial load. The heat-map scale was segmented to ensure a clear color separation for cells with values of 0%. Some closely related species/groups of species 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.