Redox Balance and Inflammatory Response in Follicular Fluids of Women Recovered by SARS-CoV-2 Infection or Anti-COVID-19 Vaccinated: A Combined Metabolomics and Biochemical Study

Abstract

To date, not many studies have presented evidence of SARS-CoV-2 infecting the female reproductive system. Furthermore, so far, no effect of the administration of anti-COVID 19 vaccines has been reported to affect the quality of oocytes retrieved from women who resorted to assisted reproduction technology (ART). The FF metabolic profiles of women who had been infected by SARS-CoV-2 before IVF treatments or after COVID-19 vaccination were examined by ¹H NMR. Immunochemical characterization of proteins and cytokines involved in the redox and inflammatory pathways was performed. The increased expression of SOD2 and NQO1, the lack of alteration of IL-6 and CXCL10 levels, as well as the increased expression of CD39, suggested that, both sharing similar molecular mechanisms or proceeding along different routes, the redox balance is controlled in the FF of both vaccinated and recovered women compared to controls. The lower amount of metabolites known to have proinflammatory activity, i.e., TMAO and lipids, further supported the biochemical results, suggesting that the FF microenvironment is controlled so as to guarantee oocyte quality and does not compromise the outcome of ART. In terms of the number of blastocysts obtained after ICSI and the pregnancy rate, the results are also comforting.

Keywords: COVID-19; NMR-based metabolomics; SARS-CoV-2; biomarkers; female fertility; follicular fluid; inflammation; oxidative stress.

Figure 1

(A) PCA Score plot obtained from the ¹H-NMR FF spectral data of all 55 women examined in this study. (B) PLS-DA score plot between control and vaccinated women. The R²Y and Q² values for the two-component model were: 0.82 and 0.71, respectively; (C) PLS-DA score plot between control women and recovered COVID-19 patients. The R²Y and Q² values for the three-component model were: 0.83 and 0.50, respectively. Data were colored by group: healthy control (N = 19, red dots), vaccinated (N = 11, blue triangles), and recovered COVID-19 (N = 25, black boxes).

Figure 2

Heat map of the most relevant metabolites (with a VIP value > 1) associated with the differentiation between control (N = 19, labeled C) and: (A) vaccinated (N = 11, V) and (B) recovered COVID-19 women (N = 25, exCov). Rows: quantification of NMR integral bin regions of metabolites with VIP > 1. Columns: different groups of women. The color scale indicates values ranging from blue (the lowest) to red (the highest). * p < 0.05.

Figure 3

Evaluation of oxidative stress in follicular fluids. (A) Measure of hydroperoxide level by d-ROMS assay; UCARR indicate “Carratelli Unit”, an arbitrary unit, and 1 U CARR corresponds to the color development caused by a H₂O₂ solution at a concentration of 0.08%; (B) measure of the antioxidant power by BAP test. The results were analyzed with Wilcoxon–Mann–Whitney test.

Figure 4

NRF2, SOD2, catalase, and NQO1 expression levels in FFs. Densitometric analysis of immunoreactive bands measured by Western Blot; protein levels were normalized to total protein, and data are expressed, as mean ± standard deviation (SD) and analyzed with Wilcoxon–Mann–Whitney test (* p < 0.05, ** p < 0.01).

Figure 5

IL6 and CXCL10 concentrations and CD39 expression levels in follicular fluids. (A) IL-6 and (B) CXCL10 concentrations were measured by ELISA sandwich. (C) CD39 expression level was evaluated by densitometric analysis of the immunoreactive band obtained by Western Blot; protein levels were normalized to total protein, and data are expressed as mean ± standard deviation (SD) and analyzed with Wilcoxon–Mann–Whitney test (** p < 0.01, *** p < 0.001).

Figure 6

ROC curves for NQO1 (A), CD39 (B), lipids (C), and TMAO (D). Sensitivity indicates the true positive, and 100% specificity indicates false positives. The curves were obtained with GraphPad Prism 8.0 software.