Are the Follicular Fluid Characteristics of Recovered Coronavirus Disease 2019 Patients Different From Those of Vaccinated Women Approaching in vitro Fertilization?

Abstract

The aim of this pilot study is to evaluate if SARS-CoV-2 infection or vaccination against SARS-CoV-2 infection induce observable metabolic effects in follicular fluid of women who are following in vitro fertilization (IVF) treatments. The possible impact of coronavirus disease 2019 (COVID-19) on fertility and IVF outcome is considered. We have selected for this study: six women vaccinated against SARS-CoV-2 infection, five recovered COVID-19 patients, and we used nine healthy women as the control group. At the time of oocytes retrieval from participants in the study, follicular fluids were collected and metabolomic analysis was performed by ¹H NMR spectroscopy in combination with multivariate analysis to interpret the spectral data. The search for antibody positivity in the follicular fluid aspirates was also carried out, together with the western blotting analysis of some inflammatory proteins, interleukin-6, tumor necrosis factor α (TNFα), and the free radical scavenger superoxide dismutase 2. Higher levels of Ala and Pro together with lower levels of lipids and trimethylamine N-oxide (TMAO) were found in follicular fluids (FFs) of vaccinated women while lower levels of many metabolites were detected in FFs of recovered COVID patients. Expression level of TNF-α was significantly lower both in recovered COVID-19 patients and vaccinated women in comparison to healthy controls.

Keywords: COVID-19; NMR; SARS-CoV-2; anti SARS-CoV-2 immunoglobulins; follicular fluid; metabolomics; proinflammatory markers.

FIGURE 1

(A) Principal Component Analysis (PCA) score plot obtained from the ¹H-NMR follicular fluid (FF) spectral data of all the 20 women examined in this study. (B) PLS discriminant analysis (PLS-DA) score plot between healthy women and vaccinated women. The R²X and Q² values for the two-component model were: 0.47 and 0.56, respectively; (C) PLS-DA score plot between healthy women and COVID-19 recovered patients. The R²X and Q² values for the two-component model were: 0.51 and 0.72, respectively. Data were colored by group: healthy control (N = 9, black dots), vaccinated (N = 6, blue triangles) and recovered COVID-19 (N = 5, red triangles). (D) Heat map of the most relevant metabolites (with a VIP value > 1) that were associated with the differentiation between healthy (N = 9, group C) and vaccinated (N = 6, group V) and recovered COVID-19 women (N = 5, exCov). Rows: quantification of NMR integral bin regions of metabolites with VIP > 1. Columns: different groups of women. Color scale indicates values ranging from blue (the lowest) to red (the highest).

FIGURE 2

Expression level of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and superoxide dismutase 2 (SOD-2) proteins in follicular fluids. (A) Representative western blot of proteins in follicular fluid (FF). (B) Densitometric analysis of the immunoreactive bands performed in three independent experiments. After densitometric analysis, western blot signals of the target proteins are normalized to the total amount of protein in each lane. The box plots show medians and whiskers. Significance (^**p < 0.01).