After a nearly complete pause at the beginning of the coronavirus disease 2019 (COVID-19) pandemic (Alviggi et al., 2020; Andrabi et al., 2020), assisted reproductive technology (ART) treatments restarted with proper screening and testing of the patients and safety precautions (Rajput et al., 2021a,b). With pandemic, new questions arose in all fields of medicine and healthcare and also in the context of in vitro fertilization (IVF) treatments, where at the moment there are not enough data on the possible effects of SARS-CoV-2 on fertility and IVF outcome (Madjunkov et al., 2020; Elsaddig and Khalil, 2021), and also on the indirect impact of COVID-19 on reproductive medicine (Kassie et al., 2021).

It has been documented the absence of viral RNA in follicular fluid (FF) of a SARS-CoV-2-positive woman and this suggests that handling of oocytes, sperm, seminal fluid, or follicular fluid in IVF practice does not constitute a significant source of infection (Demirel et al., 2021). Also, the Belgian COVART study showed that SARS-CoV-2 RNA was undetectable in follicular fluid or vaginal secretions in the cohort of women analyzed (Kteily et al., 2021). A recent work by Israeli researchers indicates that specific antibodies to the virus are present in both serum and ovarian follicles following infection or vaccination, and no apparent adverse effect on follicular function is observed (Bentov et al., 2021).

Many of the questions related to the metabolic pathway of COVID-19 disease remain to be addressed while the literature is still scarce and the infection resulting from SARS-CoV-2 is not yet fully understood at the cellular metabolite level. Various studies have been performed in the last months on serum (Bruzzone et al., 2020; Shen et al., 2020; Doğan et al., 2021; Páez-Franco et al., 2021; Shi et al., 2021), plasma (Chen et al., 2020; Fraser et al., 2020; Kimhofer et al., 2020; Wu et al., 2020; Lodge et al., 2021), and saliva (Costa Dos Santos Junior et al., 2020; Sapkota et al., 2020) of COVID-19 patients. The objectives of metabolomic works are to identify biomarkers with diagnostic potential in addition to give a description of the metabolic pathways involved in the different disorders observed in COVID-19 patients.

As far as we know, up to now no metabolomic study has been performed on FF of women infected by SARS-CoV-2 or vaccinated. The aim of the present study is to investigate whether there is a specific metabolic profile associated with a past COVID-19 positivity or vaccination in FF of women who are following IVF treatments; as a control we used FF collected before pandemic. Nuclear magnetic resonance spectroscopy (NMR) has shown its utility in various stages of ART (Asampille et al., 2020). In this study we have used NMR-based metabolomics that was shown to be well suited for the characterization of FF and to quantify and identify its metabolites (Pñero-Sagredo et al., 2010; Baskind et al., 2011; McRae et al., 2012; Wallace et al., 2012; O’Gorman et al., 2013).

Follicular fluid is an important component in the growth and development of the follicle which consists of many substances secreted from granulosa- and theca cells and transudates from blood compartment, such as hormones, growth factors, immune cells, cytokines, enzymes, anticoagulants, electrolytes, reactive oxygen species, lipids, cholesterol, and antioxidants. Therefore, changes in FF will influence the developing oocyte (Freitas et al., 2017). Cytokines in FF are important for reproduction as they modulate oocyte maturation and ovulation which influence subsequent fertilization, development of early embryo, and potential for implantation (Field et al., 2014; Bianchi et al., 2016; Robertson et al., 2018). It has been widely reported that patients with severe COVID-19 disease may undergo the so-called cytokine storm, which recalls the role of the immune system in producing an aberrant systemic inflammatory response (Chen et al., 2021). In particular, the plasma levels of some inflammatory proteins including tumor necrosis factor (TNF) and interleukin-6 (IL-6) were significantly increased in patients with severe COVID-19 (Coperchini et al., 2021). Consequently, changes in plasma composition might be also mirrored in FF composition. Therefore, we evaluated in FF of vaccinated or immunized women the possible variation in the expression levels of two proinflammatory cytokines, TNF-α and IL-6, and superoxide dismutase 2 (SOD-2), a mitochondrial isoenzyme that convert the pro-oxidant superoxide into hydrogen peroxide. Furthermore, considering that some cytokines modulate the glucose and lipid metabolism (Shy et al., 2019), possible relationships between the expression levels of these cytokines and the presence of some metabolites could be found.

The cohort of women examined consisted of 20 women. They were selected as follows: six women who received vaccination against SARS-CoV-2 infection, five recovered COVID-19 patients, and as a control, we chose nine women whose FFs were collected before the coronavirus pandemic outbreak. Patient’s demographics and steroidogenic characteristics are reported in Table 1. There were no significant differences in these parameters when comparing vaccinated women, recovered COVID-19 patients, and healthy controls.

We found that the normalized ratio of serum estradiol/oocytes changes among the groups being 380.9, 314.5, and 200.9, for vaccinated women, previously positive patients, and controls, respectively. Indeed, in both vaccinated women and recovered COVID-19 patients the oocytes retrieved/trigger day mature follicle count was 0.6 and 0.8 in healthy controls, respectively; the oocytes retrieved/serum progesterone in vaccinated women was 3.7 vs. 3.2 and 7.1 in recovered COVID-19 patients and controls, respectively. Finally, in vaccinated women the ratio of mature/total number of aspirated oocytes was 0.7 vs. 0.8 of both previously positive patients and controls, respectively. The average number of zygotes in vaccinated women (2.5 ± 1.4) and recovered COVID-19 patients (2.4 ± 1.7) is higher than in healthy controls (1.6 ± 0.7), although the difference was not significant. However, the number of blastocysts for the healthy controls remains the same of zygotes while it decreases in both vaccinated and ex-COVID-19 women (p > 0.05).

Anti-Mullerian hormone (AMH) and antral follicle count (AFC) together are defined as two valid biomarkers in the evaluation of ovarian reserve and patient response to hormonal therapies (Broer et al., 2011). No significant difference was observed between vaccinated women, previously positive patients, and controls (Table 1).

Several studies have reported that the E2/oocytes ratio is an important predictive parameter for IVF success (Bu et al., 2015; Taheri et al., 2020). However, Aslih et al. (2021) have demonstrated that the E2/M2 ratio is a better biomarker and that lower ratios produced higher quality embryos and a better pregnancy rate. We found a higher E2/M2 ratio in both vaccinated and recovered women (571 and 373, respectively) in comparison to the control one (264).

Ultimately, even if there is no statistically significant difference between the three groups of women considered, the clinical data seem to indicate some difference between them. In particular, the adequacy of the follicular response to ovulation triggering tends to decrease in vaccinated women and recovered COVID-19 patients compared to controls.

In this study we have found some metabolic differences in FFs of vaccinated women and previously infected patients in comparison with healthy controls. In particular, for vaccinated women we found higher levels of the amino acids Ala and Pro and lower levels of lipids and TMAO. TMAO is a metabolite produced from dietary choline and carnitine by the gut microbiota and, successively, by hepatic flavin monooxygenases. In a recent study Nagy et al. (2020) showed that TMAO is associated with unfavorable fertility outcomes: TMAO is significantly lower in FF from oocytes that underwent normal fertilization and developed into top-quality embryos. Our finding of lower levels of TMAO in FFs of vaccinated women, if confirmed on a higher number of cases, could then be reassuring about the fertility outcomes of these women undergoing ART treatment.

We showed that FFs of recovered patients were positive for IgG antibodies against SARS-CoV-2, as found elsewhere (Herrero et al., 2022). No detectable levels of anti-COVID IgG in follicular fluid of vaccinated women were found.

Besides, lower levels of many important metabolites were found in recovered COVID patients’ FFs together with the lowest number of retrieved oocytes; this last finding was also reported by Herrero et al. (2022).

Oxidative stress, referred to an imbalance between the concentrations of pro- and anti-oxidants, may play a role during folliculogenesis and oogenesis (Da Broi et al., 2018). Granulosa and cumulus cells protect the oocyte from oxidative stress damage also regulating the expression of the various SOD isoforms (von Mengden et al., 2020). SOD-2 in human follicular fluid represents a biomarker of follicle and in turn oocyte quality. Although not significantly, SOD-2 levels in FF of vaccinated and recovered women tend to be higher than in controls to indicate that cells respond with an increase in defense mechanisms to counterbalance a probable imbalance of redox homeostasis, thus preventing impairment of the oocyte quality.

Physiological expression of cytokines and chemokines is required for optimal ovarian functions; they are produced by resident and infiltrating leukocytes as well as granulosa and theca cells (Akison et al., 2018). Inflammatory processes might compromise steroidogenesis and oocyte development; in particular, it has been observed that pro-inflammatory cytokines in FF play essential roles in follicular growth and ovulation, influence subsequent fertilization, embryonic development, implantation potential, and implantation of embryos (Adamczak et al., 2021). It has been shown that patients with severe COVID-19 disease can be subjected to the so-called “cytokine storm” (Chen et al., 2021), an exaggeratedly violent reaction of the immune defenses which, instead of protecting from the virus, attack all the patient’s organs. Plasma levels of some inflammatory mediators such as interferon-gamma, TNF-α and IL-6 were also particularly high (Cabaro et al., 2021; Coperchini et al., 2021). Consequently, variations in the plasma composition could affect the cytokines composition of FF. In both vaccinated and recovered women, no significant variation in the levels of the two proinflammatory cytokines was observed, compared to those of controls. Even, the level of TNF-α expression is significantly reduced, indicating that the quality of the oocytes should not be compromised by inflammatory events attributable to vaccination and mild COVID-19 infection. Furthermore, in agreement with low expression of TNF-α we also found a lower level of TMAO to confirm a reduced inflammatory state (Yang et al., 2019).

All considered, our results do not indicate high oxidative stress and inflammation in FFs of both the vaccinated and recovered women, compared to controls and therefore neither the folliculogenesis nor the quality of the oocytes would seem to be compromised.

Our study has two main limitations, that is the small number of women examined and the different time interval passed between infection or vaccination and oocyte collection, that did not consider properly the time-dependency of the effect of immune response induced by infection or vaccination. Further studies should then be performed with a larger cohort of women and potential long-term effects of vaccination and COVID-19 infection on fertility should be also evaluated.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Comitato Etico Unico Regionale per la Basilicata, approval number: 82/2015 of October 7, 2015. The patients/participants provided their written informed consent to participate in this study.

SS, AI, and AO designed the study. AI selected the patients and executed oocyte retrieval. AF identified oocytes in follicular fluid and executed their fertilization. PC selected the follicular fluids to be used for metabolomic analyses. MC performed the analysis of NMR data and multivariate analysis. LV run the NMR experiments. IM performed western blotting analyses and Ig search. AO, AI, and MC were responsible for conducting the study and wrote the manuscript which was critically discussed, edited, and approved by all co-authors. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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