Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection poses a particular risk to pregnant women and their neonates, in which pregnant women have higher rates of severe coronavirus disease 2019 (COVID-19) disease than non-pregnant adults (1). Pregnant women experience immunologic and physiologic changes, such as swelling of the respiratory tract and restricted lung expansion, that make them less tolerant to viral respiratory infections, especially in the last trimester (2, 3). Maternal SARS-CoV-2 infection affects pregnancy outcomes, with increased incidence rates of intensive care admission, invasive ventilation, maternal death, iatrogenic preterm birth, and stillbirth (4–12). Furthermore, comorbidities such as obesity, diabetes, heart disease, advanced maternal age, or a lack of vaccination increase the risk of severe COVID-19 symptoms (3, 11). A recent study revealed no association between the gestational age at infection and COVID-19 morbidity and mortality, suggesting that a previously reported increase in morbidity and mortality in the third trimester may be attributable to other gestational age–affected variables for which adjustment was made in this study (4, 13). Therefore, there is a continuing challenge for clinicians to manage the SARS-CoV-2 infection during pregnancy (1, 9). In this regard, SARS-CoV-2 vaccines have shown efficacy in preventing symptomatic maternal illness and are considered safe for both the mother and infant (8, 9, 14–16). SARS-CoV-2 antibodies have been documented in umbilical cord blood and breast milk after maternal vaccination, suggesting protection for the fetus (9, 14, 17–23). On the basis of the growing evidence that supports the safety and efficacy of COVID-19 vaccination in pregnancy (8, 9, 15–25), most countries recommend full COVID-19 vaccination, regardless of pregnancy trimester.

Galectins are a family of endogenous carbohydrate-binding proteins characterized by a unique sequence motif in their carbohydrate recognition domain (CRD), with the ability to bind β-galactosidase residues (6, 26–28). Galectins are classified into three major types: prototype, which contain one CRD and form homodimers (e.g., gal-1 and gal-7); chimera containing a C-terminal CRD and a proline- and glycine-rich N terminal tail that mediate their oligomerization (gal-3); and tandem repeats that have two different CRDs in tandem connected by a linker of up to 70 amino acids (e.g., gal-9). Galectins contribute to healthy gestation by modulating multiple immune and inflammatory processes (6). In normal pregnancy, circulating maternal gal-1 levels increase from the first trimester, peaking during the second trimester, and remain similar until term (28). Being less abundant, upregulation of gal-3, gal-7, and gal-9 levels in maternal circulation occurs mainly in the second trimester (26, 27). In the extracellular compartment, galectins bind to the glycans decorating glycoproteins. One identified gal-1 ligand at the maternal–fetal interface is pregnancy-specific glycoprotein 1 (PSG1); glycans in the N- and A2 domains of PSG1 mediate the interaction between these two molecules (29). PSGs belong to the carcinoembryonic antigen family within the immunoglobulin (Ig) superfamily and are secreted to the maternal circulation by trophoblast cells (30, 31). PSG1 is one of 10 PSGs and is considered one of the most abundant trophoblastic proteins in maternal circulation during the third trimester of pregnancy (31). PSG1 interacts with soluble and membrane-bound ligands and participates in processes required for successful pregnancy (29). Functional studies with recombinant PSG1 showed that this protein has pro-angiogenic activity; inhibits the interaction of fibrinogen with platelets; and regulates extravillous trophoblast adhesion, migration, and invasion (32–34). In addition, PSG1 activates the latent form of the anti-inflammatory cytokines Transforming growth factor β (TGF-β1) and (TGF-β2) by binding to the respective latent associated peptides contributing to the establishment of maternal immune tolerance (30, 35, 36). Dynamic changes in PSG1 maternal circulation levels during virus infection remain unexplored.

A growing body of clinical data suggests that the cytokine release syndrome is one of the main reasons for the high mortality observed in COVID-19 patients (37). Changes in the cytokine profile of pregnant women correlate with the clinical severity of patients with COVID-19 (10, 38). Recent studies revealed that circulating gal-1, gal-3, and gal-9 levels are increased in non-pregnant patients with COVID-19 (39–43). Levels of gal-3 and gal-9 were reported to be upregulated only in patients with severe disease (40, 41), raising the possibility that circulating gal-3 or gal-9 can be valuable biomarkers for severe pneumonia in patients with COVID-19 (44, 45). Interestingly, exogenous gal-9 administration during acute SARS-CoV-2 infection has been reported to increase the survival rate inducing a robust innate and adaptive immune response in mice (46). More recently, an inhaled gal-3 inhibitor has been tested as a potential therapy for COVID-19 pneumonitis (47). However, despite considerable progress in dissecting the functions of individual members of the galectin family, there is no comprehensive study of the galectin signature in the maternal circulation and placenta following SARS-CoV-2 infection and vaccination during pregnancy.

Therefore, we aimed to determine the galectin fingerprint in the maternal and placental compartment during COVID-19 disease and SARS-CoV-2 vaccination in pregnancy. Our findings indicate that gal-1 is uniquely upregulated at the maternal circulation during SARS-CoV-2 infection and following vaccination. These results underscore the importance of gal-1 as a key pregnancy protective alarmin during SARS-CoV-2 infection. In addition, we observed that the concentration of PSG1, a gal-1 ligand, was increased following infection, providing the first indication of potential regulation of this trophoblast-derived protein in response to insults to maternal health.

SARS-CoV-2 infection leads to a higher risk of severe disease in pregnant than in non-pregnant women. Considering that women of reproductive age make up more than 20% of the global population, studying the effects of SARS-CoV-2 infection and vaccination in this population is of great importance. We measured the concentration of several galectins and PSG1 in the circulation of pregnant women infected with SARS-CoV-2 or vaccinated against SARS-CoV-2 and also explored their placental expression ( Figure 5 ). Our results demonstrated a dominant role of gal-1 within the galectin signature in pregnant women upon SARS-CoV-2 infection in both the PRINCE and Yale IMPACT cohorts. Our findings align with previous reports in non-pregnant individuals, indicating that SARS-CoV-2 infection increased gal-1 levels (41, 42). Moreover, one study showed a positive correlation between gal-1; levels of pro-inflammatory cytokines such as Interleukin-1 beta (IL-1β), IL-6, and IL-23; and COVID-19 severity, suggesting that gal-1 acts as an alarmin (42). In non-pregnant patients, gal-3 and gal-9 were found to be increased and correlated with COVID-19 severity (45, 53). However, we did not find increased levels of gal-3 or gal-9 in pregnant women with SARS-CoV-2 infection. This difference in results may be explained, at least, in part, by the fact that most of our cohort patients had mild or non-symptomatic COVID-19 disease. Furthermore, our results showed positive correlations between gal-1 and gal-3 or gal-9 levels only in healthy pregnancies. This positive correlation was expected because successful pregnancy is associated with a rise in maternal circulating levels of these three galectins from the first to the third trimester (28, 54, 55). The observation that only gal-1 was increased as a result of SARS-CoV-2 infection is likely responsible for the observed loss of correlation with the other two galectins in this group of patients.

The immune system plays a key role in mediating a successful pregnancy because a fine balance is necessary to promote maternal tolerance to the allogeneic fetus while protecting both the mother and the fetus from pathogens (56, 57). Several studies demonstrated that the maternal immune response tend to restrain inflammation through regulatory and anti-inflammatory mediators (58, 59), and galectins, especially gal-1, are involved in this process (6). Moreover, gal-1 has been described not only as a damage-associated molecular pattern molecule, amplifying the immune response, but also as a member of a diverse group of mediators collectively referred to as resolution-associated molecular pattern molecules with the capacity to resolve an acute inflammation process by counteracting the synthesis of pro-inflammatory cytokines (60, 61). The cytokine storm in the systemic circulation is responsible for the pathophysiology of SARS-CoV-2 infection and can lead to multi-organ damage (7, 62). Both systemic and inflammatory changes at the maternal–fetal interface were previously described during SARS-CoV-2 infection in pregnancy (48, 63). The analysis of bulk RNA sequencing of placental villi from the Yale IMPACT cohort demonstrated that COVID-19 cases presented increased expression of genes associated with an immune response, indicating a robust response at the maternal–fetal interface upon SARS-CoV-2 infection, even in the absence of localized placental infection (48). Another study evaluated the maternal systemic immune response, finding increased IL-8, IL-10, and IL-15 levels as well as a reduction in T-cell subsets, particularly of T helper cell 1 (Th1) and a subset of CD8⁺ cells characterized by the production of IL-17 (Tc17)-like cells in pregnant women infected with SARS-CoV-2 (63). Gal-1 controls the fate of Th-1 and Th-17 cells through the glycan repertoire expressed by these cells that allow gal-1 binding (64). Therefore, the increase of gal-1 in maternal circulation during COVID-19 could be associated with the observed systemic decrease in Th-1 and Tc17-like cells. Gal-1 also induces apoptosis of activated CD8⁺ T cells (65), which could explain the upregulation of gal-1 in SARS-CoV-2 infection. Thus, the increase of gal-1 observed in pregnant patients with COVID-19 could be related to its anti-inflammatory activity and its potential participation in the control of inflammation and tissue damage resulting from SARS-CoV-2 infection.

In our study, healthy pregnant women exposed to SARS-CoV-2 infection often exhibit simultaneously increased serum concentrations of gal-1 and PSG1. We have previously demonstrated that PSG1 binds to gal-1 and postulated that PSG1 protects gal-1 from inactivation by oxidation in the extracellular environment. Therefore, an increase in both PSG1 and gal-1 may extend the gal-1 activity once it is released from cells into the circulation (29). However, the correlation between these two proteins disappeared in infected pregnant women, likely due to the modest increase of PSG1 relative to gal-1. Unfortunately, we were unable to determine whether the concentration of other PSG family members, which can also interact with gal-1, is also increased in the circulation of SARS-CoV-2–infected pregnant women as suggested by the RNA sequencing data because specific validated ELISAs are only available to measure the concentration of PSG1. Future studies should be performed to determine the mechanism by which syncytiotrophoblast cells, the major cell type that secretes PSG1 into the maternal circulation, regulate PSG1 secretion following maternal SARS-CoV-2 infection. In contrast, PSG1 post-vaccination levels remain unchanged, suggesting that modulation of PSG1 secretion by the syncytiotrophoblast cells requires maternal SARS-CoV-2 infection. In the future, it would be interesting to determine whether an increase in PSG1 concentration is also observed following infection with other viruses that do not infect the PSG1-producing cells and whether this increase in PSG levels may contribute to the establishment of an anti-inflammatory environment.

The extracellular activity of gal-1 relies on its capacity to bind specific glycans. To further investigate the possible role of the gal-1 upregulation during the course of COVID-19, the expression of enzymes involved in relevant N- and O-glycosylation was analyzed in bulk placentas by RNA sequencing. Our results showed that genes involved in the initiation, hybrid complex formation, and elongation of N-glycans and in initiation and core modification of O-glycans were upregulated in patients with COVID-19. Gal-1 and gal-3 can bind β-1,6-GlcNAc–branched N-linked glycans on the cell surface or in secreted glycoproteins, and several enzymes are required for the biosynthesis of these glycotopes (51). Similar results were found for the enzymes involved in O-glycans synthesis, as initiation (GALNT1, GALNT2, and GALNT7) and core modification (C1GALT1C1 and B4GALT5) enzymes were also upregulated during SARS-CoV-2 infection. The addition of α2,6-linked sialic acids to the termini of glycans by ST6GAL1 inhibits the binding of gal-1 (6). However, our results demonstrated similar expression of ST6GAL1 in SARS-CoV-2–infected and control groups. More importantly, we observed an increase of ST3GAL1 expression signatures (responsible for the addition of α2,3-linked sialic acid, which is compatible which gal-1 high-affinity binding) within the placental compartment. These results suggest that both the expression of gal-1 and of the enzymes in the placenta involved in the N- and O-glycan modifications required for gal-1 to exert its function are increased following SARS-CoV-2 infection.

We also explored the galectin fingerprint of healthy pregnant women that were fully vaccinated. Several reports indicated that COVID-19 vaccines are safe for pregnant women and can effectively protect them from getting severe COVID-19 and prevent maternal and fetal mortality (8, 66). Our results provide evidence that the status of the maternal immune system during vaccination modulates the galectin signature and could play a role for protection against COVID-19 disease. For instance, a simultaneous increase of gal-1 and gal-7 accompanied by a decrease of gal-9 in post- vaccination sera derived from pregnant individuals suggests a role of these lectins in the establishment of a protective response against the SARS-CoV-2 virus. Gal-1 is secreted by activated B cells, T cells, and macrophages, and activated lymphocytes secrete gal-7 (67, 68). We hypothesize that the increase of these two galectins in pregnant vaccinated patients could play a role in the effective immune system activation by the COVID-19 vaccine that results in the generation of neutralizing antibodies and virus-specific T-cell responses (69). Elevation of circulating levels of gal-9 in chronic and acute SARS-CoV-2 infection has been reported in non-pregnant individuals, and it was correlated with a severe disease outcome (43). On the basis of our findings, we hypothesized that a lack of increase in gal-9 levels during vaccination likely avoids a pro-inflammatory response during pregnancy vaccination that could result in detrimental effects at the maternal–fetal interface.

In conclusion, our study revealed a clear dominance of gal-1 within the maternal galectin fingerprint during SARS-CoV-2 infection and vaccination in pregnancy. One of the pregnancy-specific gal-1 ligands, PSG1, was also upregulated during the COVID-19 disease, suggesting a potential synergistic immunomodulatory role of these proteins at the maternal–fetal interface. Further work is needed to determine the functional relevance of the galectin signature in maternal circulation during the SARS-CoV-2 infection and vaccination in pregnancy. Specifically, it would be important to understand the molecular mechanism by which gal-1 may control the immune response to a virus during pregnancy.

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 Hamburg Chamber of Physicians (Ärztekammer Hamburg) approved the study protocol under the registration numbers PV3694 (PRINCE), PV 7312-4710 (PRINCE COVID) and 2021-10647-BO-ff (PRINCE VACCINE). The study was approved by the Yale Institutional Review Board (protocol #2000027690). The patients/participants provided their written informed consent to participate in this study.

SMB designed the study and secured grant funding. FZ, A-CT, YW, YX, AD, AL-C, PV, EK, CU, JB, PCA, SFF, GSD, and MGG performed experiments and/or analyzed data. The original draft of the manuscript was written by FZ, MGG, and SMB, and further writing, review, and editing were done by all authors. All authors contributed to the article and approved the submitted version.