COVID-19-related inflammation of the placenta impedes fetal development in pregnant hamsters

Domain: neuroscience
Published: 2026-02-09
Editorial outcome: minor_revision

Abstract
Pregnant women are at higher risk of severe COVID-19, with vaccine access and hesitancy remaining a challenge. Here, we use a pregnant female hamster model of COVID-19 to explore the effects of maternal infection on pregnancy, revealing a significant increase in intrauterine growth restriction (IUGR) due to placental inflammation. Viral infection causes bronchopneumonia and weight loss in infected dams, but no vertical transmission occurs. IUGR is instead linked to placental damage, characterized by fibrin deposition, thrombosis, and elevated placental expression of IP10, IL6, and IL10, irrespective of fetal sex. Enoxaparin treatment reduces placental damage and improves fetal outcomes, while vaccination enhances viral clearance, protects the placenta, and reduces the risk of IUGR. These findings underscore placentitis as a key driver of fetal complications upon SARS-CoV-2 infection and highlight the potential of vaccination and anticoagulant therapy to protect both mother and child.

Introduction
Since its emergence in late 2019, SARS-CoV-2 has disrupted daily life and strained global health systems, causing widespread illness and death. While advanced age, male sex, and comorbidities like cardiovascular disease and diabetes have been identified as key risk factors1,2, pregnancy poses particular challenges due to unique physiological changes that increase susceptibility to severe infection3. Moreover, mother and unborn child form an indivisible dyad, whereby infections hit two vulnerable individuals at once, though sequelae may differ. Clinically, pregnancy outcomes linked to COVID-19 are influenced by factors such as gestational age, maternal comorbidities, and disease severity4,5. Compared to non-pregnant patients, expectant mothers are at a heightened risk of hospitalization, ICU admission, invasive ventilation, and maternal and neonatal mortality6,7.

Arange of viral infections is known to cause severe pathologies in the unborn8,9. The TORCH pathogens, which include rubella virus, cytomegalovirus, and herpes simplex virus, cross the placental barrier, leading to intrauterine infections with devastating consequences for the fetus. Nevertheless, the spectrum of viruses possibly interfering with fetal development also includes respiratory viruses such as influenza A virus and SARS-CoV-210,11, which affect pregnancy indirectly. While direct vertical transmission is rare12,13, SARS-CoV-2 infection appears to compromise placental function and integrity, leading to necrosis, intervillous thrombosis, and fibrin deposition, which in turn may contribute to fetal distress or demise14. Studies on the human placenta have identified the syncytiotrophoblast as the primary site of SARS-CoV-2 infection, with viral presence also reported in cytotrophoblasts, Hofbauer cells, villous stromal and endothelial cells, and maternal immune cells within the intervillous space15. Consistently, placental inflammatory and vascular lesions occur more frequently following first- and second-trimester infections than in the third trimester, independent of maternal disease severity15,16. The expression of SARS-CoV-2 entry factors ACE2 and TMPRSS2 is highest during early gestation and declines as pregnancy progresses, potentially contributing to this differential susceptibility17.

These clinical findings underscore the need for targeted research to rationalize clinical management of COVID-19 during pregnancy, in order to mitigate its harmful effects on neonatal health. However, the mechanisms by which SARS-CoV-2 impacts maternal and fetal health remain highly understudied.

Due to historical exclusion from clinical research, concerns about side effects, and insufficient communication between healthcare providers and patients, vaccine hesitancy remains common among pregnant women, with rates ranging from 26% to 57%18,19. Moreover, rumors about vaccines reducing female fertility and widespread skepticism towards new vaccine modalities have contributed to suboptimal vaccine uptake by women of reproductive age20. Importantly, SARS-CoV-2 vaccination has been shown to significantly reduce maternal mortality and morbidity without increasing the risks of adverse outcomes, such as miscarriage, stillbirth, fetal malformations, pre-eclampsia, or postpartum hemorrhage21.

As research into COVID-19 and pregnancy advances, understanding the increased risks to pregnant women and their offspring requires robust preclinical models. Animal models, including mice, hamsters, ferrets, and non-human primates22,23, offer distinct advantages and limitations for studying SARS-CoV-2 infection, particularly regarding its impact on immune responses and placental function.

The placenta plays a central role in fetal development, mediating gas exchange, nutrient transfer, and immune protection. Humans, non-human primates, and rodents all possess a hemochorial placenta in which placental trophoblast cells are in direct contact with maternal blood. However, the overall structure of this essential support organ differs, with humans and primates having a branched villous compartment, whereas in rodents, it forms a labyrinth. Such species-specific differences in placental architecture, along with variations in trophoblast invasion, vascular remodeling, and barrier composition, are key factors influencing maternal-fetal exchange and overall placental function24,25. Seeing the convenience and availability of tools, mice are still widely used as an experimental model of choice to study placental physiology and pathophysiology26. However, the murine labyrinthine placenta is characterized by a particularly shallow trophoblast invasion and limited spiral artery remodeling27; unique features that may limit their translational value for studying viral pathogenesis, associated inflammation, and immune responses at the maternal–fetal interface28. Moreover, mice are naturally resistant to clinical isolates of SARS-CoV-2, requiring exogenous expression of the human ACE2 receptor. However, in the latter model, pathology and mortality are skewed and dominated by neurotropic infection, questioning its translational relevance29,30.

In contrast to mice, Syrian hamsters (Mesocricetus auratus) naturally support SARS-CoV-2 infection, developing features of lung pathology similar to those observed in human COVID-1931. Hamsters have hence become an important model for the preclinical assessment of therapeutic (antivirals and disease-modifying treatments) and prophylactic interventions (vaccines and antibodies)32–34. Moreover, though less studied, the placentas of hamsters and humans appear to share a more similar overall anatomical structure compared to mice28. Specifically, the deeper trophoblast invasion resembling that seen in humans makes the hamster placenta better suited for studying placental dysfunction, a key factor in fetal growth restriction and placental insufficiency35. With a short gestation period (16 days) and large litters, hamsters offer further practical advantages for the study of pregnancy-related pathologies. As individual pups carried by one dam are multichorionic with separate placentas and umbilical cords, each hamster litter represents, on its own, a diverse birth cohort of multiple mother-placenta-offspring triads.

In this study, we explored the effects of SARS-CoV-2 infection on pregnancy using a hamster model, providing insight into the virus’s impact on both maternal and fetal health. Our results demonstrate that SARS-CoV-2-induced bronchopneumonia in pregnant dams leads to maternal health complications, including gestational age-dependent weight loss, although overall disease severity and lung pathology remained largely unaffected. Importantly, while vertical transmission was not observed, maternal infection significantly influenced intrauterine fetal development, markedly increasing the risk of intrauterine growth restriction (IUGR) without affecting litter size, fetal demise, or malformation rates. IUGR was linked to virus-induced placentitis characterized by pronounced fibrin deposition and tissue remodeling of the decidua, and persistent inflammation hallmarked by elevated IP10 (CXCL10), IL6, and IL10 expression in the placentas of affected pups. SARS-CoV-2-induced placental malfunction could be averted by anti-coagulative therapy, and placentitis and subsequent IUGR could be prevented by prophylactic immunization. By investigating these interactions, we highlight the risks posed by the virus and explore potential approaches, including treatments and vaccination, to protect both mother and child.

Results
Body weight loss due to SARS-CoV-2 infection in pregnant Syrian hamsters depends on gestational stage
To assess the effect of SARS-CoV-2 infection during pregnancy, after timed mating and confirmed insemination (Supplementary Fig. 1A), female Syrian hamsters were inoculated intranasally with SARS-CoV-2 (VOC Alpha; B.1.1.7) under mild anesthesia. B.1.1.7 has been extensively characterized to replicate efficiently in the lower respiratory tract of hamsters, inducing robust pulmonary pathology and a particularly strong induction of pro-inflammatory cytokines36. Infections were performed at specific embryonic ages (E), corresponding to key stages of pregnancy: insemination (E0.5), implantation (E4.5), placentation (E7.5), and a fully formed placenta (E10.5)37 (Fig. 1A) and confirmed 4 days later using buccal swabs with COVID-19 self-test kits (Supplementary Fig. 1B; specificity 100% [0/70 false positives]; sensitivity 96% [3/70 false negatives; validated by RT-qPCR). Non-infected pregnant dams (mock) were anesthetized at matched gestational time points for control, with undetectable changes in pregnancy outcome or body weight gain (Supplementary Fig. 1C, D).

Early gestational infections at E0.5 and E4.5 resulted in a significant weight loss starting at 4 days post-infection (dpi) and peaking at 7 dpi with a 5–6% reduction in body mass (Fig. 1B). However, initial body weight loss was followed by a sustained increase as pregnancy progressed. Despite general recovery, dams infected at time points before full placental development (earlier than E8) showed a significantly lower overall weight gain in the following 8 dpi (at E0.5 mean difference −24.96 g [95% CI −48.94 to −0.98], p=0.043; at E4.5 −42.85 g [−68.87 to −16.82], p=0.0047; and at E7.5 (−46.42 g [−81.11 to −11.73], p=0.015) as compared to respective mock controls (Fig. 1C). Likewise, at E15.5 (day of surgical delivery), animals subjected to infection at E4.5, E7.5, and E10.5 also appeared to be lighter with a generally reduced, though not significantly lower, net body weight gain compared to their respective mock-infected control groups (Supplementary Fig. 1E). As expected for infections with the relatively aggressive B.1.1.7. strain38, non-pregnant animals also showed a comparable pattern of weight loss, with a maximal reduction in body weight of 11% at 7 dpi. Over the 15 days of observation period, this corresponded to a significant cumulative weight loss of −13.58 g [−4.7 to −22.45] (p=0.0095, Supplementary Fig. 1F, G).

Both pregnancy and SARS-CoV-2 infection are known to influence hematological parameters. To explore this potential interaction, we analyzed the whole blood cell count in infected pregnant hamsters, comparing them to non-pregnant and non-infected pregnant females. At E15.5, pregnant hamsters exhibited a significant reduction in red blood cell counts (RBC, p=0.0458), hemoglobin (HGB, p=0.0088), and hematocrit (HTC, p=0.0458) compared to non-pregnant animals (Supplementary Fig. 2A).

Overall, these hematological adaptations in pregnant hamsters align with physiological adjustments known to support gestation in human pregnancy39,40. Also, white blood cell subsets (neutrophils, lymphocytes, basophils, monocytes, eosinophils) and platelet counts appeared altered in pregnant hamsters; however, they did not differ significantly from non-pregnant hamsters, perhaps due to the limited sample size (Supplementary Fig. 2B).

To examine the progression of SARS-CoV-2 infection in pregnant dams, we quantified viral RNA (vRNA) and infectious viral particles in the lungs on the day of surgical delivery (E15.5). In animals infected at E10.5, therefore dissected at 5 dpi, vRNA levels in lung tissue reached a median value of 6.2 Log10 [95% CI: 4.2–6.9] copies/mg. This was significantly reduced in dams infected at the earlier time points, hence dissected at 8, 11, and 15 dpi (Fig. 1D). Similarly, infectious virus was detected in all dams infected at E10.5 (5 dpi), with a TCID50/mg median value of 3.2 Log10 [95% CI: 2.3–4.3]. In line with our previous observation in non-pregnant B.1.1.7-infected hamsters38, infectious virus had become undetectable in animals infected more than 5 days ago, i.e., at the earlier time points during gestation (Fig. 1E).

Acute lung pathology was apparent at 5 dpi, characterized by alveolar hemorrhages, edema, endothelialitis, bronchopneumonia, and extensive diffuse alveolar damage affecting up to 40% of the visible lung surface (Supplementary Fig. 2C, D). These features were ameliorated in lungs from animals infected at earlier time points, resulting in an overall lower cumulative lung pathology score associated with injury; however, these differences did not reach statistical significance, likely due to the limited sample size (Fig. 1F). Features indicative of resolution of acute inflammation and of tissue healing (e.g., increased pneumocyte hyperplasia and absence of edema) were predominantly observed and significantly increased in dams infected at E4.5 (11 dpi; p=0.0067) and E7.5 (8 dpi; p=0.0025) in comparison to infection at E10.5 (5 dpi). Overall, these findings suggest that by 8 dpi, when the infectious virus had been cleared, lung pathological features shift from acute to reparative inflammation.

To confirm this, we next assessed the transcript levels of MX2, an interferon-stimulated gene as a proxy for a canonical type I interferon response, of major proinflammatory cytokines (IFNγ, IL6, TNFα, and IL1β), and of the IFNγ-induced chemokine IP10, which are upregulated in the lungs after SARS-CoV-2 infection. In line with our previous findings31,38, we observed a significant increase of these cytokines in dams infected at E10.5, with notably elevated levels of IP10 and MX2 (median fold change [95% CI] of 26.0 [7.4–396.2] and 46.7 [19.2–91.1], respectively). In contrast, after this initial peak, antiviral gene expression appeared to settle as in dams infected at E4.5, only lower levels of MX2 were detected anymore (median fold change of 1.54 [0.7–2.1]), likely due to a remnant type I IFN response induced by residual vRNA41 (Fig. 1G).

Maternal SARS-CoV-2 infection leads to intrauterine growth restriction in a gestational age-dependent manner
To assess the impact of SARS-CoV-2 infection and maternal health during pregnancy on their offspring, we performed surgical delivery of pups from infected pregnant hamsters before the onset of labor (E15.5). Despite the transient decrease in maternal weight at early gestational stages (Fig. 1B), the litter sizes of infected dams were similar to those of mock-infected controls and across experimental groups (p=0.9327; Fig. 2A). This suggests that, at least in hamsters, SARS-CoV-2 infection does not hinder implantation. Similarly, the rate of resorption of implanted embryos, a potential consequence of maternal stress and inflammation associated with infection, did not significantly differ between infected and non-infected dams, irrespective of the timing of infection (p=0.4573; Supplementary Fig. 3A).

Fetal sex has been shown to be an important confounding factor that determines fetal growth, pregnancy outcomes, and susceptibility to congenital disease in both rodents and humans8,42–44. Indeed, mock-infected dams bore male pups that were on average about 5.1% heavier than female pups (mean ± SD; 2.47 ± 0.33 vs. 2.35 ± 0.32; p=0.0128, Fig. 2B, C). Likewise, placentas from male pups were significantly heavier than female placentas (p=0.0006; Fig. 2C, E). Sex-specific pregnancy rates did not differ between mock-infected dams and those infected at different gestational ages. Moreover, these rates in infected dams were not significantly different from the expected theoretical 1:1 sex ratio, suggestive of equal spontaneous resorption rates (Supplementary Fig. 3B and Source Data).

Macroscopic examination of the pups revealed no evidence of congenital malformations. However, dams infected after placentation (after E4.5) had smaller pups of both sexes, with significantly reduced body weight (Fig. 2B, C). Notably, infection at E4.5 resulted in the most pronounced reduction in pup weight, with a mean difference of 0.47 g in males and 0.26 g in females (Fig. 2C), corresponding to 20% and 10% reductions, respectively. A significant weight reduction was also observed in female pups from dams infected at E10.5 (mean difference: 0.22 g; p=0.0122), whereas no significant effects were detected in male pups at this gestational age (Fig. 2C). Infections at E0.5 and E7.5 did not influence fetal weight in either sex. Interestingly, litters from infected dams were not uniformly affected, with low-weight pups clustered among littermates of normal weight within the same pregnancy and uterine horn (Fig. 2B).

In line with human clinical definitions, pups with birth weights below the 10th percentile of their respective mock-infected littermates were designated as small for gestational age (SGA). This classification was used to assess the risk of intrauterine growth restriction (IUGR; also referred to as fetal growth restriction, FGR) associated with maternal SARS-CoV-2 infection. We found that infection with SARS-CoV-2 at E4.5 exhibited a higher likelihood of delivering SGA pups, with the highest risk observed in male offspring (odds ratio [95% CI]: 9.8 [3.4–26.2]; females: 3.6 [1.4–8.9]) (Fig. 2D).

IUGR induced by acute viral infections often results from placental dysfunction driven by inflammation and altered cytokine profiles. These factors can lead to inadequate placental development and disruption of uterine spiral artery remodeling, reducing placental perfusion and impairing the transfer of nutrients and oxygen to the fetus45–48. Indeed, we found that placentas from dams infected at E4.5 had a significantly smaller weight in both sexes, in comparison to their respective non-infected controls. In contrast, placentas from dams infected at E10.5 were on average larger, although these differences did not reach statistical significance, with mean increases 15% (p=0.06) in males and 11% (p=0.09) in females (Fig. 2E). This trend suggests that early placental alterations may begin to emerge around this gestational stage, potentially coinciding with maternal lung pathology or localized inflammatory responses.

To test this possibility, we determined the vRNA content in whole-placenta homogenates. Interestingly, despite the rare detection of placental infection in humans49, vRNA was present in 64% of the placentas (43 out of 67) from dams infected at E10.5 (5 dpi), at concentrations up to 1000 copies/mg of tissue. There was also no difference in placental vRNA between female and male fetuses from the E10.5 infection time point (Fig. 2F). In parallel with the decline of vRNA and coinciding with the absence of infectious virus in the lungs of infected dams, vRNA became undetectable in the placentas at subsequent time points post-infection (11 dpi). To further investigate the potential of SARS-CoV-2 to cross the placental barrier, we analyzed the fetal liver, an organ identified as a potential site for viral replication50. However, vRNA was never detected in any fetal liver despite being in the immediate proximity to the fetal–maternal interface (Fig. 2F).

Altogether, an original respiratory SARS-CoV-2 infection of pregnant dams frequently results in a transient infection of the placenta. In the absence of direct transplacental transmission, the unborn is put at risk of IUGR, primarily related to pathological alterations in the placenta.

Persistent placental inflammation following viral clearance
To further investigate the impact of the maternal COVID-19-like disease and the resulting transient presence of viral RNA in the placenta, we performed a histological analysis of the E10.5 and E4.5 groups, focusing on structural integrity and morphological alterations of the placental tissues. Similar to other rodents, as well as humans and non-human primates, hamsters develop a hemochorial placenta, divided into two anatomically distinct layers. In the case of rodents, these are the junctional zone (JZ), responsible for energy (glycogen) and hormone production, and the labyrinth, which facilitates maternal–fetal exchange51,52. In non-infected placentas at late pregnancy (E15.5), only low levels of necrotic areas, fibrin deposition, and thrombi were observed, predominantly localized in the JZ. Additionally, the JZ exhibited significant congestion, characterized by enlarged sinuses filled with red blood cells (Fig. 3A, B), likely reflecting changes in blood flow and tissue remodeling associated with the progression toward parturition (at term, i.e., E16)53.

Despite the higher weight of placentas from animals infected late during gestation at E10.5 (5 dpi, Fig. 2E), we did not observe any significant change in the ratio of their JZ to labyrinth areas, a parameter associated with placental insufficiency52 (Fig. 3C). However, placentas from infected animals exhibited increased perivascular fibrin deposition, thrombosis, and necrosis, primarily localized to the JZ. These placentas also contained an increased number of nucleated red blood cells (nRBCs) within the fetal vasculature in the labyrinth, generally interpreted to result from a fetal stress response54 (Fig. 3A, B). Consequently, placentas from infected animals had significantly higher cumulative pathological scores compared to those from non-infected animals (Fig. 3D). These histopathological changes and a similarly high cumulative score were also observed in placentas from animals infected at E4.5 (11 dpi, Fig. 3A–D), indicating that tissue injury persists despite the effective elimination of vRNA from the placentas at these later time points (Supplementary Data 3), as would be expected since histological lesions often lag behind viral clearance owing to ongoing inflammation and repair processes.

Next, we investigated whether the presence of vRNA in the placenta during the acute phase of infection correlated with an increased expression of markers associated with immune cell infiltration and pro-inflammatory cytokine responses. To this end, we quantified the transcript levels of markers for NK cells (KLRG1, CD94), CD4+ (CD4) and CD8+ (CD8) T cells, as well as genes associated with type I, II, and III IFN responses (MX2, IP10, and IL28, respectively), and expression of cytokines IL6 (IL6), and IL10 (IL10). Compared to placentas from non-infected animals, those with detectable vRNA levels at 5 dpi (dams infected at E10.5) exhibited significantly increased transcript levels of CD4 and KLRG1 (Supplementary Fig. 4A). Notably, an upregulation of these cell identity markers was observed exclusively in placentas with detectable vRNA. A similar trend, yet not reaching statistical significance, was observed for CD8 and CD94 transcript levels.

Our multimodal analysis of individual mother–placenta–offspring triads revealed a certain heterogeneity. For instance, each infected dam carried fetuses representing a wide weight range (normal to SGA) and varying signs of placental involvement (infection and placentitis). We hence sought to identify whether a specific molecular signature correlated with the heterogeneity observed in placental infection and IUGR. For this, principal component analysis (PCA) was performed using key physiological and molecular variables, including fetal and placental weight, transcript levels of placental MX2, IL28, IP10, IL6, IL10, and vRNA using the compiled data from our E10.5 cohort of in total n=65 pups (6 mothers). The first two principal components (PC1 and PC2) accounted for 56.3% of the total variance in the dataset, enabling clear visualization of clustering patterns. Subsequent K-means clustering analysis identified three distinct clusters, which were clearly separated within the PCA space defined by PC1 and PC2 (Fig. 3E).

Cluster 1 predominantly comprised placentas with undetectable vRNA and increased MX2 expression. In contrast, Cluster 3 included placentas with detectable vRNA content and high expression of all assessed genes. (Fig. 3F and Supplementary Fig. 4C). Interestingly, Cluster 2, despite also containing placentas with detectable vRNA, was characterized by a low expression of all transcripts, except for a significant upregulation of IL10 (Fig. 3F and Supplementary Fig. 4C). The average fetal weight of Cluster 2 was significantly higher than Clusters 1 and 3. Notably, Cluster 2 did not contain any SGA pups, whereas the increased Odds ratio for IUGR observed at E10.5 remained unchanged for Clusters 1 and 3 (Fig. 3G). Overall, these clusters depict three different pregnancy outcomes, whereby the health of the fetus is not necessarily determined directly by the presence of the virus in the placenta, but rather indirectly by the pro-inflammatory response (immunopathogenesis).

To support this hypothesis, we performed a similar analysis in placentas from E4.5 pups (n=57, 6 mothers). At this time point after infection (11 dpi), vRNA is undetectable in the full cohort (Fig. 2F), and levels of CD4, CD8, CD94, and KLRG1 are equally downregulated in SGA pups and those above the 10th percentile (Supplementary Fig. 4F). E4.5 pups were separated into two clusters (Clusters 4 and 5), clearly defined by a significantly increased IL28 and IP10 expression in Cluster 4 (Fig. 3H, I and Supplementary Fig. 4G). Cluster 4 contained pups associated with smaller placentas (Supplementary Fig. 4H), with a smaller average fetal weight and a higher number of SGA pups (Fig. 3J), resulting in an increased OR, compared to Cluster 5 (Fig. 3K).

Importantly, these clusters contained placentas from at least three different dams, indicating that the cluster segregation was not biased by individual dams. Likewise, multivariable linear regression with covariate adjustment demonstrated that offspring sex had a negligible impact on cluster-associated effects, indicating that sex does not act as a confounder for these outcomes (Supplementary Fig. 4B, E).

Altogether, our results indicate that irrespective of the health status of the mother (acute versus resolving pneumonia) and the time of infection, the pregnancy outcome after SARS-CoV-2 infection in hamsters is intrinsically heterogeneous, worsened by a sustained innate immune response to the virus rather than by placental infection or vertical transmission.

Administration of low molecular weight heparin improves pregnancy outcomes in SARS-CoV-2-infected hamsters
In our model, we observe significant placental pathology, including fibrosis and thrombosis, mirroring complications reported during human pregnancies affected by COVID-19. Since COVID-19 is known to cause systemic coagulopathy55 and placentitis in pregnant women56, we evaluated whether low molecular weight heparin (enoxaparin)57 could mitigate adverse pregnancy outcomes in Syrian hamsters following SARS-CoV-2 infection. To that end, we focused on the E4.5 group, which showed the most distinct adverse effects on fetal outcomes. To assess the potential protective effects of anticoagulation therapy, we administered enoxaparin, a well-established anticoagulant used to prevent thromboembolism in pregnancy58, at a dosage of 3 µg/g body weight. Treatment was initiated in a post-exposure setting at 3 dpi (E7.5) around the expected peak of infection31,38 and continued for 7 consecutive days until E14.5 via daily subcutaneous injections (Supplementary Fig. 4A).

Enoxaparin treatment in B.1.1.7-infected dams resulted in significantly increased fetal and placental weights compared to untreated controls (p=0.0023 and p=0.0006, respectively; Fig. 4A, B) without affecting litter size (Fig. 4C). Similarly, in mock-infected females, pups from heparin-treated dams exhibited greater fetal weights compared to untreated counterparts, with no difference in body weight gain and litter size (Supplementary Fig. 4B–D). This indicates that heparin administration did not adversely affect pregnancy outcomes. By contrast, fetal and placental development seem to benefit from enoxaparin administration, likely by counteracting an elevated pro-coagulative tone that pregnancy may induce in hamsters, alike in humans.

Histological analysis of placentas from infected dams showed an improvement in pathology scores following anticoagulant treatment (mean difference 4.0, 95% CI: −6.7 to −1.2, p=0.0066), characterized by reduced congestion, fibrosis, and necrosis in the junctional zone and labyrinth. These results underscore the potential of enoxaparin to mitigate SARS-CoV-2-induced placental pathology and associated complications (Fig. 4D and Supplementary Data 3). The treatment showed no measurable impact on the relative proportions of placental zones (Supplementary Fig. 4E). Interestingly, placentas from enoxaparin-treated infected dams exhibited an increased number of nRBCs compared with the untreated group (Fig. 4D and Supplementary Fig. 4F). Although elevated nRBCs are typically associated with fetal hypoxia or stress and reflect compensatory erythropoiesis, their increase in this context may instead relate to the additional immune-modulatory functions of nRBCs59,60. This possibility is consistent with the observed reduction in the frequency of SGA pups following enoxaparin treatment. Nonetheless, further investigation is required to clarify the underlying mechanism (Fig. 4D and Supplementary Fig. 4F).

Despite the obvious positive effects on fetal and placental outcomes, enoxaparin treatment did not exert any antiviral effect and did not enhance the overall health of the dams. Likewise, they continued to lose weight throughout infection and gained significantly less cumulative weight than mock-infected dams (Fig. 4E, F). As expected, also viral RNA levels in their oral swabs remained unaffected, similar to those of infected, untreated controls (Fig. 4G).

COVID-19 vaccination protects from adverse pregnancy outcomes
Besides the mother herself, maternal immunity may also protect the unborn. Seroconversion kinetics to anti-SARS-CoV-2 antibodies in pregnant hamsters were monitored starting from challenge with B.1.1.7. at various time points (i.e., E0.5, E4.5, and E10.5 infection) until the end of gestation (E15.5) (Supplementary Fig. 6A). Overall, pregnancy did not impair humoral immune responses. By 5 dpi (E10.5 infection), spike-specific IgG levels in previously naïve dams had already increased above baseline (MFI 3.0 Log10; 95% CI: 2.9–3.1 vs. uninfected controls MFI 1.9 Log10; 95% CI: 1.7–2.1), as measured by flow cytometry61. Antibody levels continued to rise, reaching MFIs of 3.6 Log10 (95% CI: 3.5–3.6) at 11 dpi (E4.5) and 3.8 Log10 (95% CI: 3.7–3.9) at 15 dpi (E0.5), indicating a robust and progressive humoral response throughout pregnancy (Supplementary Fig. 5A, B). Virus-neutralizing antibody (nAb) developed with a slight delay (Supplementary Fig. 5C), whereby nAb activity remained barely detectable early at 5 dpi. Seroprotective levels33 were measured only from 11 dpi (E4.5) onwards (Supplementary Fig. 5C).

A primary infection triggers a rapid adaptive immune response in naïve individuals; however, it is not rapid enough to avert placentitis and IUGR during pregnancy (Fig. 3B). We previously established the safety, immunogenicity, and efficacy of a series of experimental YF17D-vectored COVID-19 vaccine candidates (lead YF-S0) in a range of preclinical animal models32,33,62. The updated construct YF-S0*, encoding antigenic features of the Gamma variant of concern (VOC), along with stabilizing amino acid changes to improve its immunogenicity33, has proven efficacious in protecting hamsters from experimental B.1.1.7 infection transmission, like licensed mRNA vaccines may do61, yet also from horizontal SARS-CoV-2 transmission. Here, YF-S0* was used in a proof-of-concept study, where female hamsters were vaccinated in a prime-boost regimen prior to conception to prevent COVID-19-related complications during pregnancy. Two cohorts of vaccinated animals were used for challenge infection, either at E4.5 (Fig. 5A) or E10.5 (Supplementary Fig. 5D), along with naïve controls. Seroconversion to consistently high levels of spike-specific IgG in these immunized animals prior to infection was confirmed by flow cytometry (Supplementary Fig. 5E, F).

Vaccinated animals and respective naïve controls infected at E4.5 were followed for 11 days until the end of gestation. In line with previous experience32,33,61, vaccination conferred a high degree of protection, seen as overall lower and faster decaying viral RNA loads in buccal swabs (Fig. 5B, C), accompanied by a markedly reduced lung pathology (Fig. 5D). Moreover, in vaccinated dams, infection triggered a rapid antibody recall as seen in vaccinated animals challenged from the E10.5 cohort and bled already 5 dpi for serum collection. In contrast to naïve individuals (Supplementary Fig. 5G), previously immunized animals had already seroconverted to protective levels of nAb within that time frame (Supplementary Fig. 5G). These animals were also protected from severe lung injury (Supplementary Fig. 5H), although vaccination did not prevent the adverse effects of infection at E10.5 on fetal and placental weight (Supplementary Fig. 5I) despite a significant reduction in placental damage (p=0.0016; Supplementary Fig. 5J–M). In contrast, pups of vaccinated dams that had been infected at E4.5 were significantly larger, and their placentas weighed more than those from unvaccinated controls (p=0.0022 and p=0.0259, respectively) (Fig. 5E). While vaccination did not affect overall placental zone ratios (Fig. 5F, G and Supplementary Data 3), placental damage scores were significantly reduced in vaccinated dams relative to non-vaccinated controls (p=0.0025). The most prominent histopathological improvements were reduced fibrin deposition, thrombosis, and necrosis within the placental tissue (Fig. 5H, I), further corroborating the link between maternal COVID-19, placentitis, and fetal health.

These findings, combined with an enhanced neutralizing antibody response (Supplementary Fig. 5G) and efficient viral control (Fig. 5B, C), underscore the multifaceted benefit of COVID-19 vaccine prophylaxis prior to conception, safeguarding maternal health and fetal development in case of SARS-CoV-2 exposure during pregnancy.

Methods
Study approval
Animal experiments were approved by the KU Leuven ethical committee (project number P165-2022), conducted following Federation of European Laboratory Animal Science Associations (FELASA) guidelines, and reported according to ARRIVE (Animal Research: Reporting of In Vivo Experiments).

Animals
Wild-type Syrian hamsters (8-week-old females and males) (Mesocricetus auratus, strain RjHan:AURA) were purchased from Janvier Laboratories. Animals were housed separately in individually ventilated cages (IsoCage N Biocontainment System, Tecniplast) at a temperature of 21°C, humidity of 55%, and 12:12 dark/light cycles, with ad libitum access to food and water and cage enrichment (cardboard houses and wood blocks). Assignment of the groups was blinded for operators, except for the lead investigator. Sample sizes were defined a priori by performing a power analysis with the expected effect size based on the number of pups and adjusted post-hoc based on the frequency of small for gestational age, with a statistical power of 80%, a significance level of 0.05, using Gpower and Sample size calculator Version 1.062 of the Universität Wien93. Only wild-type female hamsters were used in this study, as females are biologically required to investigate the effects of infections during pregnancy. Offspring sex was considered a variable in the assessment of embryonic outcomes.

Viruses
All research involving viruses was conducted in the high-containment BSL3 facilities of the KU Leuven Rega Institute, under licenses AMV 30112018 SBB 2192018 0892 and AMV 23102017 SBB 2192017 0589, following institutional guidelines.
SARS-CoV-2 VOC Alpha (B.1.1.7) virus stocks (derived from hCoV-19/Belgium/rega-12211513/2020; EPI_ISL_791333, 2020-12-21) were grown on Vero E6 cells, and median tissue culture infectious doses (TCID50) were defined by end-point titration, as previously described38.

SARS-CoV-2 infection model in pregnant hamsters
Females were allowed to acclimatize for two weeks and mated with a fertile male overnight during the proestrus phase (3 days after copious vaginal secretion)94. Copulation was confirmed the morning after mating (defined as E0.5) by the presence of sperm in a smear of a vaginal swab (top picture, Supplementary Fig. 1A). Brightfield images of vaginal smears mounted on glass slides were acquired using a Floid Cell Imaging Station (Invitrogen, Thermo Fisher Scientific, Cat#4471136). Image analysis was performed in Fiji95 using a custom macro with the following steps: run(“8-bit”); setAutoThreshold(“Default”); setOption(“BlackBackground”, true); run(“Convert to Mask”); run(“Despeckle”); run(“Analyze Particles…”, “size=80−Infinity circularity=0.00–0.40 show=Outlines clear summarize add”).

The area covered by sperm was normalized to the total image area and reported as sperm content (percentage of image area). Despite considerable variability in litter size at low sperm content (<15%), we observed a weak but statistically significant correlation between sperm content and litter size (R=0.39, p=0.0057). Notably, all instances of pregnancy failure occurred when sperm content was below 15% (6 out of 28; Supplementary Fig. 1A). Females with undetectable vaginal sperm were remated during the subsequent estrous cycle, while those without an estrous cycle were excluded.

Pregnant females were randomly allocated to experimental groups. Infection with SARS-CoV-2 was carried out as previously described31 at different time points during pregnancy (E0.5, E4.5, E7.5, E10.5). Briefly, the animals were anesthetized by intraperitoneal (i.p.) injection of a xylazine (16 mg/kg, XYL-M®, V.M.D.), ketamine (40 mg/kg, Nimatek, EuroVet), and atropine (0.2 mg/kg, Sterop) solution. Each animal was inoculated intranasally with 50 µL droplets in both nostrils of virus stock containing 104 TCID50 of the B.1.1.7 variant. Non-infected animals (mock) were also anesthetized but left untreated. Where indicated, the low-molecular-weight heparin derivative enoxaparin (0.3 IU/g, Clexane 10,000 IU, Sanofi) was administered subcutaneously once daily for seven days, from E7.5 to E14.5, in both the infected and mock-infected groups. Infected dams were checked daily for signs of disease (lethargy, heavy breathing, or ruffled fur). An antigen self-test (Self Test NEWGENE Covid-19, Cat#5501696) was performed 4 days post-infection (dpi) using throat swabs to confirm successful infection, as described before96 (Supplementary Fig. 1B). One day before delivery (at E15.5), females were euthanized by i.p. injection of Dolethal (500 µL, 200 mg/mL sodium pentobarbital, Vétoquinol SA). Fetuses were dissected by C-section, kept in PBS, and patted dry before weighing. Maternal blood, lungs, spleen, placentas, and fetal livers were collected for further analysis (viral RNA, infectious virus, cytokines, histopathological examination, and whole blood count). Complete blood counts were performed using an automated Element HT5 analyzer, Heska Corporation.

Immunization of hamsters
Hamsters were vaccinated i.p. with 104 PFU in 500 µL of the YF-S0* vaccine, using a prime and boost regimen (at day −28 and day −21) that induces high levels of nAb and strong antiviral cellular immune response33. Blood was drawn via the jugular vein under isoflurane anesthesia 14 days post-vaccination for serological analysis. Immunized animals were mated within 3 weeks after the booster dose. After successful mating, they were randomly allocated to experimental groups.

Genomic DNA isolation and sex genotyping
Sex determination of dissected pups was performed using placental tissue and custom-designed, validated primer sets97. For this, genomic DNA (gDNA) was isolated from ~5 to 10 mg of hamster placental tissue or 1–2 mm ear biopsies using the Platinum™ Direct PCR Universal Master Mix (Invitrogen, Thermo Fisher Scientific, Cat#A44647100) following the manufacturer’s lysis protocol.

qPCR-based genotyping was performed using the iTaq™ Universal SYBR® Green One-Step Kit (Bio-Rad, Cat#1725150) on a LightCycler® 96 system (Roche Diagnostics). Reactions (20 µL) contained 10 µL of SYBR Green master mix, 0.2 µL each of 1:10-diluted forward and reverse primers (Supplementary Data 1), 0.25 µL reverse transcriptase, 7.35 µL nuclease-free water, and 2 µL gDNA. Cycling conditions were as follows: 94°C for 2 min; 40 cycles of 94°C for 15 s, 55°C for 15 s, and 68°C for 20 s. A melt-curve analysis was performed immediately after amplification, using sequential 10 s holds at 0.5°C increments from 65 to 95°C. Male sex was assigned based on the characteristic melting-curve profile. Male samples consistently exhibited two distinct melt peaks at ~76.5 and 79.0°C. Female samples, by contrast, showed no amplification or only late, aberrant amplification, resulting in either indeterminate melt curves or inconsistent, non-reproducible profiles lacking the two characteristic peaks.

RNA extraction and RT-qPCR
Hamster lung tissues, placentas, and fetal livers were collected and homogenized using bead disruption (Precellys kit, Bertin Corp., Cat#432-0141) in TRK lysis buffer (E.Z.N.A.® Total RNA Kit, Omega Bio-tek, Cat#R6834-02) and centrifuged (16,000 × g, 5 min) to pellet the cell debris. The NucleoSpin kit (Macherey-Nagel, Cat#740956.250) was used for throat swabs. RNA was extracted according to the manufacturer’s instructions and eluted in 50 µL. From this, 4 µL were used as a template for RT-qPCR reactions. RT-qPCR was performed in duplicates on a LightCycler 96 platform (Roche Diagnostics) using the iTaq Universal Probes One-Step RT-qPCR kit (BioRad, Cat#1725141) with N2 primers and probes targeting the nucleocapsid (IDT, Cat#10006713) and specific hamster β-ACTIN, IL6, IL10, IL28, IFNγ, MX2, IP10, TNFα31 (Supplementary Data 1). SARS-CoV-2 cDNA control (IDT, Cat#10006625) was used to calculate genome copies per mg tissue or mL of oral swab. Cytokine expression levels were normalized to the mean expression of β-ACTIN transcripts. The lower limit of detection (LLOD) for SARS-CoV-2 was estimated at 7150 copies per mL, based on an analysis of repeated standard curves. Samples with values below the LLOD were considered negative.

Endpoint virus titrations
The lung tissues were homogenized using bead disruption (Precellys kit, Bertin Corp., Cat#432-0141) in 350 µL of minimal essential medium. After centrifugation (16,000 × g, 5 min, 4°C) to pellet the cell debris, endpoint titrations in triplicate were carried out on confluent Vero E6 cells seeded on 96-well plates. Viral titers were determined using the Reed and Muench method with the Lindenbach calculator98 and were expressed as 50% tissue culture infectious dose (TCID50) per mg tissue.

Serum neutralization test (SNT)
To measure SARS-CoV-2 neutralizing antibodies (nAb), serial dilutions of heat-inactivated serum samples (25 µL) were mixed with an equal volume of 100 TCID50 of SARS-CoV-2 B.1.1.7 and incubated for 1 h at 37°C. Following incubation, the serum-virus mixtures were added to 96-well plates containing Vero E6 cells that had been seeded the previous day at 104 cells per well. Cell viability was assessed after 96 h using an MTS reduction assay. To this end, the medium was aspirated from the wells, and 75 µL of an MTS/phenazine methyl sulfate (PMS) solution (2 mg/mL MTS (Promega, Cat#G1111) and 46 µg/mL PMS (Sigma-Aldrich, Cat#P9625) in PBS at pH 6–6.5) was added. Following a 2-h incubation at 37°C, absorbance was measured at 498 nm with a Spark® multimode microplate reader (Tecan, Männedorf, Switzerland).

Flow cytometry
Total anti-S-protein IgG antibodies induced by vaccination were evaluated in serum samples by flow cytometry using HEK cells stably expressing the S protein from the Wuhan-Hu-1 strain (hereafter, Mono8 cells). Mono8 cells (105 cells/well) were plated in a U-bottom 96-well plate (Corning Inc., Cat#3799) and blocked for 20 min in ice-cold FACS buffer (2% fetal calf serum in Dulbecco’s phosphate-buffered saline (Gibco) supplemented with 2.5 mM EDTA (Invitrogen, Cat#15-575-020). The cells were pelleted by centrifugation at 500 × g for 2 min and resuspended in prediluted serum samples (1:20 in FACS buffer). The cells were incubated on ice for 20 min. After two washing steps, the cells were incubated for 20 min on ice with FITC-conjugated rabbit anti-hamster IgG antibody (1:500 in FACS buffer; Jackson ImmunoResearch Labs, Cat#307-095-003). Subsequently, the cells were washed and incubated (15 min, RT, protected from light) in a 4% paraformaldehyde solution (16% Formaldehyde (w/v), methanol-free; Thermo Fisher Scientific, Cat#28908) for 10 min at room temperature, protected from light. After fixation, the cells were washed and resuspended in a FACS buffer. The acquisition was performed in a BD LSRFortessa™ X-20 Cell Analyzer (BD Biosciences). Data analysis and representation were performed in FlowJo™ v10.8 Software (BD Life Sciences). The gating strategy is depicted in Supplementary Fig. 6A.

Histology
After fixation in 4% paraformaldehyde, lung and placental tissue were paraffin-embedded and sectioned at 5 and 4 µm, respectively. Sections were stained with hematoxylin and eosin and scored blindly for lung or placenta damage. The following parameters were included in the scoring of the lungs99 by a trained pathologist blinded to the experimental groups: congestion, intra-alveolar hemorrhage, apoptotic bodies in bronchial epithelium, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular inflammation, peribronchial inflammation, vasculitis, and sinus congestion. Placentas were further stained with Masson trichrome and isolectin to visualize fibrosis and fetal blood vessels100,101, respectively.

Placental histopathology was evaluated using QuPath (version 0.5.1). Based on previously described lesions in animal placenta studies102,103, each placenta was semi-quantitatively scored in a blinded manner. The scoring system was as follows: unremarkable=0, mild=1, moderate=2, and marked=3, applied separately to the junctional zone (JZ) and labyrinth (Lab) for congestion (i.e. dilated and engorged blood vessels filled with red blood cells), fibrin deposition, thrombi, immune cell infiltration, necrosis, and perivascular fibrin deposition, based on the proportion of tissue area affected. Nucleated red blood cells (nRBCs) were quantified per histological section and scored relative to non-infected controls, using the following scores: 0: <30; 1: 30–60; 2: 60–90; 3: >90 nRBCs per section. For morphometric analysis, five regions per section were measured for JZ and Lab areas in QuPath, and the mean areas were used to calculate the JZ:Lab ratio, serving as an index of placental insufficiency52.

Statistics
All statistical analyses were performed using GraphPad Prism (version 10.6.1; GraphPad Software, Inc.). Principal component (PC) analysis and K-means clustering were performed using standardized data (mean=0, SD=1). K-means clustering was performed using the Hartigan–Wong algorithm with a maximum of 100 iterations. The Pearson correlation distance metric was used to determine similarity between samples. The maximum number of clusters was set to six, and the random seed was set to “Auto” to ensure reproducibility. Clustering results were visualized in the PCA space defined by the first two principal components (PC1 and PC2).

To evaluate whether maternal infection influenced the sex distribution of offspring, we fitted a binomial generalized linear model (GLM) with logit link using the number of female pups versus total pups per litter as the outcome and treatment group as the predictor. Estimated proportions of female pups and 95% confidence intervals were computed using the emmeans package (type=“response”). Pairwise comparisons between treatment groups were performed using Tukey-adjusted contrasts. To assess deviation from the theoretical 1:1 sex ratio, estimated proportions for each group were tested against 0.5 using model-based one-sample comparisons. Model diagnostics included dispersion assessment based on Pearson residuals.

To evaluate whether offspring sex acted as a confounding variable in the association between PCA-defined clusters and biological outcomes, we examined both its association with the exposure (Cluster) and its effect on the outcome estimates. First, independence between sex and cluster membership was assessed using contingency tables and Fisher’s exact test due to small sample sizes. For each continuous outcome (pup weight, placental weight, cytokine concentrations, and placental viral RNA levels), we specified nested linear regression models: (i) an unadjusted model including only Cluster as a predictor (Outcome~Cluster) and (ii) an adjusted model including both Cluster and Sex (Outcome~Cluster+Sex). Confounding was evaluated by comparing the estimated cluster coefficients between models and by assessing the statistical significance of the Sex term. Linear model assumptions were verified through residual diagnostics.

The analysis of sex distribution and confounding effect was performed in R (v.4.5.1), and the complete analysis scripts are available on GitHub (https://github.com/Molecular-Vaccinology-Vaccine-Discovery/Kumpanenko-et-al-Nat-Commun-2026) provide information on the sample size and statistical test being used. Unless otherwise stated, only statistically significant differences (p<0.05) are displayed in the figures. All p-values are listed in the Source Data file. The number of individual pups and placentas analyzed per experimental group is provided in Supplementary Data 2.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript investigated SARS-CoV-2 infection at different key timepoints during pregnancy in Syrian hamsters. The\nauthors showed that maternal infection leads to intrauterine growth restriction (IUGR) due to placental inflammation without\nany vertical transmission. Vaccination and anticoagulant therapies were found to protect the placenta and improve fetal\noutcomes, highlighting their importance in maternal/pregnancy care. This study is thorough and well conducted and\naddresses an important topic with significant implications for maternal and fetal health during SARS-CoV2 infection. The\narticle is clearly written, with a logical flow, thorough explanations of the results and adequate discussion with a section on\nthe limitation of the work. Overall, I believe this manuscript is well-suited for publication pending minor revisions.\nGeneral comments:\nI understand the need for the authors to perform their analysis based on days post-infection (dpi). However, dpi will\nobviously vary depending on when the infection occurred and the length of exposure will vary. Considering that the\nplacenta, unlike maternal organs, is still growing during pregnancy and its function evolves, could terminal c-section not\nhave been conducted at the same dpi for each timepoints of infection to address is the placental susceptibility/response to\ninfection? This might be beyond the scope of the work and could be addressed in the discussion.\nMinor comments:\nIntroduction: Short, concise and well structured. The authors could add more details on human placental pathology studies\nfollowing SARS-CoV-2 infection considering that there are several published studies and their work has a fair amount of\nhistopathology analysis. This would offer context for interpreting the findings from the hamster placenta.\nMethods: The manuscript is well written and provides sufficient methodological detail to allow for experimental\nreproducibility. Was fetal/placental sex considered as a variable? Given that females tend to be lighter than males, this could\nbe a relevant factor, particularly if there are discrepancies in survival rates among the pups.\nI understand that the vaccine used is YF-S0* but in Fig.5 and elsewhere in the text they write YF17D-vectored. Which might\nnot be adequate since YF-S0* is an updated construct of YF17D.\nShould the “Study Approval” section be placed before the “Animal” section at line 683?\nResults: The results are well structured, and the inclusion of the therapeutic intervention significantly strengthens the study. I\nalso appreciated the addition of the PCA analysis, which effectively highlights distinct molecular signatures and helps\nexplain the observed heterogeneity.\n- Line 152-156, this sentence should be rephrased to improve clarity\n- Line 192-194, does the authors have any data supporting this statement: “suggestive of a compensatory adaptation in\nresponse to an increased respiratory stress.”? If not I would suggest removing.\n-\n- Even if cytokine transcript levels are not elevated at later dpi, this only indicates that inflammation has been resolved by\nthat point—but it does not necessarily mean that inflammation did not occur earlier or have other effects. This is aligned with\nthe “general comment” above and would be an interesting point of discussion.\n-\n- Line 300, the authors state that analyses were performed at E4.5, but then shift to discussing E10.5 at line 312. To improve\nclarity, it might be helpful to mention both E4.5 and E10.5 at the beginning of this section.\n- Fig. 3A and Fig. 5F: the purple arrows are very difficult to see in the background. It may be helpful to choose a more\ncontrasting color to improve visibility.\n- Fig. 4F: since the graph represents only a single timepoint, could the authors clarify why lines 437–438 refer to “up to 8\ndays post-infection”? Or were several time points combined here?\n- Fig. 5: could the authors clarify why the vaccinated, non-infected group is not included in this figure? Including this group\nwould help isolate the effects of vaccination alone.\nDiscussion: The discussion is well structured and clearly presented. However, it would benefit from a deeper comparison\nwith existing literature, particularly in relation to the main findings. Currently, the focus is primarily on the treatment and\nvaccine aspects. On the other hand, the limitations section is thorough and well-articulated, effectively addressing key\nconstraints of the study.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "Title: COVID-19-related inflammation of the placenta impedes fetal development in pregnant hamsters\nSummary/Key Results: The introduction sets the stage well, establishing that, while SARS-CoV-2 infection is associated\nwith increased disease severity in expectant mothers as well as fetal complications such as compromised placental\ninsufficiency/hypoxic injury, mechanisms of SARS-CoV-2 induced maternal and fetal pathology are poorly understood.\nAdditionally, vaccine hesitancy remains common among pregnant women.\nHere, authors explored the effects of SARS-CoV-2 infection on pregnancy in hamsters, which is an appropriate animal\nmodel for this study. Hamsters are naturally susceptible to SARS-CoV-2 infection, exhibiting similar pulmonary pathology to\nhuman COVID-19 disease. Furthermore, hamsters and humans have some similarities in overall placental anatomical\nstructure- specifically hemochorial placentation. But, in contrast to the human hemomonochorial placental interface\n(characterized by deep trophoblast invasion of the maternal uterus), the labyrinthine rodent placenta generally exhibits a\nhemotrichorial interface with multiple trophoblast layers. Deeper trophoblast invasion in hamsters (vs. mice), coupled with\nthe fact that individual pups carried by one dam are multichorionic with separate placentas and umbilical cords, makes the\nhamster well-suited for studying pregnancy-related pathologies.\nTo assess the effect of SARS-CoV-2 infection during pregnancy, after timed mating and confirmed insemination,\nanesthetized female Syrian hamsters were a) inoculated intranasally with SARS-CoV-2 (VOC Alpha; B.1.1.7) at various key\nstages of pregnancy: insemination (E0.5), implantation (E4.5), placentation (E7.5), and a fully formed placenta (E10.5); or b)\nnon-inoculated pregnant dams were anesthetized at matched gestational time points as controls. Pups were surgically\ndelivered before the onset of labor (E15.5). Viral RNA and infectious viral particles in the lungs, placenta and fetal liver\n(vRNA only) were quantified (via RT-qPCR and TCID50 assay, respectively) on the day of surgical delivery (E15.5), and\npulmonary transcript levels of cytokines Ip10, Mx2, Ifnγ, Il6, Tnfα, and Il1β (previously shown to be upregulated in the lungs\nafter SARS-CoV-2 infection) were assessed (via RT-qPCR). Tissues (maternal lung from all groups and placenta from the\nE4.5 and E10.5 inoculation group) were evaluated and scored histologically. Next, the authors quantified transcript levels of\nmarkers for immune cells, including NK cells (Klrg1, Cd94), CD4⁺ and CD8⁺ T cells, genes associated with type I, II, and III\nIFN responses (Mx2, Ip10, and IL28, respectively), and cytokines IL6 and IL10 (via RT-aPCR) in order to investigate whether\nthese correlated with the presence of placental vRNA. Since individual mother-placenta-offspring triads revealed\nheterogeneity, the authors also utilized Principal Component Analysis (PCA) to see whether a specific molecular signature\ncorrelated with the heterogeneity observed in placental infection and IUGR.\nResults show that SARS-CoV-2-inoculation of pregnant hamsters was associated with:\n• Gestational age dependent weight loss (with undetectable changes in pregnancy outcome or\nbody weight gain in controls) in dams\no Early gestational infections at E0.5 and E4.5 resulted in significant (though temporary) weight\nloss starting at 4dpi and peaking at 7 dpi\no By E15.5 (surgical delivery) dams infected before E8 (full placental development) had\nsignificantly lower overall weight gain; similar though not statistically significant) trend\ntoward lower, net body weight gain (vs. controls) in dams inoculated at E4.5, E7.5, E10.5\n• Significantly higher hemoglobin levels in infected dams (no significant difference in HCT,\nRBC, WBC, PLT)\n• Detection of vRNA and infectious virus in the lung (dams) and 43/67 placentas (64%) of animals\ninfected at E10.5 (corresponds to 5 dpi); both were undetectable after 5dpi (i.e., at earlier\ngestation time points); vRNA was never detected in any fetal liver\n• Bronchopneumonia/diffuse alveolar damage (dams) affecting up to 40% lung volume, though disease\nseverity and lung pathology were not significantly affected by gestational stage\n• Upregulation of Ip10, Mx2, Ifnγ, Il6, Tnfα, and Il1β, which peaked in the lung of dams infected at\nE10.5 (with significantly elevated levels of Ip10 and Mx2), while in dams infected at E4.5 only\nlow levels of Mx2 were detected\n• Intrauterine growth restriction (IUGR)\no Dams infected after implantation (E4.5, 7.5, 10.5) had smaller pups with significantly reduced\nbody weight (most pronounced at E4.5) vs. controls; however, litters from infected dams were not\nuniformly affected (low-weight pups mixed with normal littermates in the same pregnancy and even\nthe same uterine horn)\n  birth weights below 10th percentile of their respective mock-infected littermates = small\nfor gestational age (SGA)\n  Dams inoculated with SARS-CoV-2 after implantation (E4.5, 7.5, 10.5) were more likely to\ndeliver SGA pups (with highest risk observed following infection at E4.5)\no infection at E0.5 (immediately following confirmed copulation) did not influence fetal weight\n• Placentas from dams inoculated at E4.5 and E7.5 had a significantly smaller weight (vs. non-\ninfected controls); in contrast, placentas from dams inoculated at E10.5 were significantly\nlarger… The authors interpret this finding to suggest that the onset of alterations in the\nplacenta may occur concurrently with lung pathology, possibly due to localized infection or\ninflammatory responses.\n• Pathology in placentas from infected animals characterized by increased perivascular fibrin\ndeposition, thrombosis, and necrosis, primarily localized to the junctional zone (JZ), as well as\nincreased nRBCs within the fetal vasculature (interpreted as fetal stress response).\no Placentas from infected dams had significantly higher cumulative pathological severity scores\n(both E10.5 and E4.5) compared to those from non-infected animals… Authors interpret this as an\nindication that tissue injury and subsequent remodeling persist despite the fact that vRNA has\nbeen eliminated from placentas by these later time points\n• Detectable placental vRNA at 5 dpi (dams infected at E10.5) exhibited significantly increased\ntranscript levels of Cd4 (CD4+ T cells) and Klrg1 (NK cells) vs. placentas from non-infected\nanimals\n• Heterogeneity in pregnancy outcomes- PCA resulted in clusters depicting three different pregnancy\noutcomes, overall suggesting that the health of the fetus is not necessarily determined directly\nby the presence of the virus in the placenta, but rather indirectly by the pro-inflammatory\nresponse\nSARS-CoV-2 inoculation was not associated with:\n• Vertical transmission to pups\n• Significant effects on litter size or malformation rates\n• Maternal or fetal death\nTaken together, the authors conclude from the above that SARS-CoV-2 infection of pregnant dams frequently results in a\ntransient placental infection without direct transplacental/vertical transmission, but with risk of fetal IUGR, likely secondary to\npathological alterations in the placenta (placentitis), characterized by pronounced fibrin deposition and tissue remodeling of\nthe decidua, and persistent inflammation hallmarked by elevated Ip10 (Cxcl10), Il6, and Il10 expression in the placentas of\naffected pups. Regardless of the health status of the dam (acute versus resolving pneumonia) and the timing of infection,\npregnancy outcomes following SARS-CoV-2 infection in hamsters are heterogeneous, likely worsened more by a sustained\ninnate immune response than by placental infection or vertical transmission.\nSince SARS-CoV-2 is known to cause systemic coagulopathy and placentitis in pregnant women, and significant placental\npathology, including fibrosis and thrombosis, was observed in this hamster model, the authors next evaluated whether low\nmolecular weight heparin (enoxaparin- an anticoagulant used to prevent thromboembolism in pregnancy) could mitigate\nadverse pregnancy outcomes in pregnant SARS-CoV-2 infected hamsters, with focus on the E4.5 group (which exhibited\nthe most distinct adverse effects on fetal outcomes). Post-exposure 3μg/g enoxaparin was administered subcutaneously\nonce daily to dams between 3dpi (E7.5) and 7dpi (E14.5), followed by a similar experimental design/assays to those\ndescribed above. Finally, subsequent to evaluation/monitoring of seroconversion kinetics in pregnant hamsters challenged\nwith B.1.1.7, two cohorts of dams were vaccinated with YF-S0* (safety, immunogenicity, and efficacy previously established\nby this group) in a prime-boost regimen prior to conception (along with naïve controls) and challenge at E4.5 or E10.5.\nSeroconversion to consistently high levels of spike-specific IgG in these immunized animals prior to infection was confirmed\nby flow cytometry.\nOverall, administration of low molecular weight heparin (enoxaparin) improved pregnancy outcomes and averted placental\nmalfunction in SARS-CoV-2 infected hamsters:\n• Enoxaparin treatment resulted in significantly increased fetal and placental weights (vs.\nuntreated controls), without affecting litter size\n• Histological analysis of placentas from infected dams showed improved pathology scores following\nenoxaparin treatment, characterized by reduced JZ/labyrinth congestion, fibrosis, and necrosis (no\nmeasurable impact on the relative proportions of placental zones)\n• Enoxaparin treatment did not exert any antiviral effect (oral swab vRNA levels remained similar to\ncontrols) and did not enhance the overall health of the dams- they continued to lose weight\nthroughout infection and gained significantly less cumulative weight (vs. non-infected dams)\nSARS-CoV-2 vaccination prior to conception protects from adverse pregnancy outcomes\n• Overall, pregnancy itself did not impair the humoral immune response observed in naïve dams\n• For dams challenged at E4.5, vaccination conferred protection with overall lower and faster\ndecaying vRNA loads (in buccal swabs) as well as markedly reduced lung pathology\n• Vaccination did not prevent the adverse effects of infection at E10.5 on fetal and placental\nweight despite a significant reduction in placental damage\no pups of vaccinated dams inoculated with SARS at E4.5 were significantly larger with heavier\nplacentas (vs. unvaccinated controls)\n• Placentas from hamsters challenged at both E4.5 and E10.5 had reduced pathology characterized by\nreduced fibrin deposition, thrombosis, and necrosis\nThis is an interesting, well-planned, straightforward study with clearly drawn conclusions. It is presented in a logical and\norganized manner and most of my comments are minor (see below for specific comments with line #s from the text);\nhowever, there are a few issues with the histopathology interpretation and images that are either too small\nand/or not of publication quality. Overall, I recommend publication, with minor revisions.\nValidity: No major flaws. While hamsters are a valuable model for human COVID-19 disease, this model does not fully\nreplicate human pregnancy physiology or placental architecture and is thus not fully translatable, which the authors state in\nthe section detailing “Limitations of the study.”\nOriginality and significance: Conclusions appear original and the results are of interest to several disciplines. There is a gap\nin knowledge, as stated by authors: mechanisms of SARS-CoV-2 induced maternal and fetal pathology are poorly\nunderstood, and vaccine hesitancy remains common among pregnant women (26-57% vaccinated) although studies\nindicate vaccination reduces maternal morbidity and mortality without increasing the risks of adverse fetal outcomes.\nData & methodology: For the most part (exceptions noted below) the reporting of data and methodology sufficiently detailed\nand transparent to enable reproducing the results.\nRESULTS/DATA:\nBody weight loss due to SARS-CoV-2 infection in pregnant Syrian hamsters depends on gestational stage\n(Line 136-138): Authors state that SARS-CoV-2 infections was confirmed at 4dpi using buccal swabs with a COVID-19 self-\ntest kit (presumably for humans). Was this ever validated for hamsters (i.e., using PCR) in this or previous studies that could\nbe referenced?\n(Line 146-148): This is a general comment for the entire manuscript… it seems like in some sections of the narrative results,\nstatistical means, CIs, p-values, etc. are listed, while they are left out of others. Please be consistent. I personally think it’s\nfind to just state whether something was statistically significant in the narrative sections, but it’s important to list the actual\nnumbers (p-values, etc.) in the figure/graph/legend (though I’m not sure if Nat Comm has a rule on that). Same thing for “Ns.”\nPlease list Ns in figures (i.e., experimental design graphics) and graphs (or at least in the legend) +/- in the actual narrative\n(just be consistent).\n(Line 152-154): Suggest adding this reference about the B.1.1.7. strain being relatively more aggressive in hamsters (vs.\nnewer variants) forward (maybe near line 133), as it answers the question as to why such an early variant was chosen for\nthis study.\n(Line 192) states that hemoglobin was the only blood parameter significantly affected in pregnant, SARS-CoV-2 infected\ndams, but it does not appear to be shown graphically anywhere (or maybe I’m just missing it because there are not legends\nin supplementary figures)?\n(Line 192-194): Why is this elevated hemoglobin suggestive of a compensatory adaptation in response to an increased\nrespiratory stress? Is there a reference for this? Is it supposition? Perhaps better placed in the discussion?\n(Lines 198-200): Confusing sentence, suggest rewording/clarifying\n(Line 207) states that alveolar damage affected up to 40% of lung volume. How was this measured? It is not specifically\ndescribed in the histology section of the methods. As you are evaluating 2-D glass slides it would be more accurate to list %\nof visible lung surface area affected.\n(Line 214): I think it’s an overstatement to say that lung inflammation associated with the infection was resolved by 8 dpi,\nmore like it had become more reparative/chronic in nature, but there is still some inflammation- the lung does not appear\nnormal.\nFigure 1F and lines 205-214: Were there any statistically significant differences?\n(Lines 215-216) were these cytokines evaluated in lung tissue? Are they all pro-inflammatory or are any anti? Please clarify\n(even just re-wording to say “which are pro-inflammatory CKs upregulated in..’)\nFigure 1G- show p-value/significance please\nLine 222 states that the low levels of Mx2 detected in E4.5 dams infected at E4.5 were likely due to a remnant type I IFN\nresponse induced by residual circulating vRNA… Is there a reference for this? Is it supposition? Perhaps better placed in the\ndiscussion?\nMaternal SARS-CoV-2 infection leads to intrauterine growth restriction in a gestational age-dependent manner\nFigure 2C: Please define the colored dots (small legend)\nFigure 2 (general): Ns should be listed, where applicable\n(Lines 272-275): Is there a reference for this? How would this affect placental weight?\nPersistent placental inflammation following viral clearance.\nFigure 3A: This may just be the PDF I received (vs. raw images), but these do not seem to be publication quality images, or it\nmay just be that they are too small, but when I zoom in to try to look at what the arrows are indicating, the images are too\nblurry. I can’t tell if these are accurate, representative images or not. It’s hard to even tell what the normal control should look like… is there space in the manuscript to make these pics larger in size?\n(Lines 312-314): See previous comment in methods… how did you measure ratio of JZ:labyrinth? What should the normal\nratio be? Is a higher or lower ratio associated with placental insufficiency?\n(line 317): Reference (to nRBCs representing a fetal stress response)?\n(Lines 321-323): I’m not sure this is an accurate conclusion. You haven’t really described/shown placental remodeling, and if\nit’s in the images in supplementary data 1, it’s not circled or described so the reader will not be able to tell\nAlso, it’s pretty normal to have a delay between infection (initial viral detection in tissues) and seeing histopathologic lesions\nin tissues, since it takes a little while for the inflammatory reaction to get going and for the tissue reaction to manifest\nhistologically. Similarly, histo lesions often persist for a little while (and sometimes for quite a while) after pathogen\nelimination, either due to a more chronic inflammatory response, remodeling/repair, or because of an inappropriate\ninflammatory reaction (i.e., cytokine storm) which is reported in human SARS-CoV-2 infections.\nAdministration of low molecular weight heparin improves pregnancy outcomes in SARS-CoV-2 infected hamsters\nFigure 4: Please “define” the different colored shapes for each figure component\n(Line 444): suggest removing “congestion” as a scoreable histopathologic feature\n(Line 449-451): states that “enoxaparin-treated placentas from infected dams contained an increased number of nRBCs\ncompared to the untreated group as a possible safety sign.” Earlier, nRBCs in the fetal vasculature was interpreted as a sign\nof fetal stress. Is this different because it’s not in the fetal vasculature? Please explain/clarify. Is there a reference for this?\n(Lines 464-468): Confusing sentence, please clarify and summarize the significance of this data.\nCOVID-19 vaccination protects from adverse pregnancy outcomes.\n(Lines 460-462) & Fig 1A: from what I can tell, Fig 1A does not indicate that “Seroconversion kinetics to anti-SARS-CoV-2\nantibodies in pregnant hamsters were monitored starting from challenge with B.1.1.7. at various time points;” please update\nfigure (or tell me what I missed).\nFigure 5F: This may just be the PDF I received (vs. raw images), but these do not seem to be publication quality images, or it\nmay just be that they are too small, but when I zoom in to try to look at what the arrows are indicating, the images are too\nblurry. I can’t tell if these are accurate, representative images or not. It’s hard to even tell what the normal control should\nlook like… is there space in the manuscript to make these pics larger in size?\nSupplementary Figures: A brief title for each graph/figure would be helpful.\n1. Supp Fig 1A: Define “high vs. low” sperm Supp Fig 1B: the test for E7.5 looks like it’s negative (or very faint?)… shouldn’t\nall but the mock/control have 2 lines?\n2. Supp Fig 2B: in the narrative (lines 183-185) authors state that neutrophils are significantly increased in pregnant\nhamsters (including p-value). Please add p-value to the actual graph/figure; Supp Fig 2C/D: As above, this may just be the\nPDF I received (vs. raw images), but these do not seem to be publication quality images, or it may just be that they are too\nsmall, but when I zoom in to try to look at what the arrows are indicating the images are blurry… is there space in the\nmanuscript to make these pics larger in size?\n4. Supp Fig 4 (all): Please “define” the different colored shapes for each figure components\nSupplementary Data 1 would be more useful with a legend/description\nMETHODS:\nSARS-CoV-2 infection model in pregnant hamsters:\n(line 711) What was the cutoff/definition for “abundant” sperm content? (i.e., > a certain # of sperm per 40X high power field,\netc.)\n(lines 721-722): Were non-infected control animals mock-inoculated intranasally with an inert substance (such as PBS) to\nmatch infected animals? If not, I recommend this in future, to show that iatrogenic causes (i.e., aspiration pneumonia simply\nfrom overzealous IN inoculation) is not contributing to the maternal bronchopneumonia noted histologically\n(line 731): where any length/width measurements performed? Or just weight?\nHistology: Please include a table or other summary specifically defining histopathologic scoring criteria; the general\ndescriptions in lines 805-807 (lung) & 808-812 (placenta) are not specific enough. For example, what made each of the\nfeatures listed receive the score of 1, 2 or 3? I also suggest removing congestion as a grading criteria for the placenta. This\nwill probably just end up confounding things, since, as stated in lines 307-310, increased congestion is a typical feature of\nnormal placentas near parturition. Also, did you consult with a board certified pathologist?\n(Line 803): Is isolectin specific for fetal blood vessels? Or would it stain all blood vessels, including maternal?\nPlease specify, including reference.\n(lines 810-812) How did you determine the ratio of JZ to labyrinth? A computer program like ImageJ? Something else?\nPlease specify.\nAppropriate use of statistics and treatment of uncertainties: Overall, statistics appear robust (but see note below- this is not\nmy area of expertise). There are a couple missing p-values (specified above), and the authors should be consistent about\nlisting n’s error bars, means, p-values etc. in the same place… sometimes they are written into the narrative, sometimes just\nin the figures. I suggest that they are always included in the figures/legends (+/- the narrative at the discretion of the\nauthor/editor)\nConclusions: For the most part, the discussion, conclusions and data interpretation are robust, valid and reliable.\nAdditionally, the authors clearly state the limitations of this study. There are a couple of (minor) statements that might not\nhave been supported by the data, which are detailed above/below.\nIn the first part of the discussion, suggest adding some comparisons to humans (i.e., does IUGR in hamsters recapitulate\nwhat’s seen in humans? If not, how does it differ? Etc.) This correlation with human findings is nicely done in the next part of\nthe discussion with CKs and histopathologic findings (lines 566-573).\n(Lines 547-548) As mentioned previously, I think it is an overstatement to say that infected female hamsters rapidly\nrecovered from lung damage. The lung histo images do not appear normal/resolved at later infection stages, rather the\nlesions have become more reparative/chronic in nature.\nLine 549: Suggest defining placental insufficiency (with reference). What are the features (clinical, histo, etc.)\nLine 552: Suggest changing “clearly indicating” to “suggesting”\n(Line 560): Refers to “makers” of innate/adaptive immunity- should this be “markers?”\n(lines 580-582): Please be more specific about the location of nRBCs that are associated with hypoxia/growth restriction. Is it\nwhen they are seen in the placenta? Fetal vasculature? (both are mentioned separately in text) Both? What do you think is\nthe significance of this finding? Also, approximately how many were seen (few, numerous)?\n(Lines 617-618) state that the authors did not observe the dissemination into the brains, but were additional tissues\nassessed other than the lung/placenta? I don’t remember seeing anything in the methods or results about that… Please\nclarify.\nSuggested improvements: see above.\nReferences: This manuscript references previous literature appropriately.\nClarity and context: Abstract succinctly summarizes the problem, and describes the research plan, methods (3-D vascular\nendothelial culture system, animal infections), results and conclusion.\n(lines 29-30) Suggest being more specific by saying “….linked to placental damage, characterized histologically by fibrin\ndeposition, thrombosis, and elevated Ip10, Il6, and Il10 expression in (list tissue here)”\nInflammatory material: This manuscript does not contain any language that is inappropriate or potentially libelous.", "reviewer_author_response": "REPLY TO REVIEWER’S COMMENTS\nWe sincerely appreciate the reviewers’ insightful and constructive feedback, which greatly\nhelped to strengthen the quality and clarity of our work. Here, we address all comments point\nby point. Respective revisions are all marked in red in the manuscript for ease of reference.\nWe have also provide high-resolution figures via FigShare to support thorough assessment\nand publication.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThis manuscript investigated SARS-CoV-2 infection at different key timepoints during\npregnancy in Syrian hamsters. The authors showed that maternal infection leads to\nintrauterine growth restriction (IUGR) due to placental inflammation without any vertical\ntransmission. Vaccination and anticoagulant therapies were found to protect the placenta and\nimprove fetal outcomes, highlighting their importance in maternal/pregnancy care. This study\nis thorough and well conducted and addresses an important topic with significant implications\nfor maternal and fetal health during SARS-CoV2 infection. The article is clearly written, with a\nlogical flow, thorough explanations of the results and adequate discussion with a section on\nthe limitation of the work. Overall, I believe this manuscript is well-suited for publication\npending minor revisions.\nGeneral comments:\nI understand the need for the authors to perform their analysis based on days post-infection\n(dpi). However, dpi will obviously vary depending on when the infection occurred and the\nlength of exposure will vary. Considering that the placenta, unlike maternal organs, is still\ngrowing during pregnancy and its function evolves, could terminal c-section not have been\nconducted at the same dpi for each timepoints of infection to address is the placental\nsusceptibility/response to infection? This might be beyond the scope of the work and could be\naddressed in the discussion.\nR/ Indeed, this is an interesting point and can be considered a natural continuation of\nthis work. However, placental morphology in hamsters has not yet been fully\ncharacterized. We are currently generating a temporal and spatial 3D map to define\nthe anatomical and developmental features of the hamster placenta as we did before\nfor mice (De Clercq PNAS 2019, PMID: 31249139). Building on this, we plan to\ninvestigate placental development and its response to infection in follow-up studies.\nAs noted, this falls beyond the scope of the present manuscript as now also addressed\nin the discussion (lines 591-599).\nMinor comments:\nIntroduction: Short, concise and well structured. The authors could add more details on\nhuman placental pathology studies following SARS-CoV-2 infection, considering that there are\nseveral published studies and their work has a fair amount of histopathology analysis. This\nwould offer context for interpreting the findings from the hamster placenta.\nR/ More details about placenta pathology were added (lines 61-69), including a list of\nnew references (PMID: 38897829; 40380781; 38897829; 36052328). Regarding the\nplacenta of hamsters, please refer to our previous response.\nMethods: The manuscript is well written and provides sufficient methodological detail to allow\nfor experimental reproducibility. Was fetal/placental sex considered as a variable? Given that\nfemales tend to be lighter than males, this could be a relevant factor, particularly if there are\ndiscrepancies in survival rates among the pups.\nR/ Fetal sex has now been determined (please see M&M and reference to separate\nmanuscript in preprint doi: https://doi.org/10.1101/2025.11.22.689895). All earlier\nassessed correlations have been re-analyzed considering sex as a possible\nconfounding factor (new Figure 2C, D, and F and respective narratives on pages 9-\n11). Indeed, also male pups of hamsters and their placentas tend to be larger than\ntheir female counterparts. Intriguingly, due to the generally higher average weight of\nmales, IUGR was generally more pronounced in male pups of infected mothers than\nin females when compared to matched controls from dams that had been mock-\ninfected at the same gestational age (“males had more to lose”).\nOf note, no differences were seen in viral loads between male and female pups. Most\nimportantly, gender was ruled out to play an essential role in latter OR and PCA\nanalyses linking IUGR and histopathological changes (scarification of placental\ntissues) to maternal infection and persistent inflammation in the placenta, respectively\n(new panels in Supplementary Figure 4B, E, lines 418-422).\nI understand that the vaccine used is YF-S0* but in Fig.5 and elsewhere in the text they write\nYF17D-vectored. Which might not be adequate since YF-S0* is an updated construct of\nYF17D.\nR/ YF-S0* is a second-generation vaccine candidate based on the YF17D backbone,\nin which a non-cleavable, prefusion-stabilized form of SARS-CoV-2 spike protein (S0*)\nis inserted between the prM/E and NS1 regions. This S0* version was updated from\noriginally described YF-S0 (Sanchez-Felipe et al., Nature 2021, PMID: 33260195) to\nincorporate the antigenic features of the Gamma variant of concern (VOC), along with\nadditional amino acid changes to improve protein stability and antigenicity [Sharma et\nal., Nature Commun 2022, PMID: 36333374]. In our recombinant YF17D-vectored\nvaccines, the viral backbone remains unchanged; only the inserted antigen or its\nposition within the YF17D genome is modified [Sanchez-Felipe et al., Eur J Immunol\n2024, PMID: 38571392]. For clarity, we have now added one sentence to the Results\nsection explicitly stating this (lines 507-510).\nShould the “Study Approval” section be placed before the “Animal” section at line 683?\nR/ Adapted accordingly.\nResults: The results are well structured, and the inclusion of the therapeutic intervention\nsignificantly strengthens the study. I also appreciated the addition of the PCA analysis, which\neffectively highlights distinct molecular signatures and helps explain the observed\nheterogeneity.\n- Line 152-156, this sentence should be rephrased to improve clarity\nR/ The sentence has been split into two to improve clarity (lines 164-168).\n- Line 192-194, does the authors have any data supporting this statement: “suggestive of a\ncompensatory adaptation in response to an increased respiratory stress.”? If not, I would\nsuggest removing.\nR/ We have carefully rechecked the text and made the necessary adaptations (lines\n197-202).\n- Even if cytokine transcript levels are not elevated at later dpi, this only indicates that\ninflammation has been resolved by that point—but it does not necessarily mean that\ninflammation did not occur earlier or have other effects. This is aligned with the “general\ncomment” above and would be an interesting point of discussion.\nR/ The reviewer refers to Figure 1G, where transcripts levels of IP10, MX2, IFNγ, IL6,\nTNFα, and IL1β at E4.5 are comparable to mock controls. We fully agree that this\nindicates resolution of the inflammatory response. This is consistent with our\ninterpretation, and we describe this kinetically by stating that: “… after this initial peak,\nantiviral gene expression appeared to settle as in dams infected at E4.5 only lower\nlevels of MX2 were detected anymore” (lines 231-233).\nWe further elaborate on this point in the Discussion (lines 591-599).\nAn interesting observation, however, is that neither the viral load in the lungs, nor levels\nof proinflammatory cytokines in the lungs, nor associated lung pathology appear to\ndetermine fetal outcomes. This is supported by the presence of both normal-weight\nand SGA pups within the same litter, suggesting that the determinants of fetal growth\nrestriction are likely placental-intrinsic rather than a direct consequence of the initial\nmaternal lung infection. Therefore, one should be cautious when attributing\nsubsequent fetal effects directly to the initial infection (lines 704-705).\n- Line 300, the authors state that analyses were performed at E4.5, but then shift to discussing\nE10.5 at line 312. To improve clarity, it might be helpful to mention both E4.5 and E10.5 at the\nbeginning of this section.\nR/ Adapted accordingly (line 322).\n- Fig. 3A and Fig. 5F: the purple arrows are very difficult to see in the background. It may be\nhelpful to choose a more contrasting color to improve visibility.\nR/ Adapted to green to enhance contrast with the background.\n- Fig. 4F: since the graph represents only a single timepoint, could the authors clarify why lines\n437–438 refer to “up to 8 days post-infection”? Or were several time points combined here?\nR/ Infection at E4.5 resulted in significant weight loss beginning at 4 days post-infection\n(dpi) and peaking at 7 dpi, with a 5–6% reduction in body weight (Fig. 1B and Supp.\nFig. 1F). After this point, the animals began to gain weight, particularly in pregnant\nanimals as pregnancy progressed rapidly. Therefore, cumulative body weight was\nassessed only within this time window: from the day of infection up to 8 dpi. This\nanalysis is first presented in Fig. 1C and Supp. Fig. 1G. In Fig. 4F, we applied the same\napproach. We now clearly indicate that this measurement starts from the day of\ninfection, at E4.5.\n- Fig. 5: could the authors clarify why the vaccinated, non-infected group is not included in this\nfigure? Including this group would help isolate the effects of vaccination alone.\nR/ We have previously reported that the vaccine virus is cleared at the latest by 12\ndays post vaccination, [PMIDs: 33260195, 36333374; 35313504], hence before\nmating. We did not pursue this objective, mainly due to the arduous process of mating.\nWe prioritized the allocation of pregnant hamsters into the infected groups.\nDiscussion: The discussion is well structured and clearly presented. However, it would benefit\nfrom a deeper comparison with existing literature, particularly in relation to the main findings.\nCurrently, the focus is primarily on the treatment and vaccine aspects. On the other hand, the\nlimitations section is thorough and well-articulated, effectively addressing key constraints of\nthe study.\nR/ Additional information was added (lines: 580-581, 619-622).\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nTitle: COVID-19-related inflammation of the placenta impedes fetal development in pregnant\nhamsters\nSummary/Key Results: The introduction sets the stage well, establishing that, while SARS-\nCoV-2 infection is associated with increased disease severity in expectant mothers as well as\nfetal complications such as compromised placental insufficiency/hypoxic injury, mechanisms\nof SARS-CoV-2 induced maternal and fetal pathology are poorly understood. Additionally,\nvaccine hesitancy remains common among pregnant women.\nHere, authors explored the effects of SARS-CoV-2 infection on pregnancy in hamsters, which\nis an appropriate animal model for this study. Hamsters are naturally susceptible to SARS-\nCoV-2 infection, exhibiting similar pulmonary pathology to human COVID-19 disease.\nFurthermore, hamsters and humans have some similarities in overall placental anatomical\nstructure- specifically hemochorial placentation. But, in contrast to the human\nhemomonochorial placental interface (characterized by deep trophoblast invasion of the\nmaternal uterus), the labyrinthine rodent placenta generally exhibits a hemotrichorial interface\nwith multiple trophoblast layers. Deeper trophoblast invasion in hamsters (vs. mice), coupled\nwith the fact that individual pups carried by one dam are multichorionic with separate placentas\nand umbilical cords, makes the hamster well-suited for studying pregnancy-related\npathologies.\nTo assess the effect of SARS-CoV-2 infection during pregnancy, after timed mating and\nconfirmed insemination, anesthetized female Syrian hamsters were a) inoculated intranasally\nwith SARS-CoV-2 (VOC Alpha; B.1.1.7) at various key stages of pregnancy: insemination\n(E0.5), implantation (E4.5), placentation (E7.5), and a fully formed placenta (E10.5); or b) non-\ninoculated pregnant dams were anesthetized at matched gestational time points as controls.\nPups were surgically delivered before the onset of labor (E15.5). Viral RNA and infectious viral\nparticles in the lungs, placenta and fetal liver (vRNA only) were quantified (via RT-qPCR and\nTCID50 assay, respectively) on the day of surgical delivery (E15.5), and pulmonary transcript\nlevels of cytokines Ip10, Mx2, Ifnγ, Il6, Tnfα, and Il1β (previously shown to be upregulated in\nthe lungs after SARS-CoV-2 infection) were assessed (via RT-qPCR). Tissues (maternal lung\nfrom all groups and placenta from the E4.5 and E10.5 inoculation group) were evaluated and\nscored histologically. Next, the authors quantified transcript levels of markers for immune cells,\nincluding NK cells (Klrg1, Cd94), CD4 and CD8 T cells, genes associated with type I, II, and\nIII IFN responses (Mx2, Ip10, and IL28, respectively), and cytokines IL6 and IL10 (via RT-\n⁺ ⁺\naPCR) in order to investigate whether these correlated with the presence of placental vRNA.\nSince individual mother-placenta-offspring triads revealed heterogeneity, the authors also\nutilized Principal Component Analysis (PCA) to see whether a specific molecular signature\ncorrelated with the heterogeneity observed in placental infection and IUGR.\nResults show that SARS-CoV-2-inoculation of pregnant hamsters was associated with:\n• Gestational age dependent weight loss (with undetectable changes in pregnancy outcome\nor body weight gain in controls) in dams\n• Early gestational infections at E0.5 and E4.5 resulted in significant (though temporary) weight\nloss starting at 4dpi and peaking at 7 dpi\n• By E15.5 (surgical delivery) dams infected before E8 (full placental development) had\nsignificantly lower overall weight gain; similar though not statistically significant) trend toward\nlower, net body weight gain (vs. controls) in dams inoculated at E4.5, E7.5, E10.5\n• Significantly higher hemoglobin levels in infected dams (no significant difference in HCT,\nRBC, WBC, PLT)\n• Detection of vRNA and infectious virus in the lung (dams) and 43/67 placentas (64%) of\nanimals infected at E10.5 (corresponds to 5 dpi); both were undetectable after 5dpi (i.e., at\nearlier gestation time points); vRNA was never detected in any fetal liver\n• Bronchopneumonia/diffuse alveolar damage (dams) affecting up to 40% lung volume, though\ndisease severity and lung pathology were not significantly affected by gestational stage\n• Upregulation of Ip10, Mx2, Ifnγ, Il6, Tnfα, and Il1β, which peaked in the lung of dams infected\nat E10.5 (with significantly elevated levels of Ip10 and Mx2), while in dams infected at E4.5\nonly low levels of Mx2 were detected\n• Intrauterine growth restriction (IUGR)\n• Dams infected after implantation (E4.5, 7.5, 10.5) had smaller pups with significantly reduced\nbody weight (most pronounced at E4.5) vs. controls; however, litters from infected dams were\nnot uniformly affected (low-weight pups mixed with normal littermates in the same pregnancy\nand even the same uterine horn)\n• birth weights below 10th percentile of their respective mock-infected littermates = small for\ngestational age (SGA)\n• Dams inoculated with SARS-CoV-2 after implantation (E4.5, 7.5, 10.5) were more likely to\ndeliver SGA pups (with highest risk observed following infection at E4.5)\n• infection at E0.5 (immediately following confirmed copulation) did not influence fetal weight\n• Placentas from dams inoculated at E4.5 and E7.5 had a significantly smaller weight (vs. non-\ninfected controls); in contrast, placentas from dams inoculated at E10.5 were significantly\nlarger… The authors interpret this finding to suggest that the onset of alterations in the\nplacenta may occur concurrently with lung pathology, possibly due to localized infection or\ninflammatory responses.\n• Pathology in placentas from infected animals characterized by increased perivascular fibrin\ndeposition, thrombosis, and necrosis, primarily localized to the junctional zone (JZ), as well as\nincreased nRBCs within the fetal vasculature (interpreted as fetal stress response).\n• vPlacentas from infected dams had significantly higher cumulative pathological severity\nscores (both E10.5 and E4.5) compared to those from non-infected animals… Authors\ninterpret this as an indication that tissue injury and subsequent remodeling persist despite the\nfact that vRNA has been eliminated from placentas by these later time points\n• Detectable placental vRNA at 5 dpi (dams infected at E10.5) exhibited significantly increased\ntranscript levels of Cd4 (CD4+ T cells) and Klrg1 (NK cells) vs. placentas from non-infected\nanimals\n• Heterogeneity in pregnancy outcomes- PCA resulted in clusters depicting three different\npregnancy outcomes, overall suggesting that the health of the fetus is not necessarily\ndetermined directly by the presence of the virus in the placenta, but rather indirectly by the\npro-inflammatory response SARS-CoV-2 inoculation was not associated with:\n− Vertical transmission to pups\n− Significant effects on litter size or malformation rates\n− Maternal or fetal death\nTaken together, the authors conclude from the above that SARS-CoV-2 infection of pregnant\ndams frequently results in a transient placental infection without direct transplacental/vertical\ntransmission, but with risk of fetal IUGR, likely secondary to pathological alterations in the\nplacenta (placentitis), characterized by pronounced fibrin deposition and tissue remodeling of\nthe decidua, and persistent inflammation hallmarked by elevated Ip10 (Cxcl10), Il6, and Il10\nexpression in the placentas of affected pups. Regardless of the health status of the dam (acute\nversus resolving pneumonia) and the timing of infection, pregnancy outcomes following\nSARS-CoV-2 infection in hamsters are heterogeneous, likely worsened more by a sustained\ninnate immune response than by placental infection or vertical transmission.\nSince SARS-CoV-2 is known to cause systemic coagulopathy and placentitis in pregnant\nwomen, and significant placental pathology, including fibrosis and thrombosis, was observed\nin this hamster model, the authors next evaluated whether low molecular weight heparin\n(enoxaparin- an anticoagulant used to prevent thromboembolism in pregnancy) could mitigate\nadverse pregnancy outcomes in pregnant SARS-CoV-2 infected hamsters, with focus on the\nE4.5 group (which exhibited the most distinct adverse effects on fetal outcomes). Post-\nexposure 3μg/g enoxaparin was administered subcutaneously once daily to dams between\n3dpi (E7.5) and 7dpi (E14.5), followed by a similar experimental design/assays to those\ndescribed above. Finally, subsequent to evaluation/monitoring of seroconversion kinetics in\npregnant hamsters challenged with B.1.1.7, two cohorts of dams were vaccinated with YF-S0*\n(safety, immunogenicity, and efficacy previously established by this group) in a prime-boost\nregimen prior to conception (along with naïve controls) and challenge at E4.5 or E10.5.\nSeroconversion to consistently high levels of spike-specific IgG in these immunized animals\nprior to infection was confirmed by flow cytometry.\nOverall, administration of low molecular weight heparin (enoxaparin) improved pregnancy\noutcomes and averted placental malfunction in SARS-CoV-2 infected hamsters:\n• Enoxaparin treatment resulted in significantly increased fetal and placental weights (vs.\nuntreated controls), without affecting litter size\n• Histological analysis of placentas from infected dams showed improved pathology scores\nfollowing enoxaparin treatment, characterized by reduced JZ/labyrinth congestion, fibrosis,\nand necrosis (no measurable impact on the relative proportions of placental zones)\n• Enoxaparin treatment did not exert any antiviral effect (oral swab vRNA levels remained\nsimilar tocontrols) and did not enhance the overall health of the dams- they continued to lose\nweight throughout infection and gained significantly less cumulative weight (vs. non-infected\ndams)\n• SARS-CoV-2 vaccination prior to conception protects from adverse pregnancy outcomes\n• Overall, pregnancy itself did not impair the humoral immune response observed in naïve\ndams\n• For dams challenged at E4.5, vaccination conferred protection with overall lower and faster\ndecaying vRNA loads (in buccal swabs) as well as markedly reduced lung pathology\n• Vaccination did not prevent the adverse effects of infection at E10.5 on fetal and placental\nweight despite a significant reduction in placental damage\n• pups of vaccinated dams inoculated with SARS at E4.5 were significantly larger with heavier\nplacentas (vs. unvaccinated controls)\n• Placentas from hamsters challenged at both E4.5 and E10.5 had reduced pathology\ncharacterized by reduced fibrin deposition, thrombosis, and necrosis.\nThis is an interesting, well-planned, straightforward study with clearly drawn\nconclusions. It is presented in a logical and organized manner and most of my comments\nare minor (see below for specific comments with line #s from the text); however, there are a\nfew issues with the histopathology interpretation and images that are either too small\nand/or not of publication quality. Overall, I recommend publication, with minor revisions.\nValidity: No major flaws. While hamsters are a valuable model for human COVID-19 disease,\nthis model does not fully replicate human pregnancy physiology or placental architecture and\nis thus not fully translatable, which the authors state in the section detailing “Limitations of the\nstudy.”\nOriginality and significance: Conclusions appear original and the results are of interest to\nseveral disciplines. There is a gap in knowledge, as stated by authors: mechanisms of SARS-\nCoV-2 induced maternal and fetal pathology are poorly understood, and vaccine hesitancy\nremains common among pregnant women (26-57% vaccinated) although studies indicate\nvaccination reduces maternal morbidity and mortality without increasing the risks of adverse\nfetal outcomes.\nData & methodology: For the most part (exceptions noted below) the reporting of data and\nmethodology sufficiently detailed and transparent to enable reproducing the results.\nRESULTS/DATA:\nBody weight loss due to SARS-CoV-2 infection in pregnant Syrian hamsters depends\non gestational stage\n(Line 136-138): Authors state that SARS-CoV-2 infections was confirmed at 4 dpi using buccal\nswabs with a COVID-19 self-test kit (presumably for humans). Was this ever validated for\nhamsters (i.e., using PCR) in this or previous studies that could be referenced?\nR/ We did not perform a formal validation of the COVID-19 self-test kit in this study, as\nits ability to detect the SARS-CoV-2 nucleoprotein in buccal swabs of Syrian hamsters\nhas been previously demonstrated, with detection sensitivity down to 0.1 TCID and\nup to 6-7 days post-infection [PMID: 36064749]. This reference has now been added\n₅₀\nto the Methods section (line 792). Furthermore, the test has been reported to detect\nSARS-CoV-2 nucleoprotein in saliva samples from multiple animal species, including\ncats, ferrets, cattle, sheep, swine, and bats [PMID: 34563884].\nIn our study, data from animals infected at E10.5 (where the rapid test was performed\nat 4 dpi and lung viral RNA quantified at 5 dpi) indicate that the test achieved a\nspecificity of 100% (i.e., no false positives) and a sensitivity of 86.7% (i.e., 2 false\nnegatives among 15 PCR-confirmed infected animals). Across all 70 experimental\nanimals tested, the rapid test yielded no false positives (100% specificity) and only\nthree false negatives, corresponding to an overall sensitivity of 96%. Notably, all three\nfalse-negative animals had lung viral RNA levels exceeding 10³ copies/mg at the\nexperimental endpoint (line 149-150).\n(Line 146-148): This is a general comment for the entire manuscript… it seems like in some\nsections of the narrative results, statistical means, CIs, p-values, etc. are listed, while they are\nleft out of others. Please be consistent. I personally think it’s fine to just state whether\nsomething was statistically significant in the narrative sections, but it’s important to list the\nactual numbers (p-values, etc.) in the figure/graph/legend (though I’m not sure if Nat Comm has a\nrule on that). Same thing for “Ns.” Please list Ns in figures (i.e., experimental design\ngraphics) and graphs (or at least in the legend) +/- in the actual narrative (just be consistent).\nR/ We have revised the document and harmonized the report of the p-values. As stated\nabove, for clarity of the figures, we display only statistically significant differences (p <\n0.05), except in two cases (Fig. 2F and Supplementary Fig. 3B), where showing the\nabsence of differences is important. This is now indicated in the Statistics section in\nthe Methods (line 936-137). In addition, all p-values are provided in the Source Data\nfile.\n(Line 152-154): Suggest adding this reference about the B.1.1.7. strain being relatively more\naggressive in hamsters (vs. newer variants) forward (maybe near line 133), as it answers the\nquestion as to why such an early variant was chosen for this study.\nR/ New sentence and the reference have been added after the first mention of B.1.1.7.\n(line 143-145).\n(Line 192) states that hemoglobin was the only blood parameter significantly affected in\npregnant, SARS-CoV-2 infected dams, but it does not appear to be shown graphically\nanywhere (or maybe I’m just missing it because there are not legends in supplementary\nfigures)?\nR/ The legend of Supplementary Figure 2 has been adapted to define the abbreviations\nof the Y-axis. The corresponding narrative in the main manuscript has been carefully\nrevised and modified accordingly (lines 173-174; 197-202).\n(Line 192-194): Why is this elevated hemoglobin suggestive of a compensatory adaptation in\nresponse to an increased respiratory stress? Is there a reference for this? Is it supposition?\nPerhaps better placed in the discussion?\nR/ Indeed, after careful review, no significant differences were observed between\npregnant mock and infected females. However, red blood cells, hemoglobin, and\nhematocrit remained notably different in pregnant dams compared to non-pregnant,\nreflecting hematological adaptations that align with physiological adjustments known\nto support gestation in humans [PMID: 23997449]. This has now been adapted\naccordingly (lines 172-174, 197-202).\n(Lines 198-200): Confusing sentence, suggest rewording/clarifying\nR/ Adapted accordingly (lines 205-208).\n(Line 207) states that alveolar damage affected up to 40% of lung volume. How was this\nmeasured? It is not specifically described in the histology section of the methods. As you are\nevaluating 2-D glass slides it would be more accurate to list % of visible lung surface area\naffected.\nR/ As well noted, this should have been % of the visible lung surface. The sentence\nhas now been adapted (line 215).\n(Line 214): I think it’s an overstatement to say that lung inflammation associated with the\ninfection was resolved by 8 dpi, more like it had become more reparative/chronic in nature,\nbut there is still some inflammation- the lung does not appear normal.\nR/ We agree that this may have been an overstatement. The sentence has now been\nrevised to suggest a shift from an acute pro-inflammatory profile toward reparative\ninflammatory features (lines 219-224).\nFigure 1F and lines 205-214: Were there any statistically significant differences?\nR/ Statistical information is now included in the text and the Figure Legend (lines 221-\n222).\n(Lines 215-216) were these cytokines evaluated in lung tissue? Are they all pro-inflammatory\nor are any anti? Please clarify (even just re-wording to say “which are pro-inflammatory CKs\nupregulated in..’)\nR/ Adapted to clearly state their function (lines 225-227).\nFigure 1G- show p-value/significance, please\nR/ Adapted accordingly.\nLine 222 states that the low levels of Mx2 detected in E4.5 dams infected at E4.5 were likely\ndue to a remnant type I IFN response induced by residual circulating vRNA… Is there a\nreference for this? Is it supposition? Perhaps better placed in the discussion?\nR/ MX2, along with other interferon-stimulated genes, remains detectable in lung tissue\nas long as viral RNA is present, with expression levels declining as viral RNA\ndecreases [PMID: 34381043], as now also mentioned and included as a new reference\n(line 234). At 14 dpi, when viral RNA is no longer detectable, MX2 transcripts are also\nabsent.\nWe therefore infer that the continued presence of viral RNA in the lungs of hamsters\ninfected at E4.5 (11 dpi, Figure 1D) maintains type I interferon signaling, thereby\nsustaining MX2 expression. For clarity, we have now included the reference in this\nsection of the manuscript and removed the word ‘circulating’, as both the viral RNA\nand MX2 are measured in dissected lungs (line 234).\nMaternal SARS-CoV-2 infection leads to intrauterine growth restriction in a gestational\nage-dependent manner\nFigure 2C: Please define the colored dots (small legend)\nR/ The new Figure 2C now displays the experimental groups on the x-axis.\nFigure 2 (general): Ns should be listed, where applicable\nR/ For clarity of the figures, we display only statistically significant differences (p <\n0.05), except in two cases (Fig. 2F and Supplementary Fig. 3B), where showing the\nabsence of differences is important. This is now indicated in the Statistics section in\nthe Methods (lines 936-937). All p-values are provided in the Source Data file.\n(Lines 272-275): Is there a reference for this? How would this affect placental weight?\nR/ We apologize for the omission. The relevant references on IUGR induced by viral\ninfections and associated abnormal placental development and dysfunction have now\nbeen added (PMID: 24395887, 35150886, 31507551, 29305462).\nPersistent placental inflammation following viral clearance.\nFigure 3A: This may just be the PDF I received (vs. raw images), but these do not seem to be\npublication quality images, or it may just be that they are too small, but when I zoom in to try\nto look at what the arrows are indicating, the images are too blurry. I can’t tell if these are\naccurate, representative images or not. It’s hard to even tell what the normal control should\nlook like… is there space in the manuscript to make these pics larger in size?\nR/ We apologize for this inconvenience. Separate high-resolution images will be\nprovided upon submission and deposited in FigShare for detailed inspection.\n(Lines 312-314): See previous comment in methods… how did you measure ratio of\nJZ:labyrinth? What should the normal ratio be? Is a higher or lower ratio associated with\nplacental insufficiency?\nR/ The junctional zone (JZ) and labyrinth areas were measured on histological sections\nusing QuPath (version 0.5.1). For each placenta, five regions per section were\nanalyzed, the areas of the JZ and labyrinth were quantified, and the average of these\nmeasurements was used to calculate the JZ: labyrinth ratio for comparison between\nmock and infected placentas.\nUnlike mice [PMID: 34872401], there is no established atlas of the hamster placenta\nto serve as guidance. We show that in mock-infected placentas, the average\nJZ:labyrinth ratio is approximately 0.7 (Figure 3C). A reduction in labyrinth\ndevelopment, and thus an increased JZ:labyrinth ratio, is expected to result in impaired\nnutrient and oxygen exchange, ultimately leading to placental insufficiency, circulatory\nabnormalities, and fetal growth restriction [PMID: 30319550, 34872401, 29943952].\nFor example, a reduced labyrinth is observed in a diabetic mouse model and\ncontributes to intrauterine growth restriction [PMID: 21491160]. Despite the\npathological features (Figure 3A, B) and the effects on fetal outcome (Figure 2), we did\nnot observe changes in the JZ:labyrinth ratio in placentas from infected animals (Figure\n3C).\nThe corresponding Methods section was adapted (lines 889-900).\n(Line 317): Reference (to nRBCs representing a fetal stress response)?\nR/ This reference has been added (line 340).\n(Lines 321-323): I’m not sure this is an accurate conclusion. You haven’t really\ndescribed/shown placental remodeling, and if it’s in the images in supplementary data 1, it’s\nnot circled or described so the reader will not be able to tell\nAlso, it’s pretty normal to have a delay between infection (initial viral detection in tissues) and\nseeing histopathologic lesions in tissues, since it takes a little while for the inflammatory\nreaction to get going and for the tissue reaction to manifest histologically. Similarly, histo\nlesions often persist for a little while (and sometimes for quite a while) after pathogen\nelimination, either due to a more chronic inflammatory response, remodeling/repair, or\nbecause of an inappropriate inflammatory reaction (i.e., cytokine storm) which is reported in\nhuman SARS-CoV-2 infections.\nR/ We thank the reviewer for this helpful comment. We agree that our original wording\nimplied that we had demonstrated placental remodeling, which was not directly shown.\nTo address this, we revised the text and adapted it accordingly to avoid\noverinterpretation (lines 345-347).\nAdministration of low molecular weight heparin improves pregnancy outcomes in\nSARS-CoV-2 infected hamsters\nFigure 4: Please “define” the different colored shapes for each figure component\nR/ We appreciate the reviewer’s request for clarification. In Figures 4A-D and 4F, the\ninfection and heparin administration status are already indicated below each panel\nusing the “+” and “–” symbols, which we chose to avoid overcrowding the figure. For\nconsistency, we used the same colors and shapes in Figures 4E and 4G, and indicated\nthe color code in the corresponding panel.\n(Line 444): suggest removing “congestion” as a scoreable histopathologic feature.\nR/ Congestion in H&E staining refers to the presence of excess blood within tissue\nvessels, typically visualized as dilated and engorged blood vessels filled with red blood\ncells in histological sections. This feature can reflect underlying processes such as\ninflammation, impaired venous drainage, or increased blood flow, and is reported in a\nvariety of conditions, including chronic diseases, infections, and tissue injury (PMID:\n35671465, 34007347, 23300573).\nWe have now explicitly defined what we mean by congestion in the Methods section\n(lines 893-894). We consider congestion an important histopathological feature to\nscore, as it provides valuable insight into vascular alterations and tissue pathology. To\nensure consistency, congestion was scored on a semi-quantitative scale from 1 to 3,\nranging from mild to marked, based on the extent of the affected area.\nFor these reasons, we prefer to keep this prominent histopathological feature.\n(Line 449-451): states that “enoxaparin-treated placentas from infected dams contained an\nincreased number of nRBCs compared to the untreated group as a possible safety sign.”\nEarlier, nRBCs in the fetal vasculature was interpreted as a sign of fetal stress. Is this different\nbecause it’s not in the fetal vasculature? Please explain/clarify. Is there a reference for this?\nR/ We thank the reviewer for pointing out this potential inconsistency. We have revised\nthe sentences and tempered the conclusion on the increased frequency of nRBC in\nplacentas from enoxaparin-treated, SARS-CoV-2-infected animals (474-482).\n“Interestingly, placentas from enoxaparin-treated infected dams exhibited an increased\nnumber of nRBCs compared with the untreated group (Fig. 4D and Supplementary\nFig. 4F). Although elevated nRBCs are typically associated with fetal hypoxia or stress\nand reflect compensatory erythropoiesis, their increase in this context may instead\nrelate to the additional immune-modulatory functions of nRBCs [PMID: 29698529,\nPMID: 30127064]. This possibility is consistent with the observed reduction in the\nfrequency of SGA pups following enoxaparin treatment. Nonetheless, further\ninvestigation is required to clarify the underlying mechanism. (Fig. 4D and\nSupplementary Fig. 4F).”\n(Lines 464-468): Confusing sentence, please clarify and summarize the significance of this\ndata.\nR/ We have revised the sentence for clarity and to better highlight the main conclusion.\nThe text is adapted (lines 493-500).\nCOVID-19 vaccination protects from adverse pregnancy outcomes.\n(Lines 460-462) & Fig 1A: from what I can tell, Fig 1A does not indicate that “Seroconversion\nkinetics to anti-SARS-CoV-2 antibodies in pregnant hamsters were monitored starting from\nchallenge with B.1.1.7. at various time points;” please update figure (or tell me what I missed).\nR/ We thank the reviewer for this pointing this out. The seroconversion kinetics are shown\nin Supplementary Figure 6A and B, not Figure 1A. We have corrected the manuscript\nto reflect this.\nFigure 5F: This may just be the PDF I received (vs. raw images), but these do not seem to be\npublication quality images, or it may just be that they are too small, but when I zoom in to try\nto look at what the arrows are indicating, the images are too blurry. I can’t tell if these are\naccurate, representative images or not. It’s hard to even tell what the normal control should\nlook like… is there space in the manuscript to make these pics larger in size?\nR/ We apologize for this inconvenience. Separate high-resolution images will be\nprovided upon submission and deposited in FigShare for detailed inspection.\nSupplementary Figures: A brief title for each graph/figure would be helpful.\nR/ Figure titles and legends are included in the Supplementary Figures file.\n1. (a) Supp Fig 1A: Define “high vs. low” sperm.\nR/ We have now implemented an automated quantification method that measures the\narea occupied by sperm within the imaged field. This approach enables a more\naccurate correlation between sperm content and litter size. In the revised\nSupplementary Fig. 1A, we now present both the original bright-field image and the\nthresholded image, highlighting sperm cells selected based on contrast. Despite\nnotable variability in litter size at low sperm content, we observed a weak but\nstatistically significant correlation between sperm content and litter size (R = 0.39, p =\n0.0057). Importantly, all instances of pregnancy failure corresponded to sperm content\nbelow 15%. This information is now added to the Methods section (lines 765-776)\n1. (b) Supp Fig 1B: the test for E7.5 looks like it’s negative (or very faint?)… shouldn’t all but\nthe mock/control have 2 lines?\nR/ We have changed the representative test for E7.5 and enhanced the contrast to\nimprove visualization of the faint lower band.\n2. Supp Fig 2B: in the narrative (lines 183-185) authors state that neutrophils are significantly\nincreased in pregnant hamsters (including p-value). Please add p-value to the actual\ngraph/figure; Supp Fig 2C/D: As above, this may just be the PDF I received (vs. raw images),\nbut these do not seem to be publication quality images, or it may just be that they are too\nsmall, but when I zoom in to try to look at what the arrows are indicating the images are\nblurry… is there space in the manuscript to make these pics larger in size?\nR/ We initially focused on the comparison between non-pregnant hamsters and mock-\ninfected pregnant animals. However, it is more appropriate to compare all experimental\ngroups with the corresponding adjustment for multiplicity.\nWe observe that whereas white blood cell subsets (neutrophils, lymphocytes,\nbasophils, monocytes, eosinophils) and platelet counts appeared altered in pregnant\nhamsters, did formally however not differ significant from non-pregnant hamsters,\nperhaps due to the limited sample size (Supplementary Fig. 2B). We have therefore\nrevised the text accordingly (lines 172-202), and p-values >0.05 are no longer\ndisplayed in the figures, consistent with our approach of indicating only significant\ncomparisons. All p-values are provided in the Source Data file.\nSeparate high-resolution Figures will be provided upon submission and are deposited\nin FigShare for detailed inspection.\n4. Supp Fig 4 (all): Please “define” the different colored shapes for each figure components\nR/ We appreciate the reviewer’s request for clarification. In the main and\nSupplementary Figures (now labeled “5”), the infection/mock and heparin\nadministration status are indicated below each panel using “+” and “–” symbols, which\nwe included to avoid overcrowding the figure.\nSupplementary Data 1 would be more useful with a legend/description\nR/ Supplementary Data 1 legend added.\nMETHODS:\nSARS-CoV-2 infection model in pregnant hamsters:\n(Line 711) What was the cutoff/definition for “abundant” sperm content? (i.e., > a certain # of\nsperm per 40X high power field, etc.)\nR/ To support or previous semi-quantitative scoring approach, we have now\nimplemented an automated quantification method that measures the area occupied by\nsperm within the imaged field. This approach enables a more accurate correlation\nbetween sperm content and litter size. In the revised Supplementary Fig. 1A, we now\npresent both the original bright-field image and the thresholded image, highlighting\nsperm cells selected based on contrast. Despite notable variability in litter size at low\nsperm content, we observed a weak but statistically significant correlation between\nsperm content and litter size (R = 0.39, p = 0.0057). Importantly, all instances of\npregnancy failure corresponded to sperm content below 15%. This information is now\nadded to the Methods section (lines 765-776)\n(Lines 721-722): Were non-infected control animals mock-inoculated intranasally with an inert\nsubstance (such as PBS) to match infected animals? If not, I recommend this in future, to\nshow that iatrogenic causes (i.e., aspiration pneumonia simply from overzealous IN\ninoculation) is not contributing to the maternal bronchopneumonia noted histologically.\nR/ We did not include intranasally mock-inoculated controls in these experiments, as\nprevious work has shown that intranasal administration of PBS (100 µL in the\nreferenced study; PMID: 34740873) does not induce adverse histopathological\nchanges in hamster lungs, definitively not of a similar severity induced by infection as\nabundantly shown in our previously in vivo studies assessing vaccine [PMID:\n36333374, 33260195], and drug efficacy [PMID: 34609205, 34571361, 33037151].\n(Line 731): Were any length/width measurements performed? Or just weight?\nR/ The litters were documented photographically but we did not perform length or width\nmeasurements of the fetuses; only fetal weights were recorded.\nHistology: Please include a table or other summary specifically defining histopathologic\nscoring criteria; the general descriptions in lines 805-807 (lung) & 808-812 (placenta) are not\nspecific enough. For example, what made each of the features listed receive the score of 1, 2\nor 3? I also suggest removing congestion as a grading criterion for the placenta. This will\nprobably just end up confounding things, since, as stated in lines 307-310, increased\ncongestion is a typical feature of normal placentas near parturition. Also, did you consult with\na board-certified pathologist?\nR/ Lung pathology was analyzed and scored by an experienced hamster lung\npathologist. Due to limited data on placental histopathology of the hamsters, we based\nour scoring criteria on published studies in mouse [PMID: 34872401, 1249139] and\nrabbit placentas [PMID: 28046002].\nRegarding congestion, in H&E-stained sections, it refers to dilated, engorged vessels\nfilled with red blood cells, reflecting vascular alterations that may result from\ninflammation, impaired venous drainage, or increased blood flow [PMID: 35671465;\nPMID: 34007347; 23300573]. We consider congestion a relevant histopathologic\nfeature, and it was scored semi-quantitatively from 1 to 3 (mild to marked) based on\nthe proportion of affected tissue. The corresponding methods section was adapted to\nprovide additional scoring information (line 889-900).\n(Line 803): Is isolectin specific for fetal blood vessels? Or would it stain all blood vessels,\nincluding maternal? Please specify, including reference.\nR/ Isolectin B4 binds terminal α-D-galactosyl residues expressed by endothelial cells\nand is therefore a marker for both blood and lymphatic vessels. In the placenta,\nisolectin staining is a common technique used to visualize and analyze the labyrinthine\nvasculature. The labyrinth represents the fetal vascular compartment, which is\nisolectin-positive, whereas the adjacent junctional zone is isolectin-negative. This\nstaining highlights the network of fetal blood vessels within the labyrinth, enabling\nassessment of vascular branching, density, and overall architecture [PMID: 26655736,\nPMID: 37463089]. These references are now added to the manuscript (line 888)\n(lines 810-812) How did you determine the ratio of JZ to labyrinth? A computer program like\nImageJ? Something else? Please specify.\nR/ The junctional zone (JZ) and labyrinth areas were measured on histological sections\nusing QuPath (version 0.5.1). For each placenta, five regions per section were\nanalyzed, the areas of the JZ and labyrinth were quantified, and the average of these\nmeasurements was used to calculate the JZ: labyrinth ratio for comparison between\nmock and infected placentas. The corresponding Methods section was adapted (line\n889-900).\nAppropriate use of statistics and treatment of uncertainties: Overall, statistics appear robust\n(but see note below- this is not my area of expertise). There are a couple missing p-values\n(specified above), and the authors should be consistent about listing n’s error bars, means, p-\nvalues etc. in the same place… sometimes they are written into the narrative, sometimes just\nin the figures. I suggest that they are always included in the figures/legends (+/- the narrative\nat the discretion of the author/editor)\nConclusions: For the most part, the discussion, conclusions and data interpretation are\nrobust, valid and reliable. Additionally, the authors clearly state the limitations of this study.\nThere are a couple of (minor) statements that might not have been supported by the data,\nwhich are detailed above/below.\nIn the first part of the discussion, suggest adding some comparisons to humans (i.e., does\nIUGR in hamsters recapitulate what’s seen in humans? If not, how does it differ? Etc.) This\ncorrelation with human findings is nicely done in the next part of the discussion with CKs and\nhistopathologic findings (lines 566-573).\nR/ Additional information was added (lines: 591-599, 619-622, 625-627).\n(Lines 547-548) As mentioned previously, I think it is an overstatement to say that infected\nfemale hamsters rapidly recovered from lung damage. The lung histo images do not appear\nnormal/resolved at later infection stages, rather the lesions have become more\nreparative/chronic in nature.\nR/ We thank the reviewer for this valuable comment. We agree that the phrase “rapidly\nrecovered from lung damage” may overstate the extent of tissue recovery. The text has\nbeen revised and adapted (lines 583-584).\nLine 549: Suggest defining placental insufficiency (with reference). What are the features\n(clinical, histo, etc.)\nR/ While the term placental insufficiency is commonly used to describe impaired\nplacental function leading to fetal compromise, it lacks a standardized definition.\nClinically, it is associated with fetal growth restriction and preeclampsia, reflecting\nreduced oxygen and nutrient transfer to the fetus. Histopathological features often\ninclude villous fibrosis, thrombosis, infarction, and fibrin deposition, which reduce the\neffective exchange surface [PMID: 29422210, 33085318]. In our study, we observed\nsustained inflammation with necrotic and fibrotic changes in the placenta; to improve\nclarity, we have revised the text to avoid potential misinterpretation (line 585-587).\nLine 552: Suggest changing “clearly indicating” to “suggesting”\nR/ The text has been adapted (line 589).\n(Line 560): Refers to “makers” of innate/adaptive immunity- should this be “markers?”\nR/ The text has been adapted (line 602).\n(Lines 580-582): Please be more specific about the location of nRBCs that are associated with\nhypoxia/growth restriction. Is it when they are seen in the placenta? Fetal vasculature? (both\nare mentioned separately in text) Both? What do you think is the significance of this finding?\nAlso, approximately how many were seen (few, numerous)?\nR/ The nucleated red blood cells (nRBCs) were observed within the fetal vessels of the\nlabyrinth zone. To quantify this finding, we applied a semi-quantitative scoring system\nbased on the number of nRBCs per histological section and compared these scores\nwith non-infected controls (0: <30; 1: 30–60; 2: 60–90; 3: >90). These details are now\nincluded in the Methods section (line 889-900).\nWhile nRBCs are often associated with hypoxia and fetal growth restriction, no growth\nrestriction was observed in enoxaparin-treated infected dams [PMID: 28467759,\n12951889]. Notably, in rodents, nRBCs also have immune-modulatory functions,\nincluding pathogen recognition, cytokine production, and phagocytosis, which may\ncontribute to the placental response to infection [PMID: 29698529, 30127064]. The text\nwas revised to clarify the location of nRBCs (line 339).\n(Lines 617-618) state that the authors did not observe the dissemination into the brains, but\nwere additional tissues assessed other than the lung/placenta? I don’t remember seeing\nanything in the methods or results about that… Please clarify.\nR/ We thank the reviewer for this important comment. In contrast to mice (either\nhACE2-transgenic mice infected with clinical SARS-CoV-2 isolates, or WT mice\ninfected with mouse-adapted SARS-CoV-2 strains), in which neuroinvasion and\nneurovirulence is a characteristic feature of SARS-CoV-2 infection [PMID: 37161442,\n34955064; 38765009], evidence for viral replication in the brains of Syrian hamsters is\ninconsistent and to the best of all current evidence, alike in humans, not a main factor\ncontributing to disease in this model [PMID: 33577760; 35857629; 32571934].\nMoreover, based on previous data from our laboratory, little to no viral RNA was\ndetected in the blood, spleen, upper small intestine, or liver of infected WT hamsters\n[PMID: 33203860]. We also did not observe any clinical signs of encephalitis in our\ninfection model. For these reasons, we focused our analyses on the lung and placenta\nand did not include additional tissues in the present study.\nThe text was now revised to clarify that brain tissues were not evaluated in this study\n(lines 662-664).\nSuggested improvements: see above.\nReferences: This manuscript references previous literature appropriately.\nClarity and context: Abstract succinctly summarizes the problem, and describes the research\nplan, methods (3-D vascular endothelial culture system, animal infections), results and\nconclusion.\n(Lines 29-30) Suggest being more specific by saying “….linked to placental damage,\ncharacterized histologically by fibrin deposition, thrombosis, and elevated Ip10, Il6, and Il10\nexpression in (list tissue here)”\nR/ The text has been adapted to provide clarity (line 30).\nInflammatory material: This manuscript does not contain any language that is inappropriate or\npotentially libelous."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The author have adequately addressed all the points raised and I believe this manuscript is now suitable for publication.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Overall, my concerns have been addressed in the revisions. There are two very minor remaining edits:\n1) Fig3A (E10.5): The legend states that red arrows point to thrombi; however, I don't see thrombi, the arrows seem to just be\npointing to sinuses in the JZ> suggest moving them for clarification\n2) Fig5F (E4.5): There is no box on the bottom subgross image outlining the inset depicted on the right\nOnce these corrections are made, the manuscript does not need to be resent, as they are very minor"}, "author_response": "REPLY TO REVIEWER’S COMMENTS\nWe sincerely appreciate the reviewers’ constructive feedback throughout the review process.\nBelow, we address the remaining comments point by point.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have adequately addressed all the points raised and I believe this manuscript is\nnow suitable for publication.\nR/ The authors thank the reviewer for the careful assessment of this work.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nR/ The authors thank the reviewer for the careful assessment of this work.\nReviewer #3 (Remarks to the Author):\nOverall, my concerns have been addressed in the revisions. There are two very minor\nremaining edits:\n1) Fig3A (E10.5): The legend states that red arrows point to thrombi; however, I don't see\nthrombi, the arrows seem to just be pointing to sinuses in the JZ> suggest moving\nthem for clarification\nR/ We thank the reviewer for pointing this out. Figure 3A has been revised with\nrepositioned arrows for clarity.\n2) Fig5F (E4.5): There is no box on the bottom subgross image outlining the inset\ndepicted on the right\nR/ The missing box has now been added to the bottom subgross image in Figure 5F (E4.5)\nto indicate the inset shown on the right.\nOnce these corrections are made, the manuscript does not need to be resent, as they are very\nminor"}]

Decision letter