Maternal SARS-CoV-2 infection elicits sexually dimorphic placental immune responses

There is a persistent bias toward higher prevalence and increased severity of coronavirus disease 2019 (COVID-19) in males. Underlying mechanisms accounting for this sex difference remain incompletely understood. Interferon responses have been implicated as a modulator of COVID-19 disease in adults and play a key role in the placental antiviral response. Moreover, the interferon response has been shown to alter Fc receptor expression and therefore may affect placental antibody transfer. Here, we examined the intersection of maternal-fetal antibody transfer, viral-induced placental interferon responses, and fetal sex in pregnant women infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Placental Fc receptor abundance, interferon-stimulated gene (ISG) expression, and SARS-CoV-2 antibody transfer were interrogated in 68 human pregnancies. Sexually dimorphic expression of placental Fc receptors, ISGs and proteins, and interleukin-10 was observed after maternal SARS-CoV-2 infection, with up-regulation of these features in placental tissue of pregnant individuals with male fetuses. Reduced maternal SARS-CoV-2–specific antibody titers and impaired placental antibody transfer were also observed in pregnancies with a male fetus. These results demonstrate fetal sex-specific maternal and placental adaptive and innate immune responses to SARS-CoV-2.


This PDF file includes:
Supplementary Materials and Methods           S1. Demographic and clinical characteristics of participants providing maternal and cord blood by fetal sex and maternal SARS-CoV-2 status. Table S2. Timing of Influenza and pertussis vaccination relative to maternal titers drawn at delivery hospitalization. Table S3. 2-way ANOVA analysis of Fc receptor gene expression, immunoblots, and immunohistochemistry. Table S4. 2-way ANOVA analysis of inflammatory cytokine and interferon stimulated gene expression. Table S5. Taqman gene expression assays used for qPCR.
Fluorescence was acquired using an Intellicyt iQue. Relative antigen-specific antibody titer and FcR binding was reported as Median Fluorescence Intensity (MFI).

RNA extraction
Placental tissue was placed in Trizol (100 µL/10 mg) and homogenized using a TissueTearor. The resulting suspension was then centrifuged at 12,000 x g for 10 minutes after which the pellet was discarded. Chloroform was added to supernatant at a ratio of 100 µL chloroform/50 mg tissue. Tubes were shaken vigorously for 15 seconds, allowed to stand at room temperature for 10 minutes, and then centrifuged at 12,000 x g for 15 minutes. The aqueous phase was collected and the remainder of the RNA extraction procedure was performed using an RNeasy Mini Kit with on-column DNase I treatment (Qiagen) according to manufacturer instructions.
RNA quantity and purity were assessed using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific).
cDNA synthesis and quantitative polymerase chain reaction (qPCR) cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad) according to manufacturer instructions. Briefly, 800ng of RNA was mixed with iScript Reaction Mix, iScript Reverse Transcriptase, and nuclease-free water. No template control and No reverse transcriptase (RT) controls were prepared. All samples were primed at 25°C for 5 minutes, heated to 46°C for 20 minutes, and RT was inactivated at 95°C for 1 minute using a MiniAmp Plus Thermal Cycler (Thermo Fisher Scientific). qPCR was then performed using Taqman gene expression assays on a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Gene expression assays used are listed in table S5. Gene expression was normalized to the placental reference gene Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) and expressed relative to female coronavirus disease 2019 (COVID-19) negative samples to yield a relative quantity value (2 -ΔΔCt ). Importantly, the average values for female negative sample used for normalization was obtained using all female negative samples. That is, every experimental sample would have four values, as each experimental sample had four placental biopsies. Neither fetal sex or maternal SARS-CoV-2 infection altered expression of the reference gene YWHAZ or another common placental reference gene TOP1. All genes of interest were probed using gene expression assays with a dye of FAM-MGB, and YWHAZ and TOP1 were assessed using gene expression assays with a dye of VIC_PL. Two separate biopsies were each run in technical duplicate, and the average of biological replicates were then used to obtain final reported 2 -ΔΔCt values.

Protein extraction and quantification
Placental tissue was placed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific #89901) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific #78443). Samples were homogenized using a TissueTearor. The resulting mixture was centrifuged at 10,000 rpm for 10 minutes at 4°C and supernatant was kept for protein quantification. Protein was quantified using Pierce BCA Protein Assay Kit per the manufacturer's instructions (Thermo Fisher Scientific #23225).
Immunoblotting 50 µg of protein was prepared in Protein Sample Loading Buffer (Li-Cor Biosciences) with 5 mM dithiothreitol (DTT), boiled for 10 minutes at 95°C, loaded on Mini-PROTEAN 4-20% TGX Stain-Free Precast Gels (Bio-Rad), and run at 200V for 30 minutes, after which they were transferred onto low fluorescence polyvinylidene fluoride (PVDF) membrane using a Trans-Blot Turbo Transfer System (Bio-Rad). Blots were then washed three times for 10 minutes in TBS (20X stock from Thermo Fisher diluted 1:20 in water) followed by imaging of Stain Free total protein with a ChemiDoc MP System (Bio-Rad). After stain free imaging, blots were washed three times for 10 min in TBS, followed by blocking in Intercept (TBS) Blocking Buffer (Li-Cor Biosciences) for 1 hour. Primary antibodies were then incubated in Intercept T20 (TBS) Antibody Diluent (Li-Cor Biosciences) overnight at 4°C. The next morning, membranes were washed six times for 10 minutes in TBS-T (20X stock from Thermo Fisher diluted 1:20 in water), followed by incubation with secondary antibodies at 1:15,000 dilution and hFAB Rhodamine Anti-Tubulin (Bio-Rad) at 1:4000 dilution for 1 hour in Intercept T20 (TBS) Antibody Diluent (Li-Cor Biosciences). Finally, blots were washed six times for 10 minutes in TBS-T, after which they briefly rinsed in and then placed in TBS. Fluorescence was then imaged with a ChemiDoc MP System (Bio-Rad) and quantified using Image Lab Software (Bio-Rad). Data are presented as volume of bands for protein of interest relative to the volume of bands contained in the total protein stain-free image.

Fc glycan analysis
Spike protein (Lake Pharma) was biotinylated and coupled to streptavidin magnetic beads (New England Biolabs, NEB). Cord and maternal plasma was heat-inactivated and spike protein-specific antibodies were isolated by incubating cord or maternal plasma with spike-coupled magnetic beads. The spike protein-specific antibody-bead complexes were incubated with IDEZ (NEB) for 1 hour at room temperature to cleave the Fc from spike protein-specific antibodies. The isolated Fc was then incubated with PNGase to remove the glycan and glycans were labelled with APTS per manufacturer's instructions (Glycan Assure APTS kit, Thermo Fisher Scientific). Glycans were analyzed using a 3500xL genetic analyzer (Applied Biosystems). The relative frequency of each glycoform was determined using the GlycanAssure Software.

Immunofluorescence (IF)
For co-labeling IF experiments, placenta tissue sections were rehydrated in an alcohol series after deparaffinization in xylene. Antigen retrieval was performed by boiling in 10 mM sodium citrate (pH 6.0) for 30 minutes and cooled at room temperature before blocking for 15 minutes with background sniper (Biocare Medical). Samples were then incubated in primary antibodies diluted in 5% BSA for 1.5 hours at room CellProfiler software (PMID:29969450) was used to quantify CD16/FcRn, CD32/FcRn, and CD64/FcRn colocalization as well as their respective intensities. Briefly, RGB pictures were converted to gray and placental villi selected to avoid measuring background. Following background removal, the pictures were filtered using the function CorrectIlluminationCalculate and aligned to evaluate colocalization. Finally, CD16, CD32 or CD64 and FcRn intensities in the placental villi previously delimitated to quantify colocalization.
CellProlifer software was similarly used to quantify immunohistochemistry for and CD163. Following isolation of each dye compound (3,3'-Diaminobenzidine (DAB) and Hematoxylin), the placental villi were manually isolated and the mean intensity of DAB signal quantified.

Fig. S1. SARS-CoV-2 infected mothers with male fetuses have lower plasma titers of SARS-CoV-2specific antibodies.
(A to C) Plots show maternal spike protein-, RBD-, S1-, S2-, and N protein-specific maternal blood IgG2 (A), IgG3 (B), and IgM (C) titers as median fluorescence intensity (MFI). Female neonates of mothers with SARS-CoV-2 are shown as white bars with an orange border and male neonates are shown as orange shaded bars with orange border. Differences across groups were assessed by two-way ANOVA followed by post-hoc analyses. Two-way ANOVA demonstrated a main effect of fetal/neonatal sex on maternal IgG1 titers (IgG2 p = 0.003 ; IgG3 p = 0.011 ; IgM p < 0.0001). *p < 0.05. Data are presented as mean ± SEM.     CoV-2 positive and SARS-CoV-2 negative mothers were stained for FCgR2 (purple), FcRn (red), and placental alkaline phosphatase (PLAP, green), a trophoblast marker, and DAPI (blue). Scale bars indicate 100 µm. (F) Box-and-whisker plots show FCgR2/FcRn co-localization in placental villi. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. (G) Box-and-whisker plots show tubulin/total protein quantification across fetal sex and maternal SARS-CoV-2 infection status. Two-way ANOVA revealed no significant effect of fetal sex or maternal infection. For box and whisker plots in D, F, and G, box extends from 25 th -75 th percentile, the whiskers depict minimum and maximum, and horizontal line depicts the median.

Fig. S7. Fc-glycan glycoforms in maternal bulk and SARS-CoV-2-specific antibodies.
Dot plots show the percentage of each glycoform in bulk maternal (purple) and spike protein-specific (orange) antibody glycan data from SARS-CoV-2-positive mothers for which matched data were available. Wilcoxon signed-rank test was performed to determine significance. * p < 0.05.

Fig. S10. Maternal SARS-CoV-2 infection does not impact placental cytokine expression. (A to M)
Luminex ProcartaPlex Immunoassays of tumor necrosis factor (TNF)-α (A), interleukin (IL)-6 (B), IL-12p70 (C), IL-13 (D), IL-17A (E), IL-8 (F), IL-1α (G), IL-1β (H), IL-4 (I), CD62E (J), CD62P (K), CCL2 (L), and CCL3 (M) in placental biopsies from SARS-CoV-2 negative (blue) or SARS-CoV-2 positive (orange) pregnancies. Two-way ANOVA followed by post-hoc analyses were performed to determine significance. Protein expression values are corrected to input protein concentration. Data are presented as mean ± SEM. Differences across groups were assessed by two-way ANOVA followed by Bonferroni's post-hoc analyses. *p<0.05. None of the regression models had statistical significance after FDR multiple comparisons correction using a corrected p-value of 0.05 as the cutoff. (C) Linear regression models were performed on female (top) and male (bottom) neonates separately to examine the effect of birthweight on placental transfer of S1-specific IgG1 (no significant effect). (D) Linear regression models were performed on pregnancies with a female fetus (top) and male fetus (bottom) separately to examine the effect of days since maternal infection on transplacental transfer of S1-specific IgG1 (no significant effect). (E) Linear regression models were performed on female (top) and male (bottom) neonates separately to examine the effect of gestational age at delivery on placental transfer of S1-specific IgG1 (no significant effect). (F) PLSDA model demonstrating that multivariate gene expression profiles of participants to not separate by labor status. (G) Heatmap depicting linear regressions of fetus sex and expression of individual ISG or FcR in placentas. None of the regression models had statistical significance after FDR multiple comparisons correction using a corrected p-value of 0.05 as the cutoff. Tables   Table S1. Demographic and clinical characteristics of participants providing maternal and cord blood by fetal sex and maternal SARS-CoV-2 status. DM/GDM, diabetes mellitus/gestational diabetes mellitus; BMI, body mass index; GA, gestational age; N/A, not applicable. a SARS-CoV-2 positive and negative status determined by nasopharyngeal RT PCR at time of sample collection. If a participant was SARS-CoV-2 positive at any time in pregnancy she was included in "SARS-CoV-2 positive" category. b Differences between groups were determined using chi-square test for categorical variables, and Kruskal-Wallis test for continuous variables presented as median [interquartile range, IQR]. c Disease severity classification based on published criteria from the National Institutes of Health. 3 (5) 1 (9) 1 (9) 1 (5) 0 (0) Chronic hypertension, n (%) 1 (2) 0 (0) 0 (0) 1 (5) 0 (0) 0.68 DM/GDM, n (%) 6 (11) 1 (9) 1 (9) 3 (14) 1 (9) 0.96 BMI ≥ 30 kg/m 2 , n (%)

Supplementary
17 ( Table S3. Two-way ANOVA analysis of Fc receptor gene expression, immunoblots, and immunohistochemistry. Two-way ANOVA followed by Bonferroni's post-hoc analyses (when interaction term was significant) were performed to determine significance. All main and interaction effects for genes and proteins are represented for both male and female placentas, in addition to all post-hoc analyses performed. Significant effects are indicated by bolded red statistics. N/A: not applicable, indicates post-hoc testing not performed due to lack of significant interaction term. n/a n/a n/a n/a Table S4. Two-way ANOVA analysis of inflammatory cytokine and interferon stimulated gene expression and CD163 immunohistochemistry. Two-way ANOVA followed by Bonferroni's post-hoc analyses were performed to determine significance. All main and interaction effects for genes of interest are represented for both fetal males and fetal females in addition to all post-hoc analyses performed. Significant effects are indicated in bolded red. n/a, not applicable. n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a IL-6 F(1,32) = 0.029, p = 0.87 F(1,32) = 1.29, p = 0.26 F(1,32) = 1.47, p = 0.24 n/a n/a n/a n/a IL-12p70 n/a n/a n/a n/a n/a n/a n/a n/a CD62E F(1,32) = 0.34, p = 0.57 F(1,32) = 0.33, p = 0.57 F(1,32) = 3.49, p = 0.071 n/a n/a n/a n/a CD62P F(1,32) = 0.043, p = 0.84 F(1,32) = 0.34, p = 0.56 F(1,32) = 1.62, p = 0.21 n/a n/a n/a n/a n/a n/a n/a n/a  Table S5. Taqman gene expression assays used for RT-qPCR. Thermo Fisher Scientific Assay ID, Gene symbol, and color dye for each gene expression assay used are shown.