Fetal cell microchimerism and susceptibility to COVID-19 disease in women

Between March 2020 and December 2020, we studied 161 COVID-19 female patients, of which 64 consecutive hospitalized patients and 97 HealthCare workers with a positive SARS-CoV-2 test. The study period was limited to December 2020 before the development and the availability of SARS-Co-V-2 vaccine, as we were interested in the natural history of the disease, that was strikingly modified by the mass vaccination campaign. After the exclusion of cases who had no pregnancy or female offspring or insufficient data, we analyzed 63 women (COVID-19 cases) with a previous male pregnancy and affected with SARS-CoV-2 infections of variable severity (Fig. 1). Among these women: 40 had a mild to severe respiratory insufficiency hospitalized in the COVID Units at our Institution, 11 were treated at home, and 12 were asymptomatic HealthCare workers diagnosed by a positive naso-pharyngeal swab (NPS) (n = 5) or by serological screening (n = 7). Blood samples were taken at the first follow-up visit one to three months after discharge. We also enrolled 60 unaffected female controls (Controls) with male offspring, among HealthCare workers (Fig. 1). They were selected among the 304 female workers screened for COVID-19 at our Institution, according with the indications of the Italian Ministry of Health, with periodical nasal swab every 15 or 30 days starting from April 2020. In addition, they were submitted to a serological screening every 3 months until the end of 2021. Noteworthy, none of the controls selected for this study had a positive test or reported fever or other symptoms suggestive for airways infections during the enrollment period and in the subsequent follow-up (January 2021-Nov 2021). The study was approved by the ethics committee of Istituto Auxologico Italiano (COV-endo study, n 05C021). The participants or their parents gave informed consent to include their clinical data in the present study.

SARS-Cov-2 infection was confirmed in all symptomatic patients by RT-PCR from NPS. For the serological screening we measured both anti-SARS-CoV-2-RDB-Spike (Elecsys Anti-SARS-CoV-2 S, Roche Diagnostics GmbH, Mannheim, Germany) and anti-SARS-CoV-2-N protein total Ig (Elecsys Anti-SARS-CoV-2, Roche Diagnostics GmbH, Mannheim, Germany), in order to discriminate between natural infection and immunization by vaccine, which was at that time administered to some Health System operators.

Genomic DNA was extracted from whole blood samples using the DNA Extraction Kit BACC2 (GE Healthcare, Buckinghamshire, UK). For the detection of the human Y chromosome, DNA was submitted to two rounds of PCR (35 cycles each) using primers specific for the SRY locus and corresponding to the region upstream of the SRY-coding region, as previously described [19]. We previously demonstrated that this method has a sensitivity of 1 male cell/1 million female cells. Each PCR analysis was repeated twice, two negative controls (DNA from two pre-pubertal girls) and two positive controls (DNA from two men) were included, and special care was taken to avoid external contamination (Supplementary Fig. 1). In particular, all of the samples were handled by a female technician, positive displacement micropipettes were used, and DNA extraction, PCR preparation, and analyses were conducted in separate rooms. Samples were electrophoresed in 2% agarose gels and visualized and photographed on an U.V. trans-illuminator (BioDoc-It Imaging System, UVP, Cambridge, UK).

Possible differences in the clinical features between COVID-19 patients and healthy controls were assessed by t-test and Chi-square analysis, as appropriate. Odds ratio (OR) and 95% confidence intervals (CI) were used to assess the strength of the association between the presence/absence of FCM and the presence/absence of the COVID-19 disease. Possible differences in associated comorbidities, in the clinical course and symptoms were assessed in COVID-19 patients by t test and Chi-square analysis, as appropriate. The difference between values was considered significant when p < 0.05. A multivariate logistic regression model was employed to determine odds ratios (OR) for hospitalization in patients affected with COVID-19. The predictive model was determined by stepwise selection using a P value = 0.05 as the entry value for the model. All tests were performed using the Version 20.014 of the Med-Calc Software (B-8400 Ostend, Belgium).

COVID-19 cases and Controls did not significantly differ with respect to the age at enrollment (mean age 57.54 and 54.1 years, respectively) and several clinical features related to pregnancy history (age at the last male pregnancy, number of pregnancies, presence and number of miscarriages, number of male children, time elapsed from the last male pregnancy to sampling, in vitro fertilization, cesarean section) (Table 1). No differences were observed also for other clinical parameters, which could be potential sources of different forms of chimerism, such as maternal miscarriages, the presence of twin brothers, blood transfusion and organ transplant (Table 1). Interestingly, the presence of male DNA of presumed fetal origin had a significantly higher prevalence in controls than in COVID-19 cases (70 vs 44.4%, P = 0.0044 by Chi-square, OR 0.3429, 95% CI 0.1631–0.7207, P = 0.0047 by logistic regression) (Fig. 2). Within both groups, women positive for the presence of FCM were not significantly different from those without FCM considering the age at enrollment and several clinical parameters related to pregnancy history and other sources of chimerism (Table 2).

Among women affected with COVID-19, the presence of male FCM did not influence the severity of the disease, regarding symptoms, clinical course and associated comorbidities (Table 3). Concerning the clinical course, women with or without FMC had comparable length of the diseases (assessed as the duration of acute symptoms), and comparable length of the viral clearance (measured as the time elapsed between the first positive and the first negative naso-pharyngeal swab-NPS). The type of ventilation for hospitalized case (none/low–high flow oxygen/pressure support) was not statistically different between FMC positive and negative women, too. The number of patients with known risk factor for severe COVID-19 infections (hypertension, chronic obstructive pulmonary disease-COPD, cardiovascular diseases-CVD, obesity, thyroid diseases, epilepsy, age older than 65 years) was not different between FMC positive and negative women. Finally, the risk of being hospitalized was not influenced by FMC, but was increased by the age older than 65 years (OR 38.64, RR 2.05, P < 0,0001), the presence of comorbidities such as hypertension (OR 11.61, RR 1.92, P = 0.0002), obesity and overweight (OR 4.02, RR 1.63, P = 0.016) (data not shown). Nevertheless, we had the opportunity to test the blood samples of 8 parous women deceased as a consequence of SARS-CoV-2 infection, and they were all FCM negative.