This is a prospective multicenter study that includes 31 women: 30 with laboratory-confirmed COVID-19 infection admitted at delivery in three COVID-19 maternity hospitals of Lombardy, Italy: the 'L. Sacco' Hospital (Milan), the S. Gerardo Hospital/MBBM Foundation (Monza), and the San Matteo Hospital (Pavia) between March 9 and April 14, 2020. A further woman, (subject n. 31), the wife of Italian patient One, was found to be SARS-CoV-2-positive at the 32 gestational weeks and delivered at Buzzi maternity Hospital, a COVID-19-free hub and was admitted in the study as well.
All women underwent clinical evaluation of vital signs and symptoms, laboratory analysis and radiological chest assessment at admission at discretion of physicians. The therapeutic management was consequently tailored according to the clinical findings and national guidelines22. Demographic and anthropometric characteristics, medical and obstetric comorbidities, were recorded at enrollment through a customized data collection form. All pregnancies were singleton, with a normal course and regular checks, until delivery.
Data on mode of delivery or pregnancy termination, maternal and neonatal outcomes, and postpartum clinical evolution (e.g. breastfeeding, skin to skin, etc.) were subsequently recorded. Data accuracy was independently verified by two study investigators.
The protocol was approved by the local Medical Ethical and Institutional Review Board.
Biological samples were collected at admission (T0), delivery (T1) and post-partum (T2), as summarized in figure 1. T0 samples included a nasopharyngeal swab in order to test the positivity for SARS-CoV-2. At T1, full thickness placental and umbilical cord biopsies were obtained and a 10 ml umbilical cord blood sample was collected in EDTA after cleaning throughout the cord with a sterile gauze and physiological solution before sampling. Both biopsies and blood samples were obtained in sterile way by a dedicated operator. In case of caesarean section, if possible, amniotic fluid was collected. Moreover, a 10 ml maternal blood sample in EDTA was collected, together with a vaginal swab before labor or cesarean section. Upon delivery, a nasopharyngeal swab was immediately performed on newborns and mothers. Approximately five days after delivery (T2), transitional breastmilk samples were collected from all breastfeeding women. For each subject, days occurring between T0 and T1 (ΔT1-T0) were calculated. Fourteen sets of samples from the Obstetrics and Gynecology Unit of “L. Sacco” Hospital (Milan) were immediately transferred to the dedicated laboratory of Clinical Microbiology, Virology and Diagnostics, “L. Sacco” Hospital, and/or to the laboratory of Immunology, University of Milan, according to the kind of specimen, to be readily processed. Seventeen samples collected at S. Gerardo Hospital/MBBM Foundation (Monza), and San Matteo (Pavia) were frozen at -80°C upon collection and transferred to the same laboratories in dry ice.
Molecular analysis was performed to detect viral RNA, using the automated Real-Time PCR ELITe InGenius® system and the GeneFinderTM COVID-19 Plus RealAmp Kit assay (ELITechGroup, France). The reaction mix was prepared according to manufacturer’s instruction. Three target genes, RNA-dependent RNA polymerase (RdRP), Nucleocapsid (N) and Envelope (E) have been simultaneously amplified and tested. A cycle threshold value (Ct-value) lower than 40 was defined as a positive test result according to the manufacturer’s instruction.
The presence of SARS-CoV-2 specific antibodies was investigated using SARS-CoV-2 IgG and IgM chemiluminescence immunoassay (CLIA) kits on fully automated iFlash1800 analyzer (Shenzen YHLO Biotech Co., Ltd., Shenzen, China): the assay uses Nucleocapsid (N) and Spike (S) viral proteins as magnetic beads coating antigens. The value of 10.0 AU/mL was used as positivity cut-off for IgM, while 7.1 for IgG23.
Placental and umbilical cord biopsies were manually dissected into few sections of approximately 2mm3. Such sections were then thoroughly homogenized and total RNA was isolated using the acid guanidium thiocyanate–phenol–chloroform method (RNAbee, Duotech, Milan, Italy), as previously described24. Alternatively, biopsies were paraffin-embedded and stored as such.
Plasma samples were collected from blood of all the enrolled subjects, as well as plasma samples from funicular blood, amniotic fluid and vaginal swabs. Moreover, as control for molecular analyses, plasma from a SARS-CoV-2 negative pregnant woman (CTR-), as well as plasma samples from funicular blood and placental tissues were included. RNA was extracted by the Maxwell® RSC Instrument with Maxwell® RSC Viral Total Nucleic Acid Purification Kit (Promega, Fitchburg, WI, USA). As result, RNA eluted in RNAse-free water was obtained.
Once RNA was reverse transcribed into cDNA24, real-time PCR was performed on a CFX96 (Bio-rad, CA, USA) using TaqMan probes specifically designed to target two regions of the nucleocapsid (N) gene of SARS-CoV-2. For such application, we employed the 2019-nCoV CDC qPCR Probe Assay emergency kit (IDT, Iowa, USA) which include also primers and probes that target the human RNase P gene.
Expression analyses of inflammatory response
The inflammatory response was analysed in four selected RNA samples extracted from placenta biopsies of one negative control (CTR-), one SARS-CoV-2 recovered (subject n. 31) subject and two SARS-CoV-2 positive subjects (subjects n. 17 and 25). Subjects n. 17 and 25 gave birth to SARS-CoV-2 positive newborns, according to the first nasopharyngeal swab (T1). Analyses were performed by a PCR array that include a set of 84 optimized real-time PCR primers plus 5 housekeeping genes on a 96-well plates; the procedures suggested by the manufacturer were followed (Qiagen, Hilden, Germania). Undetermined raw CT values were set to 35. Only variables with at least a two-fold increase in their value are presented and discussed in the manuscript.
Concentration of 27 cytokines was assessed in maternal and funicular plasma samples from the very same four subjects using immunoassays formatted on magnetic beads (Bio-rad, CA, USA) according to manufacturer’s protocol via Luminex 100 technology (Luminex, Texas, USA).
For the study variables, medians and ranges were reported for quantitative variables. The analyses were performed using SPSS Statistics, Version 26.0 (IBM Corp. Armonk, NY) together with GraphPad Prism 8.
All the procedures were carried out in accordance with the GLP guidelines adopted in our laboratories.