INTRODUCTION

In December 2019, an outbreak of a novel coronavirus occurred in Wuhan, China. Since then, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 160 million people worldwide, causing more than 3.3 million deaths1. Several groups have been identified as being vulnerable to developing serious complications after SARS-CoV-2 infection, but data on pregnant women are still sparse2. Pregnant women are considered more susceptible to coronavirus disease 2019 (COVID-19) due to the immunological and physiological adaptations inherent to pregnancy3. Clinical manifestations in pregnant women with COVID-19 range from asymptomatic to severe. Although SARS-CoV-2 has been identified in the cytoplasm of perivillous trophoblastic cells, indicating placental infection4, 5, only a few cases of vertical transmission have been reported6-8. Most cases lack analysis of adequate biological samples to rule out neonatal infection9; therefore, the potential for SARS-CoV-2 vertical transmission remains controversial.

Presence of SARS-CoV-2 immunoglobulin (Ig) M antibodies in the peripheral blood of neonates suggests fetal exposure to the virus. However, this test alone is not sufficient for diagnosing intrauterine infection, and analysis of IgM is useful only in patients with clinical manifestation of the infection for more than 11 days10, 11. Criteria to define vertical transmission of SARS-CoV-2, based on the presence of the virus in biological samples at different timepoints, have been proposed12. According to these criteria, intrauterine transmission is considered to have likely occurred if the mother, even if asymptomatic, is positive for SARS-CoV-2 from 14 days before until 2 days after delivery, the virus is detected in either the amniotic fluid (AF), the placenta or the neonate within the first 24 h postpartum, and there is viral persistence in the neonate beyond 24 h. Criteria have also been provided for intrapartum transmission and superficial exposure12. Recently, the World Health Organization (WHO) classified the timing of mother-to-child transmission (intrauterine, intrapartum or early postnatal) into the following categories: confirmed, possible, unlikely or indeterminate13. The different tests and samples required to determine the occurrence of vertical transmission have also been described by the WHO13.

The objective of this study was to investigate the presence of SARS-CoV-2 in asymptomatic pregnant women and the presence and persistence of the virus in their neonates, in order to explore the possibility of vertical transmission, according to the strict criteria of the WHO.

METHODS Ethics statement

Participation in this study was voluntary and all women signed an informed consent form. The study was approved by the ethics and research institutional review board of the National Institute of Perinatology, Mexico City, Mexico (Registration number 2020-1-32).

Study population and specimens

All pregnant women attending for delivery at the National Institute of Perinatology in Mexico City, Mexico, from March 2020 to March 2021 were tested routinely for SARS-CoV-2 infection using real-time reverse-transcription polymerase chain reaction (RT-PCR) of nasopharyngeal swabs at least 24 to 48 h before delivery, according to the Centers for Disease Control and Prevention (CDC) recommendations14. The participants were selected according to the following inclusion criteria: absence of COVID-19 symptoms; RT-PCR-confirmed SARS-CoV-2 infection; third trimester of gestation; a viable fetus; and obstetric indication for Cesarean delivery, such as previous Cesarean delivery, cephalopelvic disproportion and maternal comorbidity. Only patients with planned Cesarean delivery were invited to participate since we needed to collect AF before membrane rupture and umbilical cord blood before the umbilical cord was cut. Exclusion criteria were contaminated AF sample, incomplete clinical data for the woman or fetus or inability to obtain AF or neonatal samples. Clinical and demographic data were obtained from medical records. In addition, a follow-up phone call was made 2 weeks after discharge in order to evaluate the presence of COVID-19 symptoms in the mother or the child.

Mild COVID-19 was defined, according to the National Institutes of Health (NIH) COVID-19 treatment guideline criteria, as an individual who had any of the various signs and symptoms of COVID-19 (e.g. fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea and loss of taste or smell), but without shortness of breath, dyspnea or abnormal chest imaging15.

AF was collected during Cesarean delivery by direct puncture of the intact fetal membranes and aspiration using a sterile needle and syringe. Neonatal oral and rectal swabs were obtained immediately after birth, taking special care to avoid contact with any maternal blood or tissue. Additional neonatal oral and rectal swabs were collected 24 h after birth.

RT-PCR for detection of SARS-CoV-2

Isolation of RNA from maternal and neonatal swabs was carried out using the Quick-RNA-Viral Kit (ZYMO Research, Irvine, CA, USA) and RNA isolation from AF (140 µL) was carried out using the QIAamp Viral RNA Midi Kit (QIAGEN, GmbH, Hilden, Germany), according to the manufacturer's instructions.

For SARS-CoV-2 detection by real-time RT-PCR, the protocol described by Corman et al. was employed16. In this protocol, the RdRP and E viral genes, as well as the RNaseP human gene as a control, are amplified. The RT-PCR reactions were run on a StepOne plus instrument (Thermo Fisher Scientific, Waltham, MA, USA).

The presence of specific SARS-CoV-2 IgG antibodies was determined at birth in maternal and umbilical cord serum by chemiluminescent microparticle immunoassay using the SARS-CoV-2 IgG kit (Abbott Laboratories, Chicago, IL, USA) and the Architect instrument (Abbott Laboratories).

The SARS-CoV-2 cycle threshold (Ct) value on RT-PCR has an inverse correlation with viral load17. We analyzed Ct values in maternal nasopharyngeal swabs, neonatal swabs and AF samples. Depending on the results of the test carried out on AF samples and neonatal oral and rectal swabs at birth, women were subdivided into five categories: (1) all women; (2) women with a positive neonate (according to either oral or rectal swabs or both) and negative AF; (3) women with positive AF and a negative neonate; (4) women with a positive neonate and positive AF; and (5) women with both a negative neonate and negative AF.

Analysis of timing of mother-to-child transmission of SARS-CoV-2

Classification of mother-to-child transmission was assigned according to the WHO criteria13. For intrauterine SARS-CoV-2 infection, the WHO recommendations assess the following three criteria: (1) evidence of maternal SARS-CoV-2 infection during pregnancy at any time, demonstrated through accepted standard methods; (2) in-utero fetal exposure to SARS-CoV-2, demonstrated by positive RT-PCR of a sterile or non-sterile sample (such as AF) or placental tissue, or detection of IgA/IgM antibodies in umbilical cord blood; and (3) persistence of infection or immune response in the neonate, proved by viral detection in a sterile or non-sterile sample using RT-PCR in the 24–48 h after birth or detection of IgA/IgM antibodies in neonatal blood at 24 h to < 7 days. Intrauterine transmission can occur through the hematogenous route, when the virus crosses the placental barrier and reaches the fetus to cause infection. Depending on the findings, in-utero transmission can be categorized as: confirmed, possible, unlikely or indeterminate13. If all three of the above criteria for in-utero transmission are not fulfilled, then neonatal infection can be considered as being potentially due to intrapartum transmission (when there is evidence of lack of in-utero exposure) or early postnatal contact (when the neonatal age at infection is > 48 h to 28 days and there is evidence of lack of in-utero and intrapartum exposure).

Statistical analysis

Qualitative variables are presented as n (%) and were compared using χ2 or Fisher's exact test. Quantitative variables are presented as mean with SD or median with interquartile range. Analysis of quantitative variables was performed using parametric (Student's t-test) or non-parametric (Mann–Whitney U-test) tests, depending on their distribution. A univariate logistic regression model was used to determine the association between covariables and the outcome of having a neonate with a positive result for SARS-CoV-2 infection. All statistical analyses were performed using the Statistical Package for Social Sciences version 25 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).

RESULTS Characteristics of study population

Of the 190 pregnant women who tested positive for SARS-CoV-2 from March 2020 to March 2021, 148 did not meet the inclusion criteria. Therefore, results are presented for 42 mother–child dyads. Maternal and neonatal clinical data are summarized in Table 1. Type-1 or -2 diabetes mellitus, obesity or high blood pressure was observed in 10 (23.8%) women. A pregnancy complication, such as gestational diabetes mellitus or placenta accreta spectrum disorder, was diagnosed in four (9.5%) women. There were three (7.1%) cases of neonatal death, of which one tested positive for SARS-CoV-2 at birth. Two of the neonatal deaths were related to a congenital heart defect and the third case died on the second day postpartum, with no obvious external defects, respiratory failure or COVID-19-related symptoms. The cause of death could not be determined in the third case because the parents declined postmortem examination. As per the inclusion criteria, symptoms related to COVID-19 were absent in all women at the time of the SARS-CoV-2 test. However, the follow-up phone call revealed that 25 (59.5%) women subsequently developed mild disease, with headache being the most common symptom (Table 2).

Table 1. Clinical and demographic data in 42 asymptomatic SARS-CoV-2-positive pregnant women and their neonates Case Maternal age (years) Developed symptoms after discharge Maternal comorbidity/pregnancy complication Neonatal sex Birth weight (g) Birth length (cm) GA at birth (weeks) 1-/5-min Apgar score Neonatal outcome 1 25 Yes DM2 F 2815 47.5 38.0 8/9 A&W 2 40 Yes GDM M 3645 52.0 39.0 8/9 A&W 3 30 Yes Cholestasis F 2815 48.0 37.3 8/9 NND 4 28 Yes Obesity M 3250 50.0 38.4 8/9 A&W 5 25 Yes Obesity, metabolic syndrome F 2615 47.0 39.6 7/9 Umbilical hernia, patent urachus 6 23 Yes DM1, hypothyroidism M 3025 49.0 37.6 9/9 A&W 7 33 Yes None M 3580 53.0 38.4 8/9 A&W 8 27 Yes None M 3010 49.0 38.1 9/9 A&W 9 33 Yes Obesity M 2960 49.0 38.1 8/9 A&W 10 33 No Rheumatoid arthritis M 1965 43.5 36.2 8/9 Low birth weight 11 45 Yes None F 3060 50.0 38.2 8/9 A&W 12 37 No None M 2835 48.0 37.2 8/9 Ventriculomegaly 13 23 No None M 2790 48.0 38.4 8/9 A&W 14 37 No None M 3215 49.0 39.2 8/9 A&W 15 35 Yes DM2, hypothyroidism M 3505 48.0 38.0 8/9 A&W 16 30 Yes None F 3305 50.0 38.1 9/9 A&W 17 36 Yes Hypothyroidism M 3245 51.0 38.0 8/9 A&W 18 37 No None F 2685 48.0 39.5 8/9 A&W 19 22 No None M 3020 51.0 38.6 8/9 A&W 20 24 No None F 3125 49.0 40.0 9/9 A&W 21 33 No PAS F 2730 48.0 37.2 7/9 A&W 22 43 No High BP, obesity M 3680 50.0 38.6 8/9 A&W 23 37 Yes PAS F 3790 45.0 38.0 8/9 Dysmorphic syndrome 24 28 Yes DM2 F 3225 51.0 38.4 8/9 A&W 25 24 Yes None M 3165 50.5 38.5 8/9 A&W 26 38 No None M 3090 50.0 38.4 8/10 A&W 27 32 Yes None M 3930 53.0 39.5 8/9 A&W 28 23 Yes Anorexia, hydronephrosis F 3245 51.0 39.4 8/9 A&W 29 28 Yes None M 3360 49.0 37.1 9/9 Tuberous sclerosis 30 27 Yes None M 3320 52.0 37.1 8/9 A&W 31 22 Yes HIV F 3005 49.0 39.0 8/9 A&W 32 24 No None M 2710 49.5 40.5 8/9 A&W 33 35 Yes None F 2745 46.0 38.5 8/9 Complex CHD, NND 34 35 No Obesity F 3525 52.5 39.4 8/9 A&W 35 25 No GDM M 2985 51.0 37.6 9/9 A&W 36 35 No None M 2765 49.0 38.1 8/9 A&W 37 33 No None F 2970 49.5 39.6 8/9 A&W 38 36 Yes HIV M 3170 49.0 39.0 8/9 A&W 39 37 Yes DM2, hypothyroidism, high BP M 3690 52.0 38.3 8/9 A&W 40 24 No None M 2980 49.0 40.1 8/9 A&W 41 29 No None M 2535 48.0 40.2 8/9 HLHS, chromosomal abnormality, NND 42 26 Yes Pyelonephritis, pyelectasis F 2905 50.0 39.5 8/9 A&W Mean ± SD 30.88 ± 6.09 — — — 3094.88 ± 382.08 49.38 ± 1.99 38.52 ± 0.95 — — A&W, alive and well at discharge; BP, blood pressure; CHD, congenital heart disease; DM1, Type-1 diabetes mellitus; DM2, Type-2 diabetes mellitus; F, female; GA, gestational age; GDM, gestational diabetes mellitus; HIV, human immunodeficiency virus; HLHS, hypoplastic left heart syndrome; M, male; NND, neonatal death; PAS, placenta accreta spectrum disorder. Table 2. Frequency of COVID-19-related symptoms that developed after discharge in 25 SARS-CoV-2-positive pregnant women who were asymptomatic at the time of testing Symptom n (%) Headache 12 (48) Myalgia 8 (32) Odynophagia 6 (24) Chest pain 5 (20) Rhinorrhea 5 (20) Anosmia 5 (20) Arthralgia 5 (20) Dysgeusia 5 (20) Dyspnea 5 (20) Diarrhea 3 (12) Coughing 3 (12) Shivering 3 (12) Fever 2 (8) SARS-CoV-2 analysis of neonatal swabs

All 42 neonates were tested for SARS-CoV-2 by RT-PCR of oral and rectal swab samples at birth and 28 were retested at 24 h after birth. Eighteen (42.9%) neonates were positive at birth, 10 (23.8%) showed viral persistence at 24 h after birth, five (11.9%) had a positive test only at 24 h after birth (negative at birth) and six (14.3%) were negative at both timepoints (Figure 1a and Table S1). Of the 14 neonates that were not tested at 24 h after birth, one was positive at birth. SARS-CoV-2 RNA amplification was observed more frequently in rectal swab samples than in oral swab samples, regardless of the timing of sampling (Figure 1b,c). Only four neonates were positive for SARS-CoV-2 on RT-PCR of both oral and rectal swabs at birth (Figure 1b), of which three remained positive on RT-PCR of both oral and rectal swabs at 24 h after birth (Figure 1c).

Venn diagrams showing neonatal SARS-CoV-2 test results in asymptomatic SARS-CoV-2-positive women. (a) Number of positive neonates at birth and at 24 h after birth. (b,c) Number of positive neonatal oral and rectal swabs at birth (b) and at 24 h after birth (c). *Including one case that did not undergo testing at 24 h.

Vertical transmission according to WHO criteria

In order to identify instances of vertical transmission and classify peripartum transmission according to the classification proposed by Blumberg et al.12 and modified by the WHO13, we analyzed the detection timeline in cases with a positive neonatal SARS-CoV-2 test result. The 10 cases with a persistent positive neonatal SARS-CoV-2 result at 24 h were considered to correspond to possible intrauterine transmission, of which five (5/42; 11.9%) presented strong evidence of vertical transmission as the AF also tested positive for SARS-CoV-2 (Figure 2a and Table S1). Additionally, seven neonates were classified as having transient viremia (unlikely intrauterine transmission) because SARS-CoV-2 RNA amplification was not persistent at 24 h after birth (Figure 2b). The five neonates who were negative at birth but positive 24 h after birth were categorized as having early postnatal transmission (intrapartum transmission) (Figure 2c and Table S1). In the one neonate who was positive at birth but in whom a test was not performed at 24 h after birth, the timing of mother-to-child transmission was classified as indeterminate.