Seroprevalence analysis of SARS-CoV-2 in pregnant women along the first pandemic outbreak and perinatal outcome.

OBJECTIVES: To evaluate the progression of the seroprevalence of SARS-CoV-2 in the pregnant population of the south of Madrid during the first wave of the COVID-19 pandemic. Secondarily we aimed to evaluate maternal and perinatal outcomes. STUDY
DESIGN: Retrospective cohort study conducted at Hospital Universitario 12 de Octubre during weeks 10 to 19 of 2020, coinciding with the Spanish lockdown. We tested 769 serum samples obtained from routine serological testing during the first and third trimesters of pregnancy for specific IgG anti SARS-CoV-2 RBD and S proteins. RT-PCR tests were performed in suspected cases according to clinical practice. We compared maternal and perinatal outcomes in those with delivered pregnancies (n = 578) according to the presence or absence of specific IgG antibodies. Those with positive IgG were subdivided by the presence or absence of Covid-19 related symptoms at any time and the results of RT-PCR testing if performed. Therefore, we had 4 study groups: G1 (IgG negative), G2 (IgG positive, asymptomatic, RT-PCR testing negative or not done), G3 (IgG positive, symptomatic, RT-PCR testing negative or not done), and G4 (IgG positive, symptomatic, RT-PCR positive).
RESULTS: Seropositivity increased from 0% to 21.4% (95% CI 11.8-31.0) during the study period, of which 27.9% had an asymptomatic course. Overall outcomes were favorable with a significant increased rate of preterm birth in G4 vs G1 (21.4% vs 6.7%) and cesarean/operative delivery (50% vs 26.9%). Asymptomatic and mild cases did not have differences regarding pregnancy course when compared to seronegative women. There were no documented cases of vertical or horizontal transmission.
CONCLUSION: Seroprevalence in pregnant women in southern Madrid went up to 21.4% of which 27.9% had an asymptomatic course. Overall perinatal results were favorable, especially in those asymptomatic.

Introduction

The novel human coronavirus SARS-CoV-2 has been responsible for a global pandemic during 2020 with over 27 million confirmed cases and over 891000 deaths in more than 180 countries [1]. Spain has been one of the most severely affected by the first wave of SARS-CoV-2 pandemic with an official report of 218000 cases confirmed by real-time PCR (RT-PCR) on nasopharyngeal swabs (NFS) during the first wave, by May 10th 2020 [2]. However, the national seroepidemiological study, conducted from April 27th to May 11th, suggested that the real number of infections was about 10 times higher [3]. This gap was justified by the lack of detection of asymptomatic cases or even the incomplete ascertainment of those with symptoms during the first wave of the pandemic. This study also showed that, within Spain, Madrid was particularly affected, reaching a seroprevalence of 11.5% [4]. The activity of the infection was not uniform even within cities, with wide differences in the infection rates described in their districts.

The SARS-CoV-2 infection in pregnancy has direct and indirect consequences [5, 6]. Focusing on the direct impact, current knowledge is mainly derived of cases diagnosed by RT-PCR in women with suggestive symptoms or in those asymptomatic that are hospitalized for non-COVID-19 related reasons [7]. This shows that first, maternal complications appear to be similar to non-pregnant women in the same age group, although pregnant women might be at increased risk of admission to an intensive care unit, second, there is an increased risk of preterm birth and cesarean section, and third, vertical transmission is considered rare [8, 9]. However, the full spectrum of SARS-CoV-2 infection remains unknown because mild or asymptomatic infections in non-hospitalized women are underdiagnosed. This could be overcome by studying the seroprevalence in the population of pregnant women, which can yield key data for health care providers in the event of a second wave or a future pandemic.

The main objective of our study is to report the seroprevalence of SARS-CoV-2 specific antibody in stored serum samples from the standard follow up of pregnancies during weeks 10–19 in 2020, coinciding with the onset, peak and decline of the first wave of the pandemic in Madrid. Secondarily we aim to evaluate maternal and perinatal outcomes stratified by the presence of antibodies against SARS-CoV-2, COVID-19 related symptoms and the result of RT-PCR on NFS.

Materials and methods

This study was approved by the Institutional Review Board (Research Ethics Committee of the Hospital Universitario 12 de Octubre) and informed consent was obtained from all participants (N°:20/241).

Study design and population

We present a retrospective cohort study conducted at Hospital Universitario 12 de Octubre, a large teaching hospital in the south of Madrid covering an estimated population of 400.000 inhabitants, being the reference center for 4000 deliveries/year.

Antenatal routine care includes two serological screenings for HIV, syphilis, rubella, hepatitis B, hepatitis C and toxoplasma congenital infection during pregnancy, one in the first trimester (between 8–12 weeks) or at the first visit in pregnancy, and a second one during the third trimester (between 32–35 weeks). These analyses are performed in our clinical microbiology laboratory with the exception of first trimester screening in low-risk pregnancies that is carried out at peripheral units. Our cohort was composed by pregnant women with these routine serological analysis between February 28th to May 10th (weeks 10–19 of 2020), from the beginning to the end of lockdown in Spain.

Serum samples are stored for at least a year at the biobank of the Microbiology Department for any additional testing that may be required in the follow-up of the mother, fetus or newborn. We performed an analysis of stored serum samples obtained during the study period for specific IgG anti SARS-CoV-2 RBD and S proteins according to the protocol described by Amanat et al [10]. SARS-CoV-2 seropositivity was defined as the presence of anti-SARS-CoV-2 IgG antibodies in serum (titer >1:100), and was considered as a marker of SARS-CoV-2 infection. All tested women had complete maternal and perinatal follow-up.

The patients were informed of this seroprevalence study through telephone communication, to minimize the risk of contagion inherent in a hospital visit in a pandemic situation. It was explained to them that the study was carried out in the context of the public health need to understand the seroprevalence in our environment and to verify the efficacy of a new test recommended by the World Health Organization (WHO) in the network of seroprevalence studies Solidarity II. Their verbal consent was obtained for participation in the study and to be informed of the results, all being reflected in the clinical history. In the cases where the result was positive, a hospital visit was also arranged to provide additional information on recommendations and follow-up, and their written informed consent for their participation in the study was also obtained.

SARS-CoV-2 management

Screening

Since the report of the first case of COVID-19 in pregnant woman in Madrid on March 3rd, antenatal visits (both telephone and face-to-face consultations) include a systematic detailed questionnaire regarding most known SARS-CoV-2 infection symptoms, including fever, dry cough, sneezing, dyspnea, myalgia/asthenia, diarrhea, conjunctivitis, anosmia or ageusia. When any of the symptoms is present either during an antenatal visit or when consulting at the emergency room, women are tested by specific SARS-CoV-2 RT-PCR on NFS [7].

From March 31st to April 7th, it was decided to perform RT-PCR testing to all pregnant women with a scheduled cesarean section or labor induction, and since April 8th we also perform a universal screening with NFS on all women admitted to our maternity.

RT-PCR confirmed case

Management of pregnant women with a RT-PCR positive for SARS-CoV-2 includes a complete physical exam, blood work-up, chest X-ray if SpO2 in pulse oximetry is below 96% or tachypnea, and fetal ultrasound. In absence of signs of pneumonia or in mild forms [11], prophylactic low weight heparin treatment for two weeks is initiated, and out-patient management is proposed with telephone follow-up every 48 hours until 14 days after diagnosis. If there are signs of clinical deterioration during follow-up the patient is asked to return to the emergency room for re-evaluation. If the patient’s status does not allow out-patient monitoring she is hospitalized and treatment with hydroxychloroquine and prophylactic heparin is initiated, according to our hospital’s infectious disease protocol.

Delivery for SARS-CoV-2 reasons is only indicated when maternal deterioration requires considering mechanical ventilatory support. The preferable way for delivery is vaginal in absence of contraindications. Regardless of the SARS-CoV-2 status, skin-to-skin is not offered if delivery is via cesarean section given the lack of personnel to help the mothers in the process.

RT-PCR NFS testing is performed on all newborns of mothers with confirmed RT-PCR and in those with symptoms suggestive of SARS-CoV-2 infection.

Serological confirmed case

Clinicians and patients are aware of the results of the antibody study. Cases with a serological confirmation of SARS-CoV-2 infection, either with a history of a positive result of RT-PCR on NFS or asymptomatic with no RT-PCR testing, are considered non-contagious, not recommending isolation and no special measures are taken for their management.

Statistical analysis and data management

The main outcome was to study the seroprevalence of IgG specific antibodies in a cohort of pregnant women during the first wave of the pandemic. For our secondary analysis we stratified the population according to the results of the serological study (IgG positive vs. negative). Those with positive IgG were further subdivided depending on the presence or absence of COVID-19 related symptoms at any time before the serological study, and the results of the RT-PCR if performed. We analyzed 4 study groups: G1 (negative IgG), G2 (positive IgG, asymptomatic, negative or not done RT-PCR testing), G3 (positive IgG, symptomatic, negative or not done RT-PCR testing), and G4 (positive IgG, symptomatic, positive RT-PCR). For this analysis we excluded women who developed symptoms and had a subsequent positive RT-PCR after their serological testing. If the patient had mild symptoms but both RT-PCR and antibody testing were negative she was included in G1. G1 was considered the reference group for comparisons.

We recorded in all cases the following independent variables: (1) Main maternal basal characteristics and risk factors that have been associated to SARS-CoV-2 infection severity, including maternal age, body mass index, self-reported race, prior medical comorbidities and smoking; (2) Triage data: from March 3rd at every antenatal visit and at the emergency room women were asked for suggestive symptoms of SARS-CoV-2 infection. Any suggestive symptoms recorded in the clinical history during the month of February were included as well. At triage of symptomatic women the following variables were recorded: temperature, fever (considered as an axillary temperature ≥ 37.5°C), peripheral oxygen saturation (SpO2), lymphocyte, leucocyte, platelet, transaminases and LDH levels and chest X-ray result (normal/abnormal); (3) Maternal outcomes: need for admission, length of admission, need for oxygen support, type of oxygen support and intensive care unit; (4) Perinatal outcomes in women with completed pregnancies (n = 579): first or second trimester miscarriage, stillbirth (fetal death >22 weeks), neonatal death (death of alive newborn <28 days) preterm birth <37 weeks, small for gestational age (estimated fetal weight <10th centile), intrauterine growth restriction [12], ultrasound morphological anomalies (evaluated at least at the first, second and third trimester), mode of delivery, reason for operative or cesarean delivery, neonatal acidosis defined as an arterial umbilical cord pH<7.1, Apgar at 5 min, neonatal intensive care unit admission, neonatal complications, evidence of vertical transmission, skin-to-skin and breastfeeding. All independent variables were analyzed between subjects.

Outcome data were recorded in a database created on the Research Electronic Data Capture (REDCap) tool [22] hosted at the “imas12” research institute. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement was followed for reporting the results. Continuous variables were expressed in mean (SD) or median (interquartile range) when non-normally distributed as assessed by Shapiro-Wilks´ test. Categorical variables were expressed in percentage (%). Univariate comparisons were performed between all groups and G1 using the t-test or Mann–Whitney U-test for continuous variables and the chi-square or Fisher’s exact test for categorical variables. Two-sided P<0.05 was considered statistically significant. Data were carefully entered and analyzed after data cleansing, using statistical package STATA 14.2 (TX, USA: StataCorp LP).

The minimal anonymized data set necessary to replicate the study findings is included as a S1 Database.

Results

During the study period, 769 women were tested for SARS-CoV-2 specific antibodies. There were 97, 51 and 621 serological tests performed on serum samples from the stored first, second and third trimester, respectively. A total of 86/769 (11.2%) samples resulted positive for SARS-CoV-2 RBD-specific IgG. The first positive sample was obtained at week 11 and the proportion of positive samples increased over time to reach 23.3% (CI 95% 13.6–33.0) and 21.4% (95% CI 11.8–31.0) by weeks 18 and 19 respectively, as shown in Fig 1.

Fig 1

SARS-CoV-2 seroprevalence during weeks 10–19 of 2020.

There were 92/769 (11.9%) women who referred COVID-19 related symptoms at some time from February, of whom 36/92 (39.1%) were tested by RT-PCR and 31/36 (86.1%) were positive. All cases confirmed by RT-PCR had positive antibodies except for 10 cases in which the serological test had been performed prior to the onset of symptoms. These 10 cases were excluded for further analysis.

G1 was composed by 673/759 (88.7%) seronegative women. Of note, 23/673 (3.1%) referred at least one COVID-19 related symptom. G2 comprised 24/759 (3.1%) asymptomatic seropositive women. G3 was constituted by 40/759 (5.3%) symptomatic seropositive women in which confirmative RT-PCR was not performed since they never attended the emergency department for their NFS RT-PCR testing (n = 36), or the RT-PCR was negative (n = 6). Finally, G4 consisted of 22/759 (2.9%) symptomatic seropositive women with RT-PCR confirmation. The distribution of the different study groups is described in Fig 2.

Fig 2

Population distribution according to their microbiological status and symptoms.

*10 cases with positive PCR and symptoms after serological evaluation were excluded.

The main basal characteristics of the study population according to the serological status are depicted in Table 1.

Table 1

Maternal baseline characteristics.

	G1	G2	G3	G4	p
	n = 673	n = 24	n = 40	n = 22
Maternal age in years	32 (11)	31.5 (8)	30 (10)	29.5 (11)	0.88
Height in cm	162 (10)	160 (14)	163 (10)	159 (10)	0.26
Weight in kg	62 (16)	64 (11)	69 (14)*	70 (12)*	0.11
Body mass index in kg/m2	23 (5)	25 (5)	24.5 (7)	26 (6.5)	<0.01
Nulliparous	308 (45.8)	10 (41.7)	15 (37.5)	10 (45.5)	0.76
Race
•Non-hispanic white	353 (52.5)	11 (45.8)	19 (46.3)	10 (45.5)
•Black	13 (2.0)	0 (0)	3 (7.3)	0 (0)
•Hispanic	233 (34.6)	12 (50.0)	16 (40.0)	12 (54.6)	0.30
•North African	41 (6.1)	1 (4.2)	0 (0)	0 (0)
•Asian	31 (4.6)	0 (0)	2 (4.9)	0 (0)
•Other / unknown	2 (0.3)	0 (0)	0 (0)	0 (0)
Active smoking	39 (5.8)	2 (8.3)	3 (7.7)	0 (0)	0.32
Chronic medical pathology
•None	554 (82.3)	17 (70.8)	34 (85.0)	17 (77.3)	0.44
•Cardiovascular / cerebrovascular	14 (2.1)	1 (4.2)	0 (0)	2 (9.1)	0.11
•Respiratory system	15 (2.2)	1 (4.2)	2 (4.9)	2 (9.1)	0.17
•Digestive system	10 (1.5)	0 (0)	1 (2.4)	0 (0)	0.81
•Endocrine system	31 (4.6)	2 (8.3)	4 (9.8)	0 (0)	0.27
•Malignant tumor	1 (0.2)	0 (0)	0 (0)	0 (0)	0.99
•Nervous system	12 (1.8)	0 (0)	0 (0)	0 (0)	0.67
•Others	79 (11.7)	4 (16.7)	4 (9.8)	2 (9.1)	0.83
Symptoms
•Asymptomatic	652 (96.9)	24 (100)	0*	0*	<0.01
•Fever	12 (0.2)	0 (0)	18 (45.0)*	12 (54.6)*	<0.01
•Dry cough	13 (0.2)	0 (0)	20 (48.8)*	15 (68.2)*	<0.01
•Sneezing	5 (0.01)	0 (0)	6 (14.6)	14 (63.6)*	<0.01
•Myalgia/asthenia	8 (0.01)	0 (0)	19 (48.7)*	15 (68.2)*	<0.01
•Anosmia/ageusia	1 (0.0)	0 (0)	18 (30)*	9 (40.9)*	<0.01
•Conjunctivitis	1 (0.0)	0 (0)	2 (5.4)*	1 (4.6)*	<0.01
•Diarrhea	1 (0.0)	0 (0)	7 (17.5)*	5 (22.7)*	<0.01

Data presented as median (interquartile range) and n (%)

*Statistically significant differences when compared to G1

†Bonferroni correction was applied

G1: IgG negative; G2: IgG positive, asymptomatic, RT-PCR testing negative or not done; G3: IgG positive, symptomatic, RT-PCR testing negative or not done; G4: IgG positive, symptomatic, RT-PCR positive.

As shown, there were not statistically significant differences among them except for a lower height and a higher BMI in G4 when compared to G1. As expected by the definition of the groups there were also differences regarding the presence of COVID-19 related symptoms which were higher in G3 and G4. The most frequent symptoms in G3 and G4 were dry cough (48.8% and 68.2%, respectively) followed by myalgia (48.8% and 68.2%) and fever (45.0% and 54.6%).

Among the 86 seropositive women, 24 (27.9%) were asymptomatic, while 13 (15.1%) required admission, all of them for respiratory symptoms in the context of pneumonia with a pathological chest X-ray. Median hospital stay was of 5 days (range 1–11) and none of them required intensive care unit admission.

There was a total of 578 women with complete perinatal outcomes, depicted in Table 2. Median gestational age at delivery was 39.7 weeks (range 27.0–41.7). There were two cases of perinatal mortality in the seronegative group. The overall results showed non-significant differences between G2 or G3 and the basal group (G1). However, we found an increased risk of preterm birth among G4 when compared to G1 (21.3 vs 6.7, p = 0.03). The three cases of preterm birth in G4 were not directly related to a COVID-19 complication: one case was a monochorionic monoamniotic pregnancy in which a caesarean section was scheduled at 32 weeks and the other two were cases of preterm rupture of membranes in which labour induction was scheduled at 34 weeks according to local guidelines. We also observed a higher rate of operative delivery (either cesarean or operative vaginal) in G4 vs G1 (50% vs 26.9%, p = 0.01) with non-significant differences in the operative delivery rate for fetal distress (42.9% vs 24.8%, p = 0.28). There were no reported cases of suspected vertical nor horizontal transmission.

Table 2

Perinatal outcomes stratified by serological results and symptoms.

	G1	G2	G3	G4	p
	n = 523	n = 16	n = 24	n = 14
First trimester miscarriage	6 (1.2)	0 (0)	0 (0)	0 (0)	0.89
Second trimester miscarriagea	2 (0.4)	0 (0)	0 (0)	0 (0)	0.97
Stillbirthb	2 (0.4)	0 (0)	0 (0)	0 (0)	0.98
Pregnancy medical complicationsb
•None	457 (89.0)	15 (93.8)	23 (95.8)	10 (71.4)	0.12
•Hypertensive disorder	11 (2.1)	1 (6.3)	0 (0)	1 (7.1)	0.16
•Gestational diabetes	31 (6.1)	0 (0)	1 (4.2)	1 (7.1)	0.74
•Other	22 (4.3)	1 (5.9)	0 (0)	2 (14.3)	0.21
Small for gestational ageb	14 (2.7)	0 (0)	1 (4.2)	1 (7.1)	0.66
Intrauterine growth restrictionb	17 (3.3)	0 (0)	0 (0)	0 (0)	0.59
Fetal anomalies at standard ultrasounda	20 (3.9)	0 (0)	0 (0)	1 (7.1)	0.54
Gestational age at delivery in weeksc	39.9 (1.9)	39.6 (2.1)	39.5 (2.7)	38.5 (3.3)	0.95
Preterm birthc	34 (6.7)	1 (5.9)	0 (0)	3 (21.4)*	0.16
Mode of deliveryc
•Eutocic	376 (73.3)	10 (62.5)	18 (75.0)	7 (50.0)
•Operative	28 (5.5)	2 (11.8)	1 (4.2)	3 (21.4)	0.69
•Cesarean section	109 (21.2)	4 (23.5)	5 (20.8)	4 (28.6)
Cesarean or operative delivery for fetal distressc	34 (24.8)	1 (16.7)	3 (50.0)	3 (42.9)	0.29
Birthweight in gc	3250 (620)	3200 (560)	3430 (650)	3270 (810)	0.36
Apgar score at 5 min <7c	4 (0.9)	0 (0)	1 (2.7)	0 (0)	0.68
Arterial pH <7.1c	14 (2.7)	0 (0)	0 (0)	1 (7.1)	0.63
Neonatal intensive care unit admissionc	28 (5.5)	0 (0)	0 (0)	1 (7.1)	0.48
Neonatal SARS-CoV-2 infectionc	0 (0)	0 (0)	0 (0)	0 (0)	NA
Skin to skinc	402 (79.3)	12 (75.0)	22 (91.7)	10 (71.4)	0.40
Breastfeedingc	494 (97.2)	16 (100)	24 (100)	13 (92.9)	0.53

NA, not applicable

Data presented as median (interquartile range) and n (%)

*Statistically significant differences when compared to G1

a Calculated over completed pregnancies excluding 1st trimester miscarriages (n = 567)

b Calculated over completed pregnancies excluding 1st and 2nd trimester miscarriages (n = 565)

c Calculated over completed pregnancies with live newborns (n = 563)

G1: IgG negative; G2: IgG positive, asymptomatic, RT-PCR testing negative or not done; G3: IgG positive, symptomatic, RT-PCR testing negative or not done; G4: IgG positive, symptomatic, RT-PCR positive.

Discussion

Main findings of the study

Our study highlights the impact of the first wave of the SARS-CoV-2 pandemic on the pregnant women population in Madrid, during weeks 10–19 of 2020. The seroprevalence analysis performed in archived serum samples reflects the epidemic trend reaching over 21.3% seropositivity by week 19. The overall perinatal results of seropositive women were favorable and up to 27.9% of infected women had an asymptomatic course. We found no cases of vertical transmission in our cohort.

Interpretation of results and comparison with existing literature

The impact of SARS-CoV-2 in pregnancy has been mainly assessed from the perspective of the cases detected by RT-PCR. As far as we know, seroprevalence of COVID-19 in pregnant woman has been only reported once, in a multicenter study carried out in Barcelona in the last phase of the first wave (April 14 to May 5, 2020) and in which 14.0% were positive for anti-SARS-CoV-2 [13]. The seropositive rate for Madrid and Barcelona obtained through random sample participants by national health authorities in Spain has been of 11.5% and 6.8%, respectively [4]. Therefore, the percentages of infection obtained in pregnant women are higher in both studies. Nevertheless it is possible that the distribution of cases even within different city areas could be significantly different as has been documented for New York City [14] and the finding of a higher infection rate of pregnant women could be just a reflection of the infection rate in the area of the city that has been studied. Additionally, our study provides information about the evolution of seropositivity, showing that there is a delay of 2–3 weeks in its increment with respect to that observed in cases diagnosed by RT-PCR, until it becomes stable from week 18 [15]. This is in accordance with the expected time to develop IgG antibodies after detection of SARS-CoV-2 by RT-PCR.

Interestingly 28% of infected women were asymptomatic in our study, and up to 60% in the study from Barcelona. This could be explained, at least partially, by our lower proportion of samples from the first trimester (43% vs 13%), since they found a significantly lower prevalence of symptomatic infection and hospital admissions in women in their first trimester than those in their third trimester. Pregnant women are usually healthy patients that frequently visit health care centers and therefore should be considered as a possible source of infection transmission to more vulnerable cohorts that also attend these areas. This asymptomatic aspect of SARS-CoV-19 infection in pregnant women has been described in other series in US [16, 17] and Europe [18, 19] where universal screening upon hospital admission has been proposed for better resource allocation and protection of the woman’s contacts including other patients or health-care professionals.

Our data show good overall maternal and perinatal outcomes in seropositive women, regardless of their symptoms. We stratified seropositive women in three groups according to their symptoms and RT-PCR confirmation. Pregnant women with symptomatic COVID-19 had higher BMI than non-infected women as has been described in non-obstetric population [20], and reflecting the importance of prevention in the specific subgroup of the obese gravida. It is of interest the increased preterm birth rate when comparing the most symptomatic cases with RT-PCR confirmation (G4) with the reference population (G1) as well a higher rate of operative delivery. Our small sample size is limited to draw conclusions about the implication of SARS-CoV-2 infection in these findings, although these two outcomes had already been described in other cohorts of pregnant women with RT-PCR confirmed SARS-CoV-2 infection [7]. Almost 2/3 of seropositive women had symptoms suggestive of SARS-CoV-2 infection but did not undergo RT-PCR testing, mainly for two reasons. Firstly, these were milder cases where the patients did not want to attend the emergency room for testing as they consider this as a non-safe place at that time. Secondly, most of the times when they referred these symptoms during their antenatal visit, they had already resolved and therefore it was considered unnecessary to test them given the poorer performance of RT-PCR if performed later on the disease [21]. Their maternal results were excellent since none of them required hospital admission for support and we found no significant differences regarding perinatal outcomes. Although we only have a small sample of asymptomatic seropositive women, their maternal and perinatal results overlap with those of uninfected pregnant women.

We had no documented cases of vertical nor horizontal transmission, accordingly to recently published studies [8, 22], which reinforces our policy of promoting skin-to-skin and breastfeeding, given their known benefits for both mother and child.

Strengths and limitations

We acknowledge some limitations in our study. The results are drawn from a cohort of women from the south of Madrid, and cannot be extrapolated to other communities. Moreover, we could not analyze those pregnancies delivered before reaching the third trimester serology during the study period. RT-PCR on NFS was not systematically performed due to the abovementioned patients’ concerns as well as the progressive implementation of this test during the pandemic [17]. Finally, this study was not designed to evaluate pregnancy outcomes and it may lack power to evaluate some events, especially in the most underrepresented group which is seropositive asymptomatic women.

However, there are also several strengths. This seroprevalence study provides additional information in an issue that has been scarcely explored. Moreover, it covers the whole first wave of the pandemic showing nicely the evolution of the seroprevalence in our population. Finally, it has allowed us to evaluate the rate of asymptomatic pregnant women affected by SARS-CoV-2 and to obtain a first glimpse into its consequences in pregnancy.

Clinical perspective

Our data can help to better understand the evolution of the pandemic both in Madrid and worldwide, with the finding that up to 28% of infected patients had no symptoms at all. Assessing the cumulative prevalence of SARS-CoV-2 infection can help trace contacts, identify hosts with viral reservoir, estimate viral propagation in certain communities, know the rate of asymptomatic infections and obtain better estimates on both morbidity and mortality.

These results can also help obstetricians when counseling pregnant women. Although data are still limited, we can reassure them of the good maternal and perinatal results, especially in those with an asymptomatic course. We also found no evidence of impaired fetal growth, fetal anomalies related to the infection nor any signs of vertical nor horizontal transmission, which have been some of the major concerns in pregnancy care.

From an epidemiological-public health point of view, future research should focus in the follow-up of antibody levels and their efficacy in any upcoming waves. This information will be key for health-care system planning in any future events.

From a perinatal view, further investigation should focus first, in the impact of the SARS-CoV-2 infection in pregnancy at any trimester and secondly, in the follow-up of infected mothers and their newborns given the lack of knowledge on the infant’s development or long-term outcomes for the mothers.

Conclusions

A high prevalence of COVID-19 has been detected in pregnant women in Madrid, Spain, during the outbreak of the first epidemic wave. The asymptomatic profile of 27.9% of cases warrants specific protocols for diagnosis and could have important clinical and epidemiological implications. Maternal and perinatal outcomes seem favorable, especially in those asymptomatic or with milder presentation. Nevertheless SARS-CoV-2 infection warrants specific follow up for both mothers and newborns.

(CSV)

Click here for additional data file.

27 Oct 2020

PONE-D-20-28445

Seroprevalence analysis of SARS-CoV-2 in pregnant women along the pandemic outbreak

PLOS ONE

Dear Dr. Herraiz,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 11 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Rogelio Cruz-Martinez, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please amend your current ethics statement to address the following concerns:

a) Did participants provide their written or verbal informed consent to participate in this study?

b) If consent was verbal, please explain i) why written consent was not obtained, ii) how you documented participant consent, and iii) whether the ethics committees/IRB approved this consent procedure.

3. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. Please update your Methods and Results sections accordingly.

4. Thank you for stating the following in the Funding Section of your manuscript:

"This work was supported by grants to RD by the Instituto de Investigación Carlos III (grants FIS

PI 1801007, by the European Union Commission Horizon 2020 Framework Programme: Project

VIRUSCAN FETPROACT-2016: 731868. and by Fundación Caixa-Health Research (Project

StopEbola). This work was supported by grant to MDF by the Instituto de Investigación Carlos III

(FIS PI1800740). The funders had no role in study design, data collection and analysis, decision

to publish, or preparation of the manuscript."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

5. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Overall, the results of this study are very timely and relevant. There are in my opinion a few of issues that need to be clarified/addressed.

Is the highest number of preterm and cesarean births due to SARS-CoV2 positive RT-PCR? maternal respiratory impairment, obstetric or fetal cause? Can you discuss this point?

Reviewer #2: The manuscript by Ignacio Herraiz et al. addressed the issue on Seroprevalence analysis of SARS-CoV-2 in pregnant women along the pandemic outbreak

The article is interesting: it is well written, the methodology used is consistent and results are important. They provide information in the the seroprevalence of SARS-CoV-2 in the pregnant population of the south of Madrid during the outbreak and in the evaluation of maternal

and perinatal outcomes.

There is currently uncertainty regarding the impact of the infection caused by the severe acute respiratory distress syndrome coronavirus 2 (SARS‐CoV‐2) in pregnancy .

I consider that the information provided by the authors is useful and should be known by the scientific community

TITLE:

The title is clear and appropriate. "Seroprevalence analysis of SARS-CoV-2 in pregnant women along the 1 pandemic outbreak" However, it can be clarified more in the following way "Seroprevalence analysis of SARS-CoV-2 in pregnant women along the 1 pandemic outbreak and perinatal outcome."

The abstract appropriately summarizes the manuscript without omitting important results.

INTRODUCTION

The topic is of clinical importance. Most of the previous work is adequately discussed and referenced. The rationale provided by the authors is sufficient and the purpose of the study is clearly defined.

However line 54 " Regarding the impact of SARS-CoV-2 infection in pregnancy " It is important to clarify that the impact can be direct or indirect (psychological stress and problems with public health programs)

please add these two references

Ref - Miguel Parra-Saavedra et al, Attitudes and collateral psychological effects of COVID‐19 in pregnant women in Colombia ,International Journal of Gynecology and Obstetrics 2020

Ref - Roberton T, Carter ED, Chou VB, et al. Early estimates of the indirect

effects of the coronavirus pandemic on maternal and child

mortality in low‐ and middle‐income countries. Lancet Glob Health.

2020;8:e901–e908.

METHODS

The methodology is appropriate and sufficient for the stated purpose.

Reviewer #3: Dr Herraiz et al made a good study of SARS-COV-2 seroprevalence, their results are in line with previous reports about the subject.

The only comment about this manuscript is: how would you explain the relationship between higher BMI and seropositivity and symptomatology (groups G1 vs G4)? This is described also in non-obstetric population, and highlighting this finding would add interesting information about SARS-COV-2 and pregnancy.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

8 Nov 2020

Journal requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We have adjusted our manuscript to PLOS ONE´s style requirements. We have followed the instructions for file naming.

2. Please amend your current ethics statement to address the following concerns:

a) Did participants provide their written or verbal informed consent to participate in this study?

b) If consent was verbal, please explain i) why written consent was not obtained, ii) how you documented participant consent, and iii) whether the ethics committees/IRB approved this consent procedure.

We have now amended the ethics statement to address these concerns:

- We have included the following statement a at the beginning of the Methods section as requested by the editors:

“This study was approved by the Institutional Review Board (Research Ethics Committee of the Hospital Universitario 12 de Octubre) and informed consent was obtained from all participants (Nº:20/241).”

- We have included the following additional information in the Methods section, line 91:

“The patients were informed of this seroprevalence study through telephone communication, to minimize the risk of contagion inherent in a hospital visit in a pandemic situation. It was explained to them that the study was carried out in the context of the public health need to understand the seroprevalence in our environment and to verify the efficacy of a new test recommended by the World Health Organization (WHO) in the network of seroprevalence studies Solidarity II. Their verbal consent was obtained for participation in the study and to be informed of the results, all being reflected in the clinical history. In the cases where the result was positive, a hospital visit was also arranged to provide additional information on recommendations and follow-up, and their written informed consent for their participation in the study was also obtained.”

3. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. Please update your Methods and Results sections accordingly.

We have now updated our Methods and Results sections according to the PLOS ONE´s requirements.

4. Thank you for stating the following in the Funding Section of your manuscript:

"This work was supported by grants to RD by the Instituto de Investigación Carlos III (grants FIS PI 1801007, by the European Union Commission Horizon 2020 Framework Programme: Project VIRUSCAN FETPROACT-2016: 731868. and by Fundación Caixa-Health Research (Project StopEbola). This work was supported by grant to MDF by the Instituto de Investigación Carlos III (FIS PI1800740). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Yes, thank you for your support. We have now removed the funding-related text from the manuscript and included such statement in the cover letter.

5. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

Thank you again for your support. We have now uploaded the minimal anonymized data set necessary to replicate our study findings as supporting information.

Reviewer #1: Overall, the results of this study are very timely and relevant. There are in my opinion a few of issues that need to be clarified/addressed.

Is the highest number of preterm and cesarean births due to SARS-CoV2 positive RT-PCR? maternal respiratory impairment, obstetric or fetal cause? Can you discuss this point?

Yes, indeed. As stated in the discussion, several studies on pregnant women suffering from COVID-19 with positive RT-PCR have described an excess in the number of caesarean sections and preterm births. Unfortunately, our limited sample size of pregnant women with symptoms and positive RT-PCR (G4, n=14) prevent us to provide further evidence in this regard. Although we have certainly observed a higher rate of prematurity in G4, we had only 3 cases. We have examined our three cases of preterm birth in G4: one case was a monochorionic monoamniotic pregnancy in which a caesarean section was scheduled at 32 weeks and the other two were cases of preterm rupture of membranes in which labour induction was scheduled at 34 weeks according to local guidelines. In the case of caesarean sections, our results were not statistically significant, probably due to the small sample size.

We have now included this detailed information in the results section (line 225).

Therefore, we are unable to draw firm conclusions, as we have now discussed. Accordingly, we have replaced the following sentence in the discussion section (line 281):

“These two outcomes had already been described in other cohorts of pregnant women with RT-PCR confirmed SARS-CoV-2 infection.”

By:

“Our small sample size is limited to draw conclusions about the implication of SARS-CoV-2 infection in these findings, although these two outcomes had already been described in other cohorts of pregnant women with RT-PCR confirmed SARS-CoV-2 infection [7].”

Reviewer #2: The manuscript by Ignacio Herraiz et al. addressed the issue on Seroprevalence analysis of SARS-CoV-2 in pregnant women along the pandemic outbreak

The article is interesting: it is well written, the methodology used is consistent and results are important. They provide information in the the seroprevalence of SARS-CoV-2 in the pregnant population of the south of Madrid during the outbreak and in the evaluation of maternal and perinatal outcomes.

There is currently uncertainty regarding the impact of the infection caused by the severe acute respiratory distress syndrome coronavirus 2 (SARS‐CoV‐2) in pregnancy.

I consider that the information provided by the authors is useful and should be known by the scientific community.

TITLE: The title is clear and appropriate. "Seroprevalence analysis of SARS-CoV-2 in pregnant women along the 1 pandemic outbreak" However, it can be clarified more in the following way "Seroprevalence analysis of SARS-CoV-2 in pregnant women along the 1 pandemic outbreak and perinatal outcome."

Thank you for your suggestion. We have changed the title accordingly.

The abstract appropriately summarizes the manuscript without omitting important results.

INTRODUCTION: The topic is of clinical importance. Most of the previous work is adequately discussed and referenced. The rationale provided by the authors is sufficient and the purpose of the study is clearly defined.

However line 54 " Regarding the impact of SARS-CoV-2 infection in pregnancy " It is important to clarify that the impact can be direct or indirect (psychological stress and problems with public health programs) please add these two references

Ref - Miguel Parra-Saavedra et al, Attitudes and collateral psychological effects of COVID‐19 in pregnant women in Colombia ,International Journal of Gynecology and Obstetrics 2020

Ref - Roberton T, Carter ED, Chou VB, et al. Early estimates of the indirect

effects of the coronavirus pandemic on maternal and child

mortality in low‐ and middle‐income countries. Lancet Glob Health.2020; 8: e901–e908.

Yes, indeed. We thank the reviewer for this important remark. We have incorporated the two references reflecting the indirect impact of COVID-19 in pregnant women. We have also replaced the sentence (Introduction, line 54):

“Regarding the impact of SARS-CoV-2 infection in pregnancy, the current knowledge is…”

By:

“The SARS-CoV-2 infection in pregnancy has direct and indirect consequences (two new references inserted here). Focusing on the direct impact, current knowledge is…”

METHODS. The methodology is appropriate and sufficient for the stated purpose.

Reviewer #3: Dr Herraiz et al made a good study of SARS-COV-2 seroprevalence, their results are in line with previous reports about the subject.

The only comment about this manuscript is: how would you explain the relationship between higher BMI and seropositivity and symptomatology (groups G1 vs G4)? This is described also in non-obstetric population, and highlighting this finding would add interesting information about SARS-COV-2 and pregnancy.

Yes, thank you for arising this interesting point. We have highlighted this finding in the discussion section adding the following sentence (line 282):

“Pregnant women with symptomatic COVID-19 had higher BMI than non-infected women as has been described in non-obstetric population (new reference: Azzolino D, Cesari M. Obesity and COVID-19. Front Endocrinol (Lausanne). 2020;11:581356.), and reflecting the importance of prevention in the specific subgroup of the obese gravida”

Submitted filename: Response to reviewers_PONE-D-20-28445.docx

Click here for additional data file.

16 Nov 2020

Seroprevalence analysis of SARS-CoV-2 in pregnant women along the first pandemic outbreak and perinatal outcome

PONE-D-20-28445R1

Dear Dr. Herraiz,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Rogelio Cruz-Martinez, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors should be congratulated for the effort to publish this very-well designed study during this Sars-Cov2 pandemia

18 Nov 2020

PONE-D-20-28445R1

Seroprevalence analysis of SARS-CoV-2 in pregnant women along the first pandemic outbreak and perinatal outcome

Dear Dr. Herraiz:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr Rogelio Cruz-Martinez

Academic Editor

PLOS ONE