COVID-19 and its relation to pregnancy and neonates: a systematic review

Objectives: this study systematically reviewed the literature in order to better understand the association among COVID-19, pregnancy and neonates. Methods: MEDLINE, EMBASE, Web of Science, BVS and SCOPUS were assessed, considering the terms: (covid 19 OR covid-19 OR novel coronavirus OR 2019 novel coronavirus OR 2019-nCoV OR sarscov 2 OR sars-cov-2 OR sarscov2 OR sars cov-2) AND (preg-nancy OR pregnant OR pregnant women OR gestation OR gestational) AND (infant OR fetal OR neonatal). Thirty full-text were included (408 pregnant women, 11 non-pregnant women and 279 neonates). Results: fever (45.83%) and cough (31.61%) were the main symptoms of COVID-19 during the pregnancy. Low levels of lymphocytes (32.10%), elevated levels of C-reactive protein (32.35%); leukocytosis (29.41%); neutrophil (5.88%); and radiographic alterations on chest CT, x-ray or ultrasound (45.84%) were the main laboratorial findings. Cesarean delivery and preterm were registered in 239 and 49 cases, respectively. Ten neonates tested positive for SARS-CoV-2. Conclusion: when COVID-19 pneumonia affects women during pregnancy, the symptoms are similar to those experienced by non-pregnant women. In addition, there is still no plausible evidence suggesting vertical transmission of SARS-CoV-2 virus from mother to child.


Introduction
Currently known as COVID-19, SARS-CoV-2 virus (Severe Acute Respiratory Syndrome-related coronavirus 2) is a public health emergency of international concern. It has been suggested that previous complications may be risk factors for adverse outcomes related to COVID-19 such as cardiovascular disease, diabetes, hypertension, and chronic obstructive pulmonary disease. [1][2][3] Regarding symptomatology, similarities of clinical features between 2019-nCoV and previous beta coronavirus infections has been found. Most patients present fever, dry cough, dyspnea, and bilateral patchy pattern and/or ground-glass opacities on chest computerized tomography (CT) scans. 1 Nonetheless, the report from Huang et al. 1 mainly focused on nonpregnant women. Contrasting data are found on maternal and neonatal outcomes of pregnant women with COVID-19 pneumonia.
Prior December 2019, others six coronaviruses were known to infect people: 229E, OC43, NL63, HKU1, SARS-CoV, and MERS-CoV (Middle East Respiratory Syndrome coronavirus). Among these, 229E, OC43, NL63, and HKU1 can be vertically transmitted from mothers to neonates through the placenta, 4,5 whilst there is no evidence of vertical transmission of SARS-CoV and MERS-CoV. 5,6 Nevertheless, an epidemiological research presented that 2019-nCoV was more contagious than SARS-CoV, although, its nucleotide sequences were very similar (82% homologous with SARS-CoV). 7 Moreover, it has been stated that COVID-19 infections in pregnant women generally result in mild or moderate symptoms, and many infected pregnant women do not present any symptoms, 8,9 which may also lead to a high risk of neonatal infection during labor. However, the clinical characteristics and potential vertical transmission of COVID-19 pneumonia in pregnant women is still new and has not been entirely clarified. 8,10 Therefore, it is important to report the most frequent symptoms of this disease in pregnant women and neonates, as well as to understand the risk of infection that labor imposes to the neonates' health. Based on this rationale, this study aimed to systematically review the available literature in order to better understand the association among COVID-19, pregnancy and neonates.

Methods
This systematic review was conducted without patient involvement. Patients were not involved in any way during the review of the available literature, nor during the writing or editing of this document for readability or accuracy.
The search strategy followed the indications of the National Health Service Center for Reviews and Dissemination and Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Also, this systematic review was registered in International Prospective Register of Systematic Reviews (PROSPERO) with protocol CRD42020177354 (https://www.crd.york.ac.uk/ prospero).
A systematic revision of relevant studies published in the literature was conducted on May 22 nd , 2020, considering the period between December 1 st , 2019 to May 22 nd , 2020. The following database were explored: MEDLINE database (Entrez PubMed, www.ncbi.nim.nih.gov), EMBASE, Web of Science, BVS/LILACS and SCOPUS, considering the main MeSH (Medical Subject Headings) terms: (covid 19 OR covid-19 OR novel coronavirus OR 2019 novel coronavirus OR 2019-nCoV OR sarscov 2 OR sars-cov-2 OR sarscov2 OR sars cov-2) AND (pregnancy OR pregnant OR pregnant women OR gestation OR gestational) AND (infant OR fetal OR neonatal). To identify relevant article, titles and abstracts of retrieved papers were exported to Endnote Web where duplicates were identified and removed by two reviewers (GAFJ and VM). It is important to highlight that, in order to identify missing studies, we also hand searched the reference lists of the studies included.
As eligibility criteria, we considered case reports, case series and clinical researches in English, Portuguese and Spanish published in peerreviewed journals, that have addressed epidemiological, clinical features of COVID-19 and its association with pregnancy and neonates. Duplicate publications and articles that did not correspond to the objectives of this review were excluded. Similarly, publications without a research or case report, such as: review articles; descriptive studies; opinion article; correspondence; editorials; letters; and with only children's cases were also excluded. Figure 1 shows the steps of the literature search.
Two highly experienced researchers (GAFJ and VM) were previously trained regarding to criteria that were defined in this study and after they were responsible for searching the literature, applying inclusion and exclusion criteria, selecting the studies and independently extracting the data. There was a good concordance coefficient between reviewers (Kappa index=0.91). Discordances between the authors were discussed and agreed upon with a third pregnancy  After, nine sources were manually inserted by analyzing the references list of previous studies. Fifty-five sources remained on screening step after duplicates exclusion.
Following screening of titles and abstracts, 216 sources were excluded due to the following reasons: reviews; author reply/ comment/ correspondence/ expert comment/ guideline/ letter to editor/ in websites; not correspond to the objectives of this review; not peer-reviewed; language. Besides that, one full-text study was not found.

Results
A total of 408 pregnant women was considered. The mean age of all pregnant women in the 30 studies included in this review was 32.46 years old. Also, among the studies included in this review, 11 nonpregnant women and 279 neonates were considered.
The most frequent laboratorial features were elevated levels of C-reactive protein (32.35%); low levels of lymphocytes (32.10%); leukocytosis (29.41%); neutrophil levels above the normal range (5.88%); radiographic alterations on chest CT or xray (45.34%), because of viral pneumonia that is characterized by decreased diffuse and unilateral or bilateral ground-glass opacities, patchy lung consolidation and blurred borders ( Table 2).
The main findings of this study point out that, of the 279 neonates, only ten tested positive for SARS-CoV-2, indicating a low risk of vertical transmission of the disease (Table 3). Delivery outcomes, characteristics of infants at birth and evidence of vertical transmission are also displayed in Table 3.

Discussion
Due to their immunosuppressed state and physiological and adaptive changes, pregnant women are more susceptible to pneumonia and other respiratory pathogens. Nonetheless, our results highlighted that fever and cough were the main clinical features of COVID-19 during pregnancy, but asymptomatic cases are common. Several studies reported false negative diagnosis by RT-PCR, whilst chest CT and x-rays analyses seem to be a fast and highly reliable diagnostic method. Also, vertical transmission from mother to neonates seems unlikely, yet, the available data addressing this topic is still scarce. Overall, data are limited in relation to the association of the outcomes, since population-based clinical studies are still scarce and most studies have low strength of scientific evidence.
Chen et al. 10 reported that the main symptoms of SARS-CoV-2 infection in pregnant women are fever and cough, with lower frequency for myalgia, malaise, sore throat, shortness of breath and diarrhea. Laboratorial findings also report that lymphopenia and increased ALT and AST levels are frequent. These findings are in accordance with the findings of this systematic review, where fever (45.83%) and cough (31.61%) were the most frequent symptoms among the 408 pregnant women analyzed in this review, followed by myalgia (15.19%), dyspnea (11.76%), sore throat (3.92%) and diarrhea (1.96%). 5, Laboratorial tests reported decreased levels of lymphocytes (32.10%) and increased levels of C-reactive protein (32.35%) as frequent conditions, followed by leukocytosis (29.41%) and increased neutrophil ratio (5.88%).
Medonza et al. 37 reported that preeclampsia-like syndrome is one symptom of COVID-19, but its cause is different from obstetric preeclampsia and therefore not connected with placental failure. In addition, preeclampsia-like syndrome can resolve spontaneously following recovery from severe pneumonia and may not be an obstetric indication for delivery.
Other main findings seen in the reviewed studies was alterations on chest CT. 5,9,12,13,15,20,21,[23][24][25]30,31,[33][34][35] More than 45% of patients included in this review presented alterations upon chest CT, xray or ultrasound. These findings, along with the study of Liu et al. 27 suggest than CT images seem to  continue Table 1 Description of pregnant women's clinical characteristics in the selected studies.
Author ( Table 1 Description of pregnant women's clinical characteristics in the selected studies.
Author ( Table 2 Description of pregnant women's laboratorial data and radiological characteristics in selected studies.
Author ( Table 2 Description of pregnant women's laboratorial data and radiological characteristics in selected studies.
Author ( Table 2 Description of pregnant women's laboratorial data and radiological characteristics in selected studies.
Author ( Table 2 Description of pregnant women's laboratorial data and radiological characteristics in selected studies.
Author ( Table 2 Description of pregnant women's laboratorial data and radiological characteristics in selected studies.
Author ( Table 3 Description of childbirth, babies' characteristics at birth and vertical transmission in selected studies. Author (          be helpful to diagnose virus pneumonia. 27 In their study, some patients tested by real-time reverse transcription polymerase chain reaction assay (RT-PCR) retrieved negative results and presented alterations on chest CT images, such as patchy shadows and/or ground-glass opacities. In this case, chest CT seems to have a high accuracy to diagnose SARS-CoV-2 infections and present low levels of false negative results, 9,10,13,15,19,23,33 unlike RT-PCR. In addition, relevant clinical examinations such as blood cell counts should be performed together with a comprehensive assessment of the patient's medical history, epidemiological exposure and symptoms in general. [8][9][10]13,24,33 It is important to highlight that, despite chest CT not being frequently conducted on pregnant women due to its level of radiation, in Liu et al. 27 the dose was adjusted to pregnant women to allow the acquirement of images that could present signals of viral infection. This adjustment was responsible for lower-resolution images, but the quality of these images was sufficient to diagnose viral pneumonia and other alterations, such as ground-glass opacities.
All studies included in this review diagnosed patients with SAR-CoV-2 infection by using RT-PCR. However, it is important to highlight that several studies reported the possibility of a false negative result for patients who might had been infected by SARS-CoV-2. 5,10,27,30,33,34 Previous evidence has argued that false negative results could have happened due to insufficient viral load, sampling at early or late stage of the illness, and inappropriate swabbing sites. 30 In Fan et al. 18 study a pregnant patient was positive diagnosed with SARS-CoV-2 after two negative samplings, and Liu et al. 27 conducted several analyses in 25 pregnant women clinically-diagnosed but they had not received the laboratorial-confirmation.
Out of the 279 neonates, 17 were delivered by an emergency cesarean section. 10,11,15,16,19,21,25,28,29,31,32 For these mothers, an emergency cesarean section was elected due to some complications such as pre-eclampsia, 19,21 gestational hypertension, 10,19 fetal bradycardia, 15 fetal distress, 5,10,16,28 PROM 5,10,28 and decreased fetal movement and oxygen decompensation. 21 Previous studies have reported the pregnant women with viral pneumonia are more likely to have complications during pregnancy and can also lead to spontaneous miscarriage, preterm birth, low birth weight infants, intrauterine growth restriction and five-minute Apgar score <7 compared to healthy pregnant women. 38 The results of this study have evidenced a high rate of preterm births (n=49), and out of 273 neonates were delivered and six deaths were reported. Previous studies have shown that SARS-CoV-2 can be transmitted from human-to-human through close contact of the droplets and also via aerosol. 1,2 However, the potential for transmissibility from mother-to-fetus is still unknown. In this review, among 279 neonates evaluated by different studies, only ten showed a positive result for SARS-CoV-2, after at least 30 hours of childbirth. 10,30,34 However, in some cases, the placenta, umbilical cord, amniotic fluid and breast milk were tested and no positive result for the presence of the virus was found. 10,18,25,30,31,34 It is important to consider the possibility of false-negative in these cases, since RT-PCR method was adopted, which may have limitations due to the latency period of the virus, as previously mentioned.
Although, some authors believe that vaginal delivery may increase the risk of vertical transmission of COVID-19, the hypothesis that best explains the mother's non-viral transmission to her child was demonstrated by Zheng et al., 39 who state that the angiotensin-converting enzyme 2 (ACE2) receptor of COVID-19 has very low expression in almost all cell types of the early maternal-fetal interface, suggesting that there may be no cells that are potentially susceptible to COVID-19 in the maternal-fetal interface.
Despite still being unclear and requiring future clinical studies with a larger sample size to guarantee scientific evidence, vertical transmission seems unlikely. However, protocols for the care of pregnant women and newborns should take into account that, although studies have not shown a positive test for SARS-CoV-2 in breast milk, breastfeeding may present risks of contamination due to direct physical contact. 40 Despite this, breastfeeding should be encouraged, but mothers should be properly advised on preventive protocols to avoid transmission.
Recently, the medical field has debated about the immunization of pregnant women against SARS-CoV-2, but clinical trials involving pregnant women are still scarce. [41][42][43] A shared decision-making between the mother, her family, and the obstetrician is essential. The discussion should focus on the risks of infection, risk of morbidity and mortality, and the uncertainty regarding the safety of the vaccine in pregnancy and lactation. But it must be emphasized that at this point we do not have biologic reasons to believe that the currently approved vaccines are harmful to pregnant women or infants. 41,43 As well known, antibodies can be transmitted from mother to the fetus via placental transport. 44,45 The transportation of antibodies, such as Immunoglobulin-G (IgG), protect the baby from virus infections after birth, and this process is known as passive immunization. 44,45 However, in Wang et al., 40 the IgG levels of infants decreased considerably in the first two months of life, suggesting that the passive immunization of IgG for SARS-CoV-2 provided limited protection for the infants, especially if the mother was infected with the virus for less than two weeks before delivery.
The key question to be answered at this point is whether there is transmissibility of antibodies from the pregnant woman who received the vaccine to the fetus. Rottenstreich et al. 46 indicated that immunization of pregnant women may provide maternal and neonatal protection, since they found antibodies (IgG) in umbilical cord serum. This is still unclear, so clinical trials should be conducted to understand the association of these outcomes.
Our study has some limitations. Because of the unprecedented nature of the subject due to the recent pandemic, few population-based clinical studies have been published in the literature assessing the health condition of pregnant women and newborns relating to the involvement of COVID-19. Most of the studies included in this review were case reports and retrospective case report analyzes with a small sample size. We suggest that longitudinal clinical studies with a representative sample be conducted in order to ensure a better understanding of the relationship among COVID-19, pregnancy and neonate outcomes. Also, the percentages of each symptom during pregnancy were calculated based on a ratio between the total number of cases and the studies that have reported such condition. If a paper had not reported a determined symptom, it was inferred that that condition was not present. Moreover, most of the studies published so far have evaluated patients in the 3 rd trimester of pregnancy, which makes it impossible to understand the cause-and-effect relationship of the 1 st and 2 nd trimesters and COVID-19. Finally, future studies should answer some other questions, such as whether vaginal delivery increases the risk of vaginal transmissibility and whether uterine contractions increase the possibility of the virus ascending. As well as, the role of women's vaccination on infants' protection.
Despite the limitations of this study, this review carried out an extensive search of the literature in different databases, with well-defined criteria and in a standardized protocol, which ensured the inclusion of studies published in high-impact journals.
Based on this review, it can be concluded: 1-COVID-19 in women during pregnancy results in symptoms similar to those experienced by non-pregnant women when affected by the same disease. There is a high rate of asymptomatic positive cases during pregnancy. When the cases are symptomatic, the disease is revealed in its mildest form. Fever and cough are the clinical signs commonly seen in pregnancy; 2-There is still no plausible evidence suggesting vertical transmission of SARS-CoV-2 virus from mother to child. However, in this review it was possible to observe that cesarean sections have been performed more frequently. High rates of premature births also have been reported; 3-As COVID-19 maternal illness does not appear to be as severe as SARS and MERS, the high rate of cesarean section is unreasonable. Obstetricians should assess case by case in order to avoid the indication of unnecessary cesarean sections; 4-Although there is no evidence of the virus in the infected woman's breast milk, breastfeeding might lead to the baby's infection due to the close contact. Nevertheless, breastfeeding should be encouraged, but mothers should be given adequate guidance on preventive protocols to avoid transmission.

Authors' contribution
Foratori-Junior GA and Mosquim V contributed with literature search, figures, study design, data collection, data analysis, data interpretation and writing of this manuscript. Machado MAAM, Oliveira TM and Sales-Peres SHC contributed with study design, data analysis, data interpretation and writing of this manuscript. All authors approved the final version of the article.