SARS-CoV-2 Vaccines during Pregnancy and Breastfeeding: A Systematic Review of Maternal and Neonatal Outcomes

(1) Objective: This systematic review summarizes current knowledges about maternal and neonatal outcomes following COVID-19 vaccination during pregnancy and breastfeeding. (2) Study design: PubMed, Cochrane Library, and the Education Resources Information Center (ERIC) were searched up to 27 October 2021. The primary outcome was to estimate how many pregnant and lactating women were reported to be vaccinated and had available maternal and neonatal outcomes. (3) Results: Forty-five studies sourcing data of 74,908 pregnant women and 5098 lactating women who received COVID-19 vaccination were considered as eligible. No major side-effects were reported, especially during the second and third trimester of pregnancy and during breastfeeding. Conversely, available studies revealed that infants received specific SARS-CoV-2 antibodies after maternal vaccination. (4) Conclusions: Vaccination against the SARS-CoV-2 virus should be recommended for pregnant women, after the pros and cons have been adequately explained. In particular, given the still limited evidence and considering that fever during the first months of gestation increases the possibility of congenital anomalies, they should be carefully counseled. The same considerations apply to breastfeeding women, also considering the immune responses that mRNA vaccines can generate in their human milk.


Introduction
Physiological, mechanical, and immunologic changes in pregnant women could influence their possibility of being attacked by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. Main symptoms of the disease (COVID-19) are related to an impaired microcirculatory function; indeed, infected pregnant women are at increased risk of preeclampsia (PE)-like symptoms [2] and of need for hospitalization and intensive care unit admission [3]. Given that the incidence of obstetrical complications, such as preterm birth, appears to be proportional to the severity of the infection, infants born to infected mothers with a more severe clinical course may have a worse outcome, mainly due to neonatal morbidity and mortality associated with prematurity. Therefore, the immunization of pregnant women against SARS-CoV-2 appears to be justified [4].
Although no conclusive evidence is yet completely available regarding the effectiveness and safety of COVID-19 vaccines in pregnancy, due to the non-inclusion of these women in clinical trials evaluating vaccines, the studies performed to date have allowed detecting a significant lower risk of contracting SARS-CoV-2 infection among vaccinated than unvaccinated pregnant women. The number of pregnant women vaccinated against COVID-19 to date, globally, has exceeded hundreds of thousands with no adverse event reports in excess of the nonpregnant population [5]. The same goes for the efficacy of vaccination during breastfeeding, which is considered to be similar to that among nonpregnant women. Currently there is unanimous consensus that there is no biological plausibility in support of a possible harm to infants nursed by vaccinated mothers [6]. Regarding the fertility of women who undergo vaccination against COVID-19, public health agencies and scientific societies internationally ruled out a possible association between vaccine and fertility problems [7].
Our aim was to summarize the evidence in the literature on the outcomes of vaccination against SARS-CoV-2 in pregnant women and in women who have recently delivered, as well as the possible effects of vaccines currently used ( Figure 1) on their newborns.
Viruses 2022, 14, x FOR PEER REVIEW 2 of 20 SENSITIVE nonpregnant women. Currently there is unanimous consensus that there is no biological plausibility in support of a possible harm to infants nursed by vaccinated mothers [6].
Regarding the fertility of women who undergo vaccination against COVID-19, public health agencies and scientific societies internationally ruled out a possible association between vaccine and fertility problems [7]. Our aim was to summarize the evidence in the literature on the outcomes of vaccination against SARS-CoV-2 in pregnant women and in women who have recently delivered, as well as the possible effects of vaccines currently used ( Figure 1) on their newborns.

Search Strategy and Study Selection
This systematic review was performed following PRISMA guidelines [8]. Search terms included "SARS-CoV-2" OR "COVID-19" AND "vaccination" OR "vaccine" AND "pregnancy" OR "pregnant" OR "breastfeeding". We considered studies providing information about maternal and/or child outcomes after maternal SARS-CoV-2 vaccination with a mRNA-based vaccine published after 1 January 2021. We did not apply limitations on study design to include all available literature, but preprint studies were not considered. The

Search Strategy and Study Selection
This systematic review was performed following PRISMA guidelines [8]. Search terms included "SARS-CoV-2" OR "COVID-19" AND "vaccination" OR "vaccine" AND "pregnancy" OR "pregnant" OR "breastfeeding". We considered studies providing information about maternal and/or child outcomes after maternal SARS-CoV-2 vaccination with a mRNA-based vaccine published after 1 January 2021. We did not apply limitations on study design to include all available literature, but preprint studies were not considered. DNA vaccines: modified plasmids that carry genes that typically code for the spike protein, which is then produced in the vaccinated individual, as in Indian ZyCoV-D ® COVID vaccine.

How do current available vaccines work?
The Pfizer-BioNTech BNT162b2 vaccine and Moderna mRNA-1273 vaccine contain mRNA encoding for spike membrane proteins, encapsulated in lipid nanoparticles. Oxford-AstraZeneca ChAdOx1 contains SARS-CoV-2 virus cDNA fragments encapsulated in a viral vector. Both are non-replicants and self-destroying. When administered, macrophages and dendritic cells grab particles and convert them into proteins which are degraded into peptides and then exposed as antigens. Antigenic viral peptides are presented to major histocompatibility complexes (MHC) I and II and identified by helper T lymphocytes, causing B cells to synthesize neutralizing antibodies and cytotoxic T lymphocytes that kill infected cells.
Neutralizing antibodies directed to the virus membrane glycoproteins such as the spike protein and nucleocapsid proteins drive the humoral immune response to the SARS-CoV-2 virus. These antibodies prevent the virus from entering cells and, hence, its infectious capacity. However, not all antibodies have neutralizing activity, and some can increase the activity of the virus. Therefore, to solve this problem, mRNA fractions were isolated and encapsulated within lipid nanoparticles to create mRNA vaccines. COVID-19 infection is the first infection to be prevented with mRNA vaccines, and the vaccine underwent many tests quickly before being administered to humans. Both viral-vector vectors and mRNA vaccines induce a cell-mediated response by cytotoxic Th1 and CD8 + lymphocytes that recognize and digest viral antigens exposed by MCH class I molecules.
As of yet, the induction of regulatory T cells in the vaccine-induced immune response has not been reported. Other COVID-19 vaccine types are now being studied in phase 1, 2, and 3 clinical trials (https://covid19.trackvaccines.org/vaccines/).

Assessment of Risk of Bias
We evaluated the quality of included cohort studies and the risk of bias using the Newcastle-Ottawa Scale (NOS). The NOS comprises "participant selection", "comparability of study groups," and "assessment of outcome or exposure". A score above 6-7 denotes a reasonable quality.

SENSITIVE
The searches identified 1345 potentially relevant papers and studies, while 576 after duplicates were removed. After title and abstract screening, 60 full-text studies were considered potentially eligible for inclusion. A flowchart of study selection process is reported in Figure 2. We considered 46 records: 30 of them included pregnant mothers , two studies included both pregnant and lactating mothers [39,40], and 14 studies included lactating mothers [41][42][43][44][45][46][47][48][49][50][51][52][53][54]. Most studies were conducted in the United States (41.3%) and Israel (26.1%), along with three in Italy (6.5%), two in Spain (4.3%) and in Poland (4.3%), and the remaining (17.2%) in the United Kingdom, Qatar, Belgium, Germany, Norway, Portugal, the Netherlands, and Singapore. Most cohort studies including pregnant women had a reasonable quality, as reported in Figure 3 (case series and case reports without a nonexposed group could not be considered). Most cohort studies including pregnant women had a reasonable quality, as reported in Figure 3 (case series and case reports without a nonexposed group could not be considered).
Most studies including breastfeeding mothers reported only data about exposed women, without a control group; the remaining six studies had a reasonable quality, as reported in Figure 4.

Synthesis of Results in Pregnant Women
The characteristics and most relevant findings of the included studies about vaccination during pregnancy are reported in Table 1. The most relevant question concerns the safety of vaccination administered during pregnancy. No major adverse effects during pregnancy were reported.
To date, the largest study on the safety profile of mRNA vaccines during pregnancy is a cross-sectional survey published by Shimabukuro et al. in the New England Journal of Medicine, who reported preliminary findings from three US vaccine safety monitoring systems [34]. From 14 March 2020 to 28 February 2021 the authors surveyed over 35,000 women aged 16 to 54, identified as pregnant from their inclusion in various pregnancy registries. Among those women, 4000 provided information on the outcome; 827 of these delivered, and 29,000 reported post-vaccination symptoms. The limitation of the study is that all pregnant women were vaccinated in the third trimester, and this ruled out the possibility of evaluating some of the possible adverse effects of vaccinations on the progress of pregnancy and on newborns. However, although not fully comparable, the estimate of obstetrical and neonatal complications of maternal vaccination (n = 827) is similar to that described in studies on pregnant women conducted in the pre-COVID-19 period [30]. Most studies including breastfeeding mothers reported only data about exposed women, without a control group; the remaining six studies had a reasonable quality, as reported in Figure 4.  Most studies including breastfeeding mothers reported only data about exposed women, without a control group; the remaining six studies had a reasonable quality, as reported in Figure 4.        Comparing adverse effects in pregnant women and nonpregnant women, according Bookstein Peretz's data, there were no additional adverse effects of vaccination during pregnancy. Furthermore, the rate and the severity of adverse effects were unaffected by the timing of immunization during pregnancy [12].
Two very large Israeli cohort studies provided data that support the safety of vaccination in pregnancy. The first is a retrospective study by Goldshtein et al. that looked at the relationship between immunization with Pfizer vaccine and the risk of infection in pregnant women who were vaccinated in the second and third trimesters of gestation. The primary outcome was SARS-CoV-2 infection demonstrated by the positivity of the molecular swab at 28 days after the initial vaccination dose. The cumulative incidence of infection in vaccinated women was significantly lower than in unvaccinated women. SARS-CoV-2 hospitalization rates were 0.2% in the protected women and 0.3% in the unprotected peers. During the study's follow-up period, 18% of the vaccinated group and 18.9% of the unvaccinated group completed the pregnancy [19]. Dagan et al. in the second Israeli study compared a cohort of 10,861 pregnant women vaccinated with BNT162b2 mRNA matched 1:1 with 10,861 unvaccinated pregnant women. Twenty-six percent were immunized during the first trimester, 48% were immunized during the second trimester, and 26% were immunized during the third trimester. The results reflect the effectiveness of the vaccine against the alpha variant, which was predominantly circulating in Israel during the study period. The primary outcome was to determine the incidence of SARS-CoV-2 infection documented by a positive molecular swab and the incidence of symptoms and hospitalization. The periods in which the cumulative incidence was calculated were 14-20 days and 21-27 days following the first administration and 7-56 days following the second injection. The incidence of infection, as well as the risk of severe illness and hospitalization, was considerably greater in the unvaccinated group compared to the vaccinated group [15].
A very relevant question relates to the protective capacity of vaccination or natural infection for newborns. Several studies on this question have been performed. The most interesting was carried out in Boston on a cohort of 103 women protected with mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) vaccine, as well as 28 women with confirmed SARS-CoV-2 infection [39]: 30 vaccinated women were pregnant; 16 vaccinated women were post breastfeeding; 57 vaccinated women were neither pregnant nor breastfeeding. Among the 28 unvaccinated women, 22 were infected with SARS-CoV-2, while six were infected with SARS-CoV-2 and were breastfeeding. The authors measured the anti-SARS-CoV-2 receptor-binding domain (RBD) antibody titer and the neutralizing and non-neutralizing antibody titer after vaccination and after the natural infection. The presence of neutralizing and non-neutralizing antibodies and the response of CD4 + and CD8 + T-cells were detectable in all subgroups and were also observed in the umbilical cord blood and human milk following immunization. The level of antibodies against variants B.1.1.7 and B.1.351 was decreased, but the cellular T response was conserved against these variants.
The presence of neutralizing antibodies in pregnant women and newborns following vaccination was confirmed in all cohorts. Interestingly, a surprising discovery is that the transfer ratio appears to rise with latency from immunization; these findings imply that earlier immunization, at least among women in their third trimester, may create a stronger protection in the baby, the immunobiology of which deserves additional study [26].

Synthesis of Results in Lactating Women
Questions are often asked about the presence of antibodies and mRNA in breast milk after COVID vaccine. The characteristics and most relevant findings of the included studies on vaccination during breastfeeding are reported in Table 2. No major adverse reactions needing emergency treatment or hospitalization were described in mothers and infants. Binding, neutralizing, and functional non-neutralizing antibody responses, as well as CD4 and CD8 T-cell responses, were present in pregnant, lactating, and nonpregnant women following vaccination. Binding and neutralizing antibodies were also observed in infant cord blood and breast milk. Binding and neutralizing antibody titers against the SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern were reduced, but T-cell responses were preserved against viral variants. In human milk, a biphasic response was observed, with SARS-CoV-2 specific IgA starting to increase between days 5 -7 after the first dose and declining after day 15, on average. After the second dose, an accelerated immune reaction was observed. The anti-SARS-CoV-2 vaccines used were well tolerated by mothers and breastfed infants.
Breastfeeding must not be interrupted after vaccination.
They showed a positive correlation between antibody levels in serum and breast milk samples (lower in who received AstraZeneca  It seems clear that, in the human milk of women protected with mRNA vaccines (currently preferentially indicated in Italy for women of reproductive age), there is a constant presence of anti-spike antibodies, particularly of the IgA and IgG type, as also shown by preliminary data from a study still in progress at the "Bambino Gesù" Children's Hospital in Rome [53].
The relationship between milk IgA antibodies and immunization timing during breastfeeding has to be researched further, considering the drop in IgA levels recently observed of up to 25% in a cohort of lactating women (probably deriving from the different timing of withdrawals) [55].
To date, the largest study is a cross-sectional survey performed by McLaurin-Jiang et al., who recruited 4455 vaccinated breastfeeding mothers. According to their findings, vaccination appears to have few negative effects on lactation despite the reported shortterm side-effects (such as fever, fatigue, or headache) [46], with resolution within 72 h after vaccination [51].
An increase in milk IgA levels is seen 2 weeks after the first dose of vaccine, and they are detectable in 86% of cases 2 weeks after the second dose; IgG was already detected just 1 week after the second dose [40,48,52].
When compared to detectable levels following the first dose, maternal blood IgG levels rose sixfold following the second dose; a similar pattern of increase was observed for milk IgG levels, although whether this could stimulate adequate infant immunity or not still requires further studies [55].
No mRNA presence was detected, highlighting the fact that there is no transfer of mRNA to the baby via breast milk and no reason to discontinue breastfeeding for this reason at the time of vaccine administration [44].
To exclude that mRNA vaccine components could get into milk after immunization, polyethylene glycol was measured by Golan et al. in milk samples collected before and after vaccine administration, and its concentration did not significantly change [55].

Discussion
Primary prevention of infections in pregnancy through vaccination is one of the most successful public health programs of the last 10 years. It has led to a significant decrease in maternal and perinatal morbidity and deaths from influenza and whooping cough [56]. The frequency of COVID-19 vaccination is still relatively low among pregnant women worldwide. Shamshirsaz et al. demonstrated that vaccination acceptance was significantly associated with the history of influenza or pertussis vaccine administration during pregnancy (OR = 3.03; 95% CI: 1.37-6.73; p = 0.006) [57].
Therefore COVID-19 infection in pregnancy has a course that is certainly more severe than in other periods of women's life. It can cause adverse effects on the course of pregnancy, such as preterm birth, even if the real possibility and frequency of maternal-fetal transmission are still under study.
According to Ciapponi et al., COVID-19 pregnant women had a twofold higher risk of requiring mechanical ventilation, whereas their neonates had a threefold higher risk of being hospitalized in the neonatal intensive care unit (NICU) [58]. Kazemi et al. noted a marked risk of abortion in infected mothers; one possible mechanism could be placental inflammation induced by viral particles [59]. Most infections in neonates and children arise from family clusters [60], linked to infected adult patients; they often exhibit only milder clinical signs [61]. Recently, a few case reports of neonates presenting with a multisystem inflammatory syndrome after maternal SARS-CoV-2 infection (MIS-N) were reported [62].
Lumbreras-Marquez et al. assessed that, if all pregnant women received immunization during the two months of May and June 2021, the total expected maternal deaths in Mexico would have firmly decreased [63].
Data from mRNA vaccine studies show that the vaccine safety, tolerability, and efficacy in immunization are similar in pregnant women and their nonpregnant peers [4]. Since mRNA vaccines appear to stimulate Toll-like receptor 3 and that such activation has been associated with negative gestational outcomes [5], Shanes et al. examined the placentas of 84 women who got a SARS-CoV-2 vaccine during pregnancy and 116 unvaccinated women. The authors found no raised incidence of deciduous arterial disease, fetal vascular dysfunction, chronic low-grade villitis, or chronic histiocytic intervillositis in the vaccinated group [33].
However, the speed with which these vaccines were developed and authorized caused some concerns in the communities, starting from the possible effects on fertility or child outcomes. Long-term statistics are obviously scarce and will remain so for some years. The COVID-19 Vaccines International Pregnancy Exposure Registry (C-VIPER) will systemically estimate the risk of obstetric outcomes, neonatal outcomes, and infant outcomes in pregnant women exposed to a COVID-19 vaccine from 30 days before to the first day of their last menstrual period to end of pregnancy compared to a matched unexposed group. To date, the estimated study completion date is 31 December 2025 [64].
Although pregnant women were excluded from the first trials, at the time of writing, there is absolutely no evidence or theoretical reason to believe that any of the COVID-19 vaccinations could impair future fertility to date [7].
Concerns have also been raised about the chance of spontaneous abortion (pregnancy loss that occurs at fewer than 20 weeks of gestational age), a frequent event that affects 11 to 22 percent of identified pregnancies. Analyzing the data of the CVC v-safe COVID-19 pregnancy registry, the cumulative risk was within the expected risk range when data were compared to two historical cohorts that represent the "normal" lower and upper ranges of spontaneous abortion risk [37].
This systematic review is limited by the statistical heterogeneity in results of previous studies (ranging from single case reports to multicenter studies) and the relatively small sample size of pregnant women available in the literature, considering that more than 9.5 billion shots of COVID-19 vaccines have already been administered globally, at the time of writing. However, we described the outcomes of a global cohort of 74,908 pregnant women and 5082 lactating women who received COVID-19 vaccination, and this can be considered an adequate sample to rule out any adverse effect in them and their infants.
Scientific societies agree in declaring these vaccines safe during pregnancy and breastfeeding.
The American College of Obstetricians and Gynecologists (ACOG) recommends that individuals over the age of 12, including pregnant and lactating women, receive a COVID-19 vaccine or a series of vaccines [65]. The European Board and College of Obstetrics and Gynecology (EBCOG) also said that immunization should be offered to all pregnant women, after they have been adequately informed of the benefits, especially in pregnant women at high risk for the presence of comorbidities (obesity, diabetes, heart disease, lung disease). Furthermore, the EBCOG recommends immunization against COVID-19 for all breastfeeding women, in the absence of a particular contraindication [6].
Considering the still limited evidence of vaccine safety during the first trimester of pregnancy, the Italian National Institute of Health and the Italian Obstetric Surveillance System recently updated guidelines for COVID-19 vaccinations during pregnancy; they cautiously stated that pregnant women who desire to be vaccinated should consider benefits and risks, given that fever is one of the possible reactions to the vaccine [66], and that this can cause an increased risk of birth defects [67], more so than the vaccine itself.

Conclusions
In this review we summarize the current knowledge about maternal and neonatal outcomes after COVID-19 vaccination, in order to help clinicians be thoroughly informed and fight misinformation. Benefits of COVID-19 vaccination outweigh the risks during pregnancy for both mothers and infants. Indeed, available studies report a good maternal immune response, as well as the transfer of maternal antibodies to confer passive protection against SARS-CoV-2 in newborns following maternal vaccination. The existence of anti-SARS-CoV-2 antibodies in breast milk suggests a possible specific protective effect on the newborn-infant after both maternal infection and vaccination, even if only clinical trials can provide scientific evidence.
It should be noted that the ability to protect oneself is not limited to the presence of circulating antibodies; next to these there are memory cells, which play a fundamental role in the response against SARS-CoV-2 in the event of virus exposure. Therefore, the potentiality of the cellular component (primarily lymphocytes) of breast milk to react to SARS-CoV-2 must be investigated.