Breastfeeding vs. breast milk transmission during COVID-19 pandemic, which is more important?

The catastrophic coronavirus disease 2019 (COVID-19) pandemic has raised many health questions, and whether breast milk from SARS-CoV-2 infected mothers may be a vector for SARS-CoV-2 transmission has become a hot topic of concern worldwide. Currently, there are extremely limited and conflicting data on the risk of infection in infants through breastfeeding. For this reason, we investigated almost all current clinical studies and systematically analyzed the presence of SARS-CoV-2 and antibodies in the breast milk of mothers infected with SARS-CoV-2, their effects on newborns, and the mechanisms involved. A total of 82 studies were included in this review, of which 66 examined the presence of SARS-CoV-2 in breast milk samples from mothers diagnosed with COVID-19, 29 reported results of antibody detection of SARS-CoV-2 in breast milk, and 13 reported both nucleic acid and antibody test results. Seventeen studies indicated the presence of detectable SARS-CoV-2 nucleic acid in breast milk samples, and only two studies monitored viral activity, both of which reported that infectious viruses could not be cultured from RNA-positive breast milk samples. All 29 studies indicated the presence of at least one of the three antibodies, IgA, IgG and IgM, in breast milk. Five studies indicated the presence of at least one antibody in the serum of breastfed newborns. No COVID-19-related deaths were reported in all 1,346 newborns. Our study suggests that direct breastfeeding does not pose an additional risk of infection to newborns and that breast milk is a beneficial source of anti-SARS-CoV-2 antibodies that provide passive immune protection to infants. In addition, direct breastfeeding would provide maternal benefits. Our review supports the recommendation to encourage direct breastfeeding under appropriate infection control guidelines. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/#myprospero, identifier: 458043.


Introduction
During the global pandemic of COVID-19, the safety of direct breastfeeding by infected mothers to their infants has become one of the hottest topics. Few conditions are considered to be clear contraindications to breastfeeding. Known pathogens that can be transmitted in breastfeeding include HIV (1), human T-lymphotropic virus 1 (2), and cytomegalovirus (3). Previously, some studies showed the presence of SARS-CoV-2 in the breast milk of a few cases (4)(5)(6), a situation that caused considerable alarm. There is insufficient evidence as to whether breastfeeding is a possible mode of vertical mother-to-child transmission.
Breast milk is the gold standard for infant feeding. Not only does breastfeeding provide the best source of nutrition for the newborn and a powerful first barrier against infection, but it also has the emotional stimulation to enhance dynamic, two-way communication between mother and infant, laying the foundation for a physical and psychological bond between mother and child (7). In the short and long term, breastfeeding provides tremendous health benefits for both child and mother, especially the immunological properties of breast milk that make it a protective factor against infant morbidity and mortality (8). For infants, passive immunity is derived mainly from breast milk. SARS-CoV-2-specific antibodies have been reported to be detected in the breast milk of infected women (9,10). The transfer of these antibodies to infants through breast milk may protect against SARS-CoV-2 infection. So, are SARS-CoV-2 antibodies present in breast milk or not? Could they provide benefits to newborns? Although the World Health Organization declared on May 5, 2023, that the spread of COVID-19 was no longer a "public health emergency of international concern", the global interest in ensuring that mothers and infants are not separated and that breastfeeding mothers are provided with the necessary support and/or breast milk for their infants has been well established for some time, and there are very limited and conflicting data on the presence of SARS-CoV-2 in breast milk and the potential for vertical transmission in breast milk. Therefore, there is a strong need for a comprehensive analysis and discussion of the evidence regarding the potential for vertical transmission of breastfeeding and the presence of anti-SARS-CoV-2 antibodies in breast milk. Here, we plan to use this systematic review to synthesize the data published to date on the risk of SARS-CoV-2 transmission through breast milk, to provide a comprehensive summary of the presence and characteristics of antibodies in breast milk and their effects on infants, to provide evidence for assessing the risk-benefit of breast milk transmission vs. breastfeeding, and to provide a basis for the management of mother-infant dyads and optimal health strategies for breastfeeding in similar outbreaks in the future.

Identify research question
Is breast milk of SARS-CoV-2 infected mothers a possible carrier of SARS-CoV-2 transmission? What is more important, breastfeeding or breast milk transmission?

Identify relevant types of evidence
An experienced information specialist conducted a comprehensive search of PubMed, MEDLINE, CNKI, Wanfang Database, bioRxiv, medRxiv, Embase, and Cochrane Library online databases, and to maximize the scope of the review without time and language restrictions, the last data update was April 30, 2023. We used the keywords "SARS-CoV-2", "COVID-19", "breast milk", and "antibody" individually or in combination to achieve a comprehensive literature search. We also searched the gray literature of the preprint servers bioRxiv and medRxiv. In addition, we manually searched the references of the original articles included in the study to avoid missing important literature that was not noted in the initial search. Inclusion criteria were (i) the presence of SARS-CoV-2 RNA or/and the presence of anti-SARS-CoV-2 antibodies in mothers diagnosed with SARS-CoV-2 infection and (ii) breast milk samples. Studies that included the same study population but reported different data and outcomes were also included. Exclusion criteria were (i) questionnaires; (ii) studies that explored breastfeeding intentions only (psychological); (iii) studies in which mothers were not diagnosed with SARS-CoV-2 infection; (iv) artificial vaccinations or mixed groups with vaccinations that could not be grouped separately; (v) studies with severely missing data on mothers, breast milk samples, or newborns; and (vi) studies that tested only breast milk studies that detect factors other than SARS-CoV-2 RNA or antibodies in breast milk, including cytokines, chemical elements, proteomes, and nerve growth factors. None of the literature types were restricted. See Supplementary Material S1 for detailed search strategies.

Study selection
After completing the initial search, two independent reviewers conducted a screening process, and literature with quantifiable evidence was included in our review. We excluded duplicate publications and duplicate case studies (we identified duplicates based on author name, location, participant admission date, maternal and neonatal characteristics, and publication date). One reviewer reviewed the selected articles in their entirety, and studies that contained complete data descriptions were used for data graphing. Any conflicts that arose during the data extraction process were resolved by consensus with a third reviewer. All seven authors participated in the discussion and decided on the topic. to make meaningful comparisons between studies. Figure 1 shows a visual representation containing the workflow.

Results
A total of 82 studies were finally included (4,6,, including three studies published in Chinese (25, 27, 28). They came from 21 countries, with 26 studies from China (31.7%). The earliest study start time is December 2019 (24), and the longest study span is 12 months (86). Thirteen studies reported both nucleic acid test results and antibody test results. Of 82 studies, 33 case reports and nine case series were included. A total of 1,755 mothers and 1,346 newborns and at least 2,043 breast milk samples were included in the studies. The maximum sample size of mothers included in the study was 165, the maximum sample size of breast milk was 316, and the maximum sample size of newborns was 165. The follow-up period ranged from three days to 11 months. The characteristics of the included studies are listed in Table 1.

Results of SARS-CoV-2 nucleic acid detection in breast milk
A total of 66 studies examined the presence of SARS-CoV-2 in breast milk samples from mothers with a confirmed diagnosis of COVID-19 (4,6,9,. All included studies used RT-PCR assays. Fifty-five studies reported the timing of breast milk specimen collection, with the earliest being immediately after delivery and the longest span of breast milk specimen collection being 206 days. A summary of studies on the detection of SARS-CoV-2 in human milk is presented in Table 2. Of these 66 studies, 17 reported positive results for SARS-CoV-2 nucleic acid in breast milk samples (4,6,11,14,17,18,29,32,33,36,38,39,46,47,55,59,72), but only two studies monitored viral activity (6,47). The remaining 49 studies showed negative results for SARS-CoV-2 in breast milk. Thirty-eight studies reported full or partial use of breastfeeding for newborns. Of the 17 studies that were positive for SARS-CoV-2 RNA in breast milk, 179 newborns in 11 studies were given breast milk, and only 18 (10%) had a positive nasopharyngeal swab result

Results of SARS-CoV-2 antibody detection in breast milk
Twenty-nine studies reported results of antibody testing against SARS-CoV-2 in at least 1,279 breast milk samples from mothers with a confirmed diagnosis of COVID-19 (9, 10, 62-88), and the majority of mothers included in these studies had mild disease severity. All 29 studies showed the presence of at least one of the three antibodies, IgA, IgG and IgM, in breast milk. Twenty-seven of these studies reported the method of detection, and 23 studies used an ELISA assay. The longest duration of antibodies was ten months (80). Twenty-three studies found IgA antibodies in breast milk samples, 17 studies found IgG antibodies in breast milk samples, and 11 studies reported the presence of IgM antibodies in breast milk. Six studies indicated that all three antibodies, IgA, IgG and IgM, were detected in breast milk (10,68,69,79,87,88). Nine studies also reported serum antibodies in newborns born to mothers, five studies reported the presence of at least one antibody in the serum of newborns (63,(70)(71)(72)88), most of whom were breastfed, four studies reported negative serum antibodies in newborns (62, 69, 75, 78), with one study detecting IgA antibodies in the saliva of newborns (75). Table 3 presents a summary of studies in which antibodies against SARS-CoV-2 were detected in human milk.  The sample of the positive breast milk was retested the next day. e Two of the seven pregnant women delivered. f Missing data from 5 individuals. g Missing data from 6 individuals.
h One breast milk was tested for nucleic acid and six breast milk was tested for antibodies. i The breast milk samples were all from the same mother. Hinojosa-Velasco et al. (Continued)  The breast milk samples collected from the infected mother was negative for SARS-CoV-2 by RT-PCR.
(Continued)  Piersigilli et al.  Buonsenso et al.    The breast milk sample collected from the infected mother was negative for SARS-CoV-2 by RT-PCR.
De Socio et al.        mothers, and SARS-CoV-2 RNA was detected in the breast milk of four mothers. A study from Italy tested ten breast milk samples collected from the same mother during the first five days after the birth of her newborn, and three samples were positive for SARS-CoV-2 RT-PCR (46). The remaining 49 studies, including at least 1,229 breast milk samples, did not detect SARS-CoV-2 RNA. These data suggest that SARS-CoV-2 RNA is indeed present in the breast milk of COVID-19 mothers.

4.2.
Is breast milk a carrier of vertical transmission from mother to child?

The presence of SARS-CoV-2 nucleic acid in breast milk does not represent a replicationcompetent virus
Although SARS-CoV-2 RNA was detected in 38 breast milk from 17 studies, only two studies monitored viral activity (6,47). Krogstad et al. (47) performed viral cultures on 160 breast milk samples and did not detect the virus in any culture, although SARS-CoV-2 RNA was detectable in 9.2% of these milk samples. Notably, they artificially added virus to breast milk samples from control experiments and infectious SARS-CoV-2 could be cultured despite several freeze-thaw cycles. Chambers et al. (6) added replication-competent SARS-CoV-2 to two breast milk samples that were pasteurized. The authors failed to detect SARS-CoV-2 RNA or culturable virus in the Holder pasteurized breast milk samples. In contrast, the two unpasteurized breast milk samples from the control group were found to be positive for viral RNA. These studies suggest that SARS-CoV-2 virus particles detected in breast milk are not infectious. In fact, there are no reports of transmission of SARS-CoV and Middle East Respiratory Syndrome coronavirus (MERS-CoV) through breast milk.

Most children breastfed from nucleic acidpositive breast milk have negative results
Of the 179 children given breast milk in the 17 breast milk SARS-CoV-2 positive studies, only 18 (10%) of these children had positive nasopharyngeal swab SARS-CoV-2 nucleic acid results and the majority (90%) of newborns had negative nasopharyngeal swab results.

Children breastfed from nucleic acidnegative breast milk show positive results for SARS-CoV-2
Of the 26 studies in which breast milk samples tested negative for nucleic acid, 219 children were given breast milk, and 11 of these studies found 24 children with positive nasopharyngeal swab nucleic acid test results for SARS-CoV-2.

Feeding method is not breastfeeding despite the child being SARS-CoV-2 nucleic acid positive
Results from three of the eight formula-fed studies showed that three children tested positive for SARS-CoV-2 on nucleic acid from nasopharyngeal swabs (11, 31, 46).

Positive mother and positive child, but breast milk negative
The mothers included in this review were all SARS-CoV-2 positive mothers, and 50 children born to these positive mothers in 16 studies turned out to be SARS-CoV-2 positive, but the breast milk of these mothers tested negative for SARS-CoV-2.
In addition, the largest cohort study published to date showed no evidence of vertical mother-to-child transmission when newborns were roomed with their mothers and breastfed. On the other hand, mother-child separation does not guarantee a virusfree environment for the infant.
These findings provide strong evidence that there is no necessary link between breastfeeding and a child being nucleic acid positive, suggesting that breast milk is not a source of vertical transmission. All this evidence supports the view that mothers infected with SARS-CoV-2 do not put their newborns at additional risk of infection through breastfeeding.

4.3.
What are the reasons for positive results? 4.3.1. Contaminated or exposed to a third party infected with SARS-CoV-2 1. The first is respiratory contamination from the mother Mothers do not wear masks when collecting breast milk samples or breastfeeding, which increases the likelihood that breast milk or the newborn will be contaminated with the mother's respiratory secretions, especially during the acute symptomatic phase when the mother's respiratory viral load is extremely high (89).

The second is pollution from the environment
Because breastfeeding involves a range of intimate behaviors between mother and infant, including hand contact and skin-toskin contact with the breast and contact with the surface of the He et al. 10.3389/fped.2023.1253333 Frontiers in Pediatrics container holding the milk, the risk of environmental contamination of breast milk or the newborn is possible (90).

Exposure to a SARS-CoV-2 infected third party
Newborns were infected with SARS-CoV-2 after delivery through close contact with infected family members (43), especially considering that in these cases, parents, relatives, caregivers of newborns, and individuals in their communities were diagnosed with COVID-19 and were susceptible to horizontal human-tohuman transmission (91). We believe that even if the detectable SARS-CoV-2 RNA in breast milk samples is the result of contamination, this is still an important finding because it is a potential and unanticipated source of exposure for uninfected infants. This emphasizes the importance of proper breast hygiene before feeding.

Virus shedding
SARS-CoV-2 enters host cells by binding to the ACE2 receptor (92,93). The ACE2 receptor is expressed in both female reproductive organs and breast tissue (94). Although ACE2 receptor expression is extremely low in breast tissue (only 5%), SARS-CoV-2 could theoretically be present in any tissue with an ACE2 receptor and, therefore, SARS-CoV-2 may still be shed from the breast milk of infected mothers during lactation.

Reflux infection
Human milk has the potential to be contaminated with SARS-CoV-2 RNA from the infant's oropharynx to the breast. If the infant is infected earlier than the infant's parents, there is a possibility of "reverse" vertical transmission from the infant to the mother, a phenomenon that has been observed in other pathogens such as HIV (95, 96) and Ebola (97). In this case, one possible mechanism of maternal infection is retrograde flow, in which milk and saliva move from the infant's mouth back to the mammary gland during suckling.

Associated with higher viral load
It is known that some viruses can be transmitted through breast milk and that higher serum viral load can increase the risk of transmission (98). In the case of HIV and HTLV-1, the level of the virus in breast milk correlates with the amount of virus in the whole body (2, 99, 100).
In addition to this, Bastug et al. (59) believe that the method of detection of the virus in breast milk, the timing of sample collection, and the transportation and storage of the samples are all potential factors that could lead to a positive result.

Relationship between positive results and neonatal outcome
Although 17 studies reported the presence of detectable SARS-CoV-2 RNA in breast milk samples, however, it is reassuring, that longitudinal follow-up of most studies showed no adverse outcomes in infants who still continued to be breastfed, including those in whom viral RNA was detectable on the skin of the mother's breast (73). Of the 1,346 newborns included, only three were admitted to the NICU (60, 72). Notably, in the context of the COVID-19 pandemic at the time, most of the newborns in the study were admitted to the NICU for isolation and observation rather than for any absolute indication. No deaths due to COVID-19 were reported in all neonates.

Negative results and interpretation
There are several possible reasons why the majority of studies (74%) failed to detect SARS-CoV-2 RNA in the breast milk of COVID-19 mothers. One of the most important reasons could be due to the extremely low level of ACE2 expression in the breast (94,101,102). Indeed, ACE2 needs to be co-expressed with protease, TMPRSS2, or CTSB/L to activate the S protein and promote the entry of SARS-CoV-2 into host cells. However, only 5% of mammary cells express ACE2 (103). The second reason is the antiviral mechanism of SARS-CoV-2 by specific substances in breast milk. Breast milk contains whey protein (104), lactoferrin (105), and mucin (106), which can block virus entry and replication by binding to various receptors of SARS-CoV-2. In addition, the presence of antibodies in breast milk, especially IgA antibodies, which have neutralizing activity against SARS-CoV-2, is also believed to be one of the reasons for the absence of SARS-CoV-2 in breast milk (67). Third, if breast milk samples are collected farther back in time from the time of infection, the viral load in breast milk will be lower, especially for asymptomatic mothers, who may not be able to detect SARS-CoV-2 RNA in breast milk because it is difficult for them to determine the onset of infection. The results showed that 117 (70.9%) women were positive for anti-SARS-CoV-2 IgA in colostrum and confirmed that the presence of anti-SARS-CoV-2 IgA in colostrum was independently associated with lower clinical signs in newborns. Two (1.2%) participants were also found to be positive for anti-SARS-CoV-2 IgG in colostrum. These two studies are the largest samples of mothers included to date (80, 86). The study by Bode et al. (82) showed that detectable IgA antibodies were found in breast milk even eight months after onset. IgM antibodies were also present in breast milk, albeit at lower concentrations (9,10,62,68,87). In general, IgM and IgA-like responses often occur earlier after the onset of the disease, while IgG responses occur later (107). It is known that antibody abundance in breast milk is variable and there is a high degree of intra-and inter-individual variability in the three antibody classes (108). A study by Fox et al. (79) has demonstrated much higher titers of IgA than expected in 12/15 milk samples. The difference between IgA and IgG may be due to the fact that systemic IgG-secreting B cells are shorter-lived than IgA-secreting B cells in the submucosa of the breast. It is likely that antibody secretion also depends on the time elapsed after the onset of the disease, and that antibody levels in breast milk decline over time (84, 109). Those participants who showed only IgA antibodies in breast milk were more likely to be enrolled immediately after SARS-CoV-2 infection, whereas those who showed only IgG antibodies in breast milk were more likely to be enrolled some time after infection. Breast milk samples were collected closest to the time point of SARS-CoV-2 infection where antibody levels were likely to be highest. In addition, the severity of symptoms may have influenced the residence time of antibodies in breast milk, with more severe SARS-CoV-2 infections resulting in immune responses with higher antibody levels (110, 111).

Transfer of antibodies from breast milk
Large amounts of Igs in the mother's body can be transmitted to the newborn through breast milk (112, 113). IgG antibodies are a possible indication of immunity or resistance. Of the 29 included studies that reported breast milk antibody test results, five showed that neonates were seropositive for antibodies to SARS-CoV-2 (63, 70-72, 88). Interestingly, Conti et al. (75) also detected the presence of IgA antibodies in the saliva of newborns. In China, Gao et al. (71) conducted an observational clinical analysis of 14 mother-infant pairs and found that three of the breast milk samples tested positive for SARS-CoV-2 IgM or IgG, and the corresponding three newborns fed by breast milk tested negative for SARS-CoV-2 RNA at birth, while they tested positive for SARS-CoV-2 IgG, and one of the neonates also tested positive for IgM. No SARS-CoV-2 nucleic acid was detected in breast milk samples at different stages. This suggests that specific antibodies to SARS-CoV-2 can be transferred to the infant through breast milk and provide potential protection to the neonate. Similarly, the studies of Dong et al. (63) and Fenizia et al. (72) support this idea. At present, it is unclear which factors affect the efficiency of the transfer of these maternal antibodies, and further studies are needed.

Protective effect of breast milk antibodies
It is well known that antibodies can bind to the RBD of the surface spike protein of SARS-CoV-2, preventing the virus from binding to the ACE2 receptor of the target cell (114). This implies that in the context of COVID-19, antibodies are able to limit virus transmission. The secretory immunoglobulin A (sIgA) of anti-SARS-CoV-2 neutralizes SARS-CoV-2 before they reach and bind to epithelial cells. Secretory IgA acts directly on the mucosal surface to inhibit microbial binding to host epithelial cell receptors and to trap pathogenic microorganisms in mucus, enhancing ciliary activity and thereby eliminating invading pathogens (115, 116), providing durable passive immunity to newborns and infants (117). In addition to IgA, IgG in breast milk can attack viral envelope glycoproteins and provide the body with potent systemic antibodies. IgG also acts as an antibody that degrades intracellular viruses by binding to a crystallizable cytosolic fragment motif containing −21 tripartite receptor (TRIM21) (118). These antibodies can be passed on to offspring through breastfeeding, preventing or reducing the severity of disease in newborns. It has been found that even partially degraded IgA, IgG and IgM antibodies, if their antigen-binding fragment (Fab) fraction remains intact, still function and bind to the antigen, leading to target degradation and elimination from the intestine (119). This suggests that any neutralizing SARS-CoV-2 targeting antibodies present in breast milk may have a protective effect on infants receiving breast milk. In addition, intestinal Fc receptors may transport antibodies from the gut to the vascular circulation, further aiding systemic defense. IgA spiking antigen immune complexes would also stimulate active immunity and contribute to the maturation of the infant's immune system (75). This will facilitate the protection of the newborn from a variety of viral pathogens. This active form of protection of human milk against viruses cannot be provided to the infant through artificial formula.
Typically, maternal IgG antibodies are retained in the infant for more than six months, and infants who are unable to produce immunoglobulins are protected by maternal antibodies for up to 12 months after birth (120). Intriguingly, some studies suggest that antibodies transferred to newborns through breast milk are lower than expected (121) and decline in the infant faster (122) and for a shorter duration than expected (63).
These data highlight that preferential transfer of SARS-CoV-2 specific IgA and IgM and/or IgG in breast milk to newborns can create a non-pathological, but highly protective barrier against COVID-19 disease in breastfed infants. Unfortunately, some studies have focused on the presence and characteristics of breast milk antibodies, and incomplete information about newborns has weakened the support for the protective effect of antibodies on infant and child health. Also, these studies did not consider the effect of drugs used to treat COVID-19 on breast milk antibodies. Larger sample sizes and long-term follow-up studies with longterm follow-up of antibody class, duration, and titer changes are needed to better understand the association of SARS-CoV-2 antibodies in milk with COVID-19 outcomes in infants. Breast milk is rich in nutrients (123) and since it requires no preparation and is always at the right temperature, it helps to provide a complete, balanced, adequate, and proper diet (124). Not only is it the gold standard source of nutrition for infants, but it is the primary source of passive and active immunization for newborns (81), and breastfeeding can reduce neonatal mortality (125). The role of breastfeeding in protecting infants from infection is also crucial. The immaturity of an infant's immune system at birth increases the risk of infection from external factors, including viruses and bacteria (126), and breastfeeding can significantly reduce the risk of infection in infants (127). Breast milk contains antibodies, linoleic acid (128, 129), milk fat globule membranes, human milk oligosaccharides, osteopontin, and other antiviral components (130, 131). These nutrients bind to some of the receptors required for viral entry into host cells, limiting the ability of viruses to enter and achieving antiviral [including papillomavirus (132), human immunodeficiency virus (133), rotavirus (134), chikungunya virus (135) and Zika virus (135)], antiparasitic (136) and antifungal effects (137).
In addition, the large presence of bioactive mediators in breast milk [IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12, IL-18, IFN-γ, TNFα, TGF-b, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF)] (138, 139), which can compensate for the lack of cytokine pool in newborns. On the other hand, the benefits of breast milk are much broader, considering the emotional, cognitive, psychological, socioeconomic, and environmental development of the child and mother (140,141). Mother-infant separation hurts the physical and mental health of both mother and infant (142), and separation of mother and infant after birth is considered a source of stress, leading to an increased incidence of developmental problems in children (143). For mothers, early cessation of breastfeeding is a significant cause of maternal depressive symptoms in the postpartum period (144). In addition, a reduced risk of breast cancer or reproductive system cancers has been observed in breastfeeding women (145). Breastfeeding mothers are less likely to develop hypertension, diabetes, hyperlipidemia, cardiovascular disease, and metabolic syndrome (146, 147).

Strengths and limitations
To our knowledge, this is the largest systematic review of SARS-CoV-2 and its antibodies in breast milk to date, with the largest number of included studies, the most comprehensive assessment parameters, and the most thorough assessment of virological evidence. The studies that examined nucleic acids included different time points of breast milk collection, included cases at different gestational weeks, and had more representative findings. The geographic distribution of the included study population was globally representative (21 countries including Arab countries). In studies examining antibodies, the presence and effectiveness of antibodies in breast milk against the virus were linked to the clinical outcomes of newborns. Our review answers a series of real-world questions of great relevance, particularly those that physicians and mothers are eager to answer. These data will have implications beyond the epidemic, providing objective, evidence-based data for developing optimal strategies for breastfeeding and maternal and infant management in similar epidemic pandemics in the future.
Our study also has some limitations. First, most studies did only qualitative SARS-CoV-2 RNA analysis and lacked precise viral load assessment, and only two studies performed cell culture. Second, most studies were retrospective, observational, and cross-sectional, and some studies did not focus on the presence of nucleic acids or antibodies but simply reported nucleic acid or antibody results, and these limitations limited our ability to study the long-term health effects of breastfeeding on infants born to SARS-CoV-2-infected mothers. Third, the small size of the sample for most of the studies, which included 33 case reports and nine case series. Despite repeatedly reading and analyzing the included literature, we could not confirm that we had excluded all potentially repeatedly reported cases. Also, there is a risk of publication bias given that COVID-19-positive cases in infants and breast milk are most likely to be reported and published. Finally, given the importance of summarizing all available cases, we did not assess the quality of the studies included in this review.

Conclusion
In conclusion, regardless of whether the breast milk of confirmed COVID-19 mothers was positive or negative for SARS-CoV-2 RNA, direct breastfeeding did not pose an additional risk of infection to the neonate, and in any case, neonatal outcomes were favorable, with no neonatal COVID-19related deaths and good health outcomes for all infants in all studies included in this review. Also, breast milk secreted by infected mothers is a beneficial source of anti-SARS-CoV-2 antibodies that neutralize the activity of SARS-CoV-2. Not only does direct breastfeeding not exacerbate the severity of the disease, but it may provide passive immune protection to the infant. In addition, direct breastfeeding would benefit the mother by reducing depression and anxiety in the mother. Notably, given the potential for respiratory or contact transmission (horizontal transmission) of SARS-CoV-2 during breastfeeding, breastfeeding should be conducted under appropriate infection control guidelines.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Funding
The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.