Records of antibodies in breast milk in postpartum women who have been vaccinated or exposed to COVID-19: A systematic review

Background: Breast milk is a critical element in developing a baby’s immunity through immune transfer. Antibodies are an essential unit of immunity against infection with the SARS-CoV-2 virus. This paper explores antibodies in breast milk in postpartum women who have been vaccinated or exposed to coronavirus disease 2019 (COVID-19). Duration of antibody appearance was studied to determine the adequate time in transferring antibodies by breastfeeding. Methods: Three databases, PubMed, Google Scholar, and ScienceDirect, were used as sources of articles. Inclusion criteria applied in selecting articles were prospective observational study or experimental design study in English, evaluating antibodies in breast milk, and conducted between 2019–2021. Article quality and risk of bias were assessed with Critical Appraisal Skills Programme (CASP). The data found were synthesized in a narrative manner. Results: This systematic review included 20 articles. A total of 306 postpartum women who were infected with COVID-19, 20 postpartum women who had viral symptoms and 495 postpartum women who had been vaccinated were studied. Immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies were found in the breast milk of infected and vaccinated postpartum women. SARS CoV-2 infection is associated with the presence of IgA dominant, whereas vaccination is related to the presence of IgG dominant. Antibodies persisted from day 10 of onset to 10 months in infected postpartum women and started from three days to six weeks in vaccinated postpartum women. Meta-analysis could not be carried out due to the variety of articles. Conclusions: Antibodies found in breast milk in infected and vaccinated postpartum women have different dominant types. Further research needs to be done regarding the mechanism of antibody transfer in breast milk, longer research duration and studies that directly examine the comparison of antibodies in breast milk in vaccinated and infected postpartum women. Registration: PROSPERO ( CRD42022340859, 23 June 2022).

Introduction SARS CoV-2 became a worldwide pandemic and caused changes in all aspects of life including in mothers with postpartum and neonatal period. 1 Coronavirus disease 2019 (COVID-19) infection during pregnancy, birth and postpartum is associated with a significant increase in morbidity and mortality in mothers and babies.New mothers and their infants are more susceptible to infection compared to the general population. 2 COVID-19-related postpartum deaths have been reported in Brazil, with an estimated 106 deaths in 2020. 3In addition, babies are also vulnerable to COVID-19 infection.The babies born to mothers infected with COVID-19 are more likely to be admitted to the neonatal unit. 4Case overview of COVID-19 in children in previous studies were seen to be less frequent, less severe, and the mortality rate very low, but there is growing evidence that they are as susceptible as adults. 5Infants with severe respiratory failure and the prolonged clinical course associated with SARS-CoV-2 exposure may be due to extreme prematurity, immature lungs, and immunocompromised status. 6east milk is the only food needed in the first 6 months of life. 7Breastmilk also the best source protection for babies. 8reast milk contains various kinds of antibodies that provides protection to the baby.One way for babies to get additional antibody protection is through vaccination.In the case of COVID-19, vaccination is one of the main protections but the provision of a COVID-19 vaccine for newborns and babies are not yet available.A recent study found that a COVID-19 vaccination regimen consisting of BNT162b2 was found to be safe, immunogenic, and efficacious in children aged five to 11 years. 9When compared with infants, postpartum mothers have the opportunity to get antibodies through vaccination.The COVID-19 vaccine is an effective way to prevent COVID-19. 10In influenza outbreaks in previous years, influenza vaccines have been shown to increase serum antibodies and reduce disease severity in both mother and baby.
Breastfeeding can protect for at least six months because breast milk contains consistently high levels of actively produced anti-influenza immunoglobulin A (IgA).The results of this research found that breastfeeding can provide protection for at least the first 6 months because breast milk contains consistently high levels of actively produced antiinfluenza immunoglobulin A (IgA).Infants with fever have fewer episodes of respiratory illness, which implies that breastfeeding may provide local mucosal protection. 11The risk of COVID-19 can be reduced through breastfeeding among children as has been documented for other infections compared to formula feeding. 12e risk of being exposed to the virus and providing nutrition is a dilemma, especially regarding the content of substances contained in breast milk.Breastfeeding mothers do not understand the substances contained in breast milk, whether viruses or protective substances when giving it to their babies.This condition also exacerbated by the rise of misinformation on social media about the content of the SARS-CoV-2 virus in breast milk, causing breastfeeding issues during the pandemic. 13Breastfeeding mothers will be distraught and ask themselves whether the coronavirus can be transmitted through breast milk and what they can do to protect themselves and their babies. 8The World Health Organization (WHO) said breastfeeding does not need to be stopped during COVID-19 infection or after the mother's vaccination. 14Research conducted by United Nations Children's Fund (UNICEF) in five countries in South Asia found that less than 25% of interviewees understood that it was safe to continue breastfeeding.They preferred to give formula milk, 15 whereas a meta-analysis conducted in 2020 showed that the SARS-CoV-2 genome is generally not found in the breast milk of breastfeeding mother infected with COVID-19. 16After the virus content information has been clearly explained, the protection points become an essential part to know.Previous studies have found that there are antibodies in the breast milk of COVID-19-infected mothers, but the duration of the presence of these antibodies is unknown. 17nfected and vaccinated postpartum women allow the formation of an antibody against COVID-19, but the long-term impact on antibody composition and functional activity is unclear. 18Given this context, the purpose of this research is to determine the status of antibodies in breast milk following COVID-19 infection and vaccination.After finding records of the presence of antibodies, the duration of the appearance of antibodies will also be studied.Knowledge about the duration of antibodies in breast milk determines the adequate time in transferring antibodies by breastfeeding.The current results can be used as a guideline for recommendations to continue breastfeeding and health promotions that emphasize the presence of antibodies in breast milk in vaccinated postpartum mothers.

REVISED Amendments from Version 3
There is an addition to the discussion regarding the effect of post-vaccination examination time on antibody levels.We also add a discussion on the relationship between antibodies in the blood and human milk.
Any further responses from the reviewers can be found at the end of the article

Study design and search strategy
The status of antibodies in breast milk after exposure to COVID-19 and after vaccination and the duration of antibodies appearing in breast milk were explored by compiling a systematic review.The preparation of this report follows the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. 19,39This systematic review is registered in PROSPERO (CRD42022340859, 23 June 2022).Data were collected in July-September 2021.English-language research conducted from 2020 to 2021 according to topics was searched in PubMed (RRID: SCR_004846), Google Scholar (RRID:SCR_008878), and Science Direct.The keywords used were a combination of Medical Subject Heading (MeSH) terms and relevant keywords in a different order: "breast milk", "COVID-19", "antibody", "immunoglobulin", "vaccine", "severe", "acute respiratory syndrome coronavirus 2", "coronavirus disease 2019", "SARS-CoV-2".The author also uses synonyms in the search.In the articles found, the researcher also examined the literature in the bibliography, including manuscripts that were not captured in the electronic literature search.

Study selection
The inclusion criteria applied in selecting articles were prospective observational study or experimental design study in English, evaluating antibodies in breast milk, and the study was conducted during the COVID-19 pandemic between 2019 and 2021.The exclusion criteria for this study were case reports, animal studies, letters to editors, study reviews, abstracts without full text.Manuscripts that only discussed breast milk in healthy postpartum or breastfeeding mothers were not included.

Data collection process
Two authors (EMK and NAR) performed title and/or abstract screening independently of the included articles using standard Microsoft Excel (RRID:SCR_016137) forms.The data obtained were combined in one folder and then an assessment was carried out.Each author analyzed all existing manuscripts, and then the results were compared with each other.A third external collaborator was consulted (Hari Paraton) to address disagreements in consensus.

Data items
The outcomes from this study were the presence of antibodies and the duration of the appearance of antibodies in breast milk.The antibodies studied were IgG and IgA against COVID-19.The tool used to detect the presence of antibodies is ELISA or similar tools.The authors realized that not all manuscripts had explanations for these two antibody levels, therefore the studies that only discussed one type of antibody were also included in the systematic review.The authors also realized that not all studies that discussed antibodies also explained the duration of the appearance.Based on the consideration of the limited number of articles, the authors discussed the articles that discusses the time when antibodies can be detected.Unclear information was included in the exclusion criteria.

Study risk of bias assessment
Authors conducted a risk of bias assessment study using critical appraisal tools.The Critical Appraisal Skills Programme (CASP) was used to assess the formal article by two independent research team members, EMK and NAR.The use of CASP is based on the 1994 JAMA 'Users' guides to the medical literature, which is used for both randomized controlled trials and systematic reviews.This checklist was adapted from Guyatt GH, Sackett DL, and Cook DJ and is used by health care practitioners. 20CASP results are concluded into the category of moderate overall quality and low overall quality. 21he results of the analysis are presented in Table 1.

Effect measures
Due to the limited number of manuscripts, a meta-analysis could not be completed.The effect that was measured in this study was the number of respondents' breast milk presentations where antibodies were found in breast milk.The obtained durations were also combined descriptively.

Data abstraction and synthesis
The study selection process was through a review of the inclusion and exclusion criteria.As suggested in a systematic review of the literature, the analysis in this study was based on the findings of each study.The steps started from extracting the relevant results, sorting, and examining them to identify sub-themes and themes.The type of synthesis used is in the form of narrative synthesis.Narrative synthesis was chosen because it allowed authors to gather insight into the antibody content in breast milk and we did not perform this meta-analysis.The data are arranged in Table 2   Further information for the question "What were the results of this study?"has been presented in Table 2 and Table 3.

Reporting bias assessment
The researcher was unable to form the funnel plot due to the limited number of the manuscript.Funnel plots have a similar function to forest plots.This plot serves to assess the intervention effect of individual studies on some measure or precision of each study.The risk assessment of bias in this review was carried out using a risk approach that emerged and was reported as a research limitation.

Certainty assessment
The main domain used to assess the certainty of evidence was the risk of bias through critical appraisal and the inconsistency of the results of the research included in the review.

Results of article screening
In searching the database, 1,465 abstracts were found from searches with relevant keywords.The authors then screened results for possible inclusion.The findings of 935 articles were excluded from the eligibility list.The articles did not meet the criteria, including the type of manuscript, the language used, and the topics discussed under the proposed title.After this stage, the researcher tried to re-examine the assessment results, but the manuscript finally entered the exclusion criteria.After screening, 30 full-text articles were selected and examined in detail to determine eligibility.Furthermore, 20 articles were determined that met the requirements.There were eight studies on the antibody status of infected breastfeeding mothers and 12 studies on antibody status of vaccinated breastfeeding mothers included.Figure 1 shows the study selection flowchart.The quality of the research is in the moderate category and is arranged in Table 1.Antibody status in breast milk from infected breastfeeding mother A total of 306 breastfeeding mothers who were positive for COVID-19 and 20 of them who had viral symptoms and had a very high likelihood that they had been exposed to COVID-19 were studied to determine antibody status in breast milk.Samples were taken within 48 hours after delivery to six months after infection.Table 2 describes characteristics of research on breastfeeding mothers infected with COVID-19 and antibody status in breast milk.The samples included in this review are breastfeeding mothers with positive PCR confirmation and mothers who had a very high likelihood that they had been exposed to COVID-19 when viewed from the symptoms they had.Almost all studies used controls, namely comparisons with healthy breastfeeding mothers and breastmilk samples taken during the pre-pandemic period.One study did not include controls, namely a longitudinal study.Detection of all antibodies was performed using ELISA.Most studies found that breast milk contains immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM).IgA was the dominant antibody found, while IgG and IgM were not always found in individuals.However, infected breastfeeding mothers did not always have IgA antibodies against COVID-19, which was found in a study by Juncker et al. (2021), who found that 59% of mothers had these antibodies. 22tibody status in breast milk from vaccinated breastfeeding mother The characteristics of research on vaccinated breastfeeding mothers and antibody status in breast milk can be seen in Table 3.A total of 495 breastfeeding mothers who had been vaccinated, received the first and second doses of various types of vaccines and had never had COVID-19 were analyzed.IgG and IgA antibodies were found in the breast milk of vaccinated mothers.No studies showed IgM data.Administration of the second dose of the vaccine was associated with an increase in IgG in breast milk.Despite evidence of increased antibodies, anti-SARS-CoV-2 IgG antibodies were not detected in the plasma of infants whose mothers were vaccinated during breastfeeding. 30This study is one of 20 studies examining infant plasma.In the study, infant blood was collected with a heel stick at 5-15 weeks after breastfeeding mothers were vaccinated the second dose.Eight blood samples were collected from infants aged 68 days to 1 year.IgG levels were higher than IgA levels in the breast milk of vaccinated breastfeeding mothers.

Time when antibodies can be detected
In mothers infected with COVID-19, the appearance of antibodies was detected on day 10 and could persist for up to 10 months.IgA levels are known to decrease over time, while IgG is relatively stable.In vaccinated mothers, antibody status differed between mothers vaccinated with first dose and second dose Most studies found the presence of antibodies after the first dose, although in small amounts.Early appearance could be detected three days after vaccination up to six weeks in both first dose and second dose of vaccination.The highest levels and immune response were found about four weeks after vaccination, but at the second dose, the antibody appeared between days four and 10.After that, there may be a chance to experience a decline before the sixth week, especially on day 15 or day 43 AE 4. These studies stopped examining antibodies at the sixth week.Therefore, the data provided are limited to detection at the sixth week.

Comparison of antibody levels by type of vaccine
Not all studies address differences in antibody levels based on the type of vaccine received.There are four studies discussing antibodies in blood and breast milk samples.Research that discusses the comparison of antibodies in breast milk conducted by Lechosa-Muñiz et al. (2021), 23 Selma-Royo et al. (2021), 24 Gray et al. (2021) 25 and Valcarce et al.
(2021). 52The mean antibody titers found in breast milk were found to be different for each vaccine type, 0.41, 0.45, and 0.09 (AU) for mothers who received BNT162b2, mRNA-1273, and ChAdOx1-S (one dose), respectively.No differences could be found between those vaccinated with BNT162b2 vs. mRNA-1273. 23Research conducted by Selma-Royo et al.
(2021) found that mothers vaccinated with Moderna and BioNTech/Pfizer after the first dose had a higher increase in breast milk anti-SARS-CoV-2 IgG than Oxford/AstraZeneca (p < 0.0001) and BioNTech/Pfizer (p = 0.002 ).In addition, the number of doses received also determines the amount of antibodies present in the breastmilk.COVID-19 vaccination induced anti-SARS-CoV-2 IgA and IgG in breast milk with higher levels after the 2nd dose. 24This is consistent with the study of Gray et al. (2021) who found that booster doses of the vaccine increased SARS CoV-2 IgG, but not IgA in breast milk. 25Another study conducted by Valcarce et al. (2021) found that both mRNA vaccines produced statistically significant SARS-CoV-2-specific IgA and IgG in breast milk and plasma. 52 serum, when analyzing IgG, the mean antibody titres observed in the serum of lactating mothers were different according to the type of vaccine they received, being 0.32, 0.30, and 0.16 (AU) for mothers who received BNT162b2, mRNA-1273, and ChAdOx1-S (one dose), respectively.When analyzing IgA, the mean antibody titres observed in the serum of lactating mothers showed differences according to the type of vaccine administered: 0.12, 0.16, and 0.02 (AU) for mothers who received BNT162b2, mRNA-1273, and ChAdOx1-S (one dose) vaccines, respectively. 23In the comparison of antibodies from samples in the maternal sera and umbilical cord in a study conducted by Gray et al.
(2021) found that neutralizing antibody titers were lower in the umbilical cord than maternal sera, although this proved not to be statistically significant (p = 0.05).Vaccine booster doses increase SARS CoV-2 IgG, but not IgA, in blood. 25he mean of SARS-CoV-2 IgG in plasma was a significantly higher at post second vaccine dose in mothers vaccinated with Pfizer vs. mothers vaccinated with Moderna (p = 0.005). 52The type of vaccine, the number of doses, and the location of sampling have a role in the number of antibodies found.

Discussion
The antibody status in the breast milk of breastfeeding mothers who have been exposed to COVID-19 or are highly suspected and those who have been vaccinated against COVID-19 differs.The dominant antibody in mothers infected with COVID-19 is IgA, while the predominant antibody in vaccinated mothers is IgG, according to Young et al. (2021). 18accination is associated with an increase in dominant IgG and IgA antibodies after direct viral infection.These results differentiate antibodies in breast milk between vaccinated and infected mothers with COVID-19.Breast milk from both groups, namely mothers who are infected with COVID-19 and mothers who are vaccinated showed neutralizing activity against live SARS-CoV-2 virus, which could be attributed to SARS-CoV-2 IgA and IgG antibodies. 18The dominant IgA response in breast milk can be found according to previous studies, namely the result of natural infection. 26tibody transfer through breast milk is an evolutionary strategy for enhancing immunity early in life. 27Research conducted by Pullen et al. (2021) in which they applied a serologic system to characterize SARS-CoV-2 specific antibodies in maternal serum and breast milk found a preferential transfer of antibodies capable of causing neutrophil phagocytosis and neutralization.Distinct SARS-CoV-2-specific antibody response was observed in serum and breast milk from individuals previously infected with SARS-CoV-2, with a predominant transfer of IgA and IgM into breast milk. 27IgA is the most essential class of Ig provided by breast milk to infants because it acts in the intestines while the function of secretory IgA (SIgA) in infants is still in development. 28The presence of IgM antibodies was found in this study although not all studies examined the presence of IgM even if only in a small sample.The presence of IgM in some samples suggests the possibility that breast milk may have a protective effect on the newborn. 29though IgG are present in breast milk, they are functionally attenuated. 27Emerging data from vaccinated pregnant and lactating women suggest that vaccine-induced transfer may be altered due to unusually high levels of IgG antibody induced by mRNA vaccines approved by the current Emergency Use Authorization. 25 Vaccine gives the baby strong IgA and IgG antibodies and may increase immunity compared to natural infection. 27Nevertheless, the IgG transfer scheme still needs to be studied further because this study has not been able to find out about the transfer mechanism.A study conducted by Golan et al. (2021) found that no IgG was found in the plasma of infants whose mothers were vaccinated during lactation. 30Although high levels of IgG were found in breast milk, these antibodies may not be transferred effectively to the baby.IgA antibodies produced after vaccination with the Pfizer/BioNTech vaccine resist the gastric phase but are degraded during the intestinal phase of the infant's digestion.By contrast, IgG is more susceptible to degradation in both digestive phases. 31The results of another study found that maternal SARS-CoV-2 IgG was efficiently transferred across the placenta when infection occurs more than two months before delivery.Passive immunity inherited from the mother can last in infants for up to six months.Neonates can mount a strong antibody response to perinatal SARS-CoV-2 infection. 32Antibodies shown from breastfeeding may have a protective effect on the recipient infant, provided that the infant has not increased its immune response to infection. 33udies showing the duration of antibody persistence in breast milk are limited.Antibodies can be detected in mothers who are breastfeeding and infected with COVID-19 as early as day 10 and can last for up to 10 months.IgA levels are known to decrease over time, while IgG is relatively stable.In lactating mothers who were vaccinated, the trial was only up to six weeks in duration.Antibodies have been detected after the first dose of vaccination for three days and can last up to six weeks.Before six weeks, it can decrease.It is not known whether the antibody status will still decrease or persist at a certain point.Individual research by Young et al. (2021) showed that IgG began to decline by 90 days after the second vaccine dose. 18This suggests that further research is needed to investigate the exact duration of antibodies in breast milk after vaccination.
The type of vaccine is related to the number of antibodies detected in breast milk even though IgG status is more dominant than IgA and IgM.Post-vaccination IgG levels reached levels similar to those of terminally ill COVID-19 patients and demonstrated a decreased breadth of antibody responses targeting the endemic coronavirus. 34Another study conducted by Dashdorj et al. (2021) found that IgG levels from high to low were Pfizer/BioNTech, AstraZeneca, Sputnik V and Sinopharm, respectively. 35These different responses also allow different amounts of antibodies to be transferred into breast milk.
Based on the results of the opportunity for antibody transfer, breastfeeding is highly recommended for mothers infected with COVID-19 and mothers who have been vaccinated.Research conducted by Verd et al. (2021) found that in a sample of children visiting emergency services with potential symptoms of COVID-19, a higher prevalence of positive SARS-CoV-2 reverse transcriptase (RT)-PCR test results among those who were formula-fed exclusively compared to those who have been breastfed.Breastfeeding may reduce the risk of exposure to COVID-19 and other infections in children, compared to formula feeding. 12It is vital to continue to breastfeed according to the available time duration.Women with confirmed COVID-19 should be advised to adhere standard precautions for contact with breastfeeding. 36ere are clear and up-to-date recommendations on the duration of breastfeeding from the American Academy of Pediatrics, UNICEF and WHO.WHO and UNICEF recommend: early initiation of breastfeeding within 1 hour of birth; exclusive breastfeeding for the first 6 months of life; and the introduction of complementary (solid) foods that are nutritionally adequate and safe at the age of 6 months along with continuous breastfeeding until the age of 2 years or older. 55,56In the first 6 months this is important because the baby's production of sIgA is low during that period. 37Apart from direct breastfeeding, every mother has the right to choose to breastfeed her baby and health workers need to help ensure good handwashing practices before and after expressing the milk. 38e to varying collection times, certain patients' milk and serum samples may contain no Ig A in vaccinated women.The interval between sample and vaccination has a direct bearing on variations in antibody concentrations.Neutralizing anti-SARS-CoV-2 antibodies (IgA and IgG) were examined in breast milk and blood samples from women who had received vaccinations at least 20 days after the immunization cycle ended.This research was conducted by Scrimin et al. in 2022.IgA was absent from all forty-two milk samples, while anti-SARS-CoV-2 IgG was present in all of them.IgA's inherent kinetics could be connected to the quicker fall in IgA.The absence of IgA may suggest a sharp fall following immunization, even if regular breastfeeding would help it last. 57In a study conducted by Esteve-Palau et al. ( 2021), lactating mothers who were vaccinated against SARS-CoV2 with the Pfizer-BioNTech COVID-19 vaccine and who were 18 years of age or older were included.The study examined the levels of SARS-CoV-2-specific antibodies in the breast milk of mRNA-vaccinated women over time and their correlation with serum antibody levels.Each participant had serum and breast milk samples taken simultaneously at three different time points: two weeks following the first vaccination dose (time point 1), two weeks following the second vaccination dose (time point 2), and four weeks following the second vaccination dose (time point 3).Following the second dosage, breast milk's IgG(S1) levels rose and showed a positive correlation with matching serum levels. 58table is the link between antibody concentrations in blood and milk.A study conducted on animals revealed that during lactation, there were alterations in blood and milk metabolism.Milk has the potential to predict blood metabolites and metabolic condition, as confirmed by the association between blood and milk. 59The antibodies, or immunoglobulins, present in milk and colostrum are identical to those present in blood or mucosal secretions.According to Hurley (2011), they are a class of proteins with a variety of defensive bioactivities. 60

Limitations and recommendations
Most of these studies only involve a small number of samples, and they did not find a mechanism for antibody transfer during breastfeeding.We recommend that future research explores antibody testing with a more extended research duration (more than six weeks) in vaccinated postpartum women.In order to ensure that the reported time is more accurate, multiple individual studies that directly discuss the comparison of antibodies in vaccinated mothers with mothers exposed to COVID-19 and the assessment of antibody transfer mechanism by breastfeeding may be useful.In addition, the researcher was unable to compile a meta-analysis because it included various types of academic papers resulting in a lack of homogeneity.
The strength of this study is that there have not been many studies that have reviewed the comparison of antibody types and duration of antibodies in infected and vaccinated postpartum women.The results obtained can support evidencebased health promotion to support continued breastfeeding during the pandemic and allow for the right time to support antibody transfer.

Conclusions
IgA and IgG antibodies were found in the breast milk of infected and vaccinated postpartum women.Infection with SARS CoV-2 is related with the presence of IgA, but vaccination is associated with increased IgG.Antibody levels persisted from day 10 of onset to 10 months in infected breastfeeding mothers and start from three days to six weeks in vaccinated breastfeeding mothers.Antibodies produced in breast milk in infected and vaccinated postpartum women have different dominant types.Further research needs to be done regarding the mechanism of antibody transfer in breast milk, longer research duration and studies that directly examine the comparison of antibodies in breast milk in vaccinated and infected postpartum women.Be consistent throughout the manuscript and tables when describing the population.
Determining the difference between exposed to COVID-19 or infected with COVID-19 (or even suspected infection with COVID-19).This point is important as many people that are exposed may not know they are exposed and if asymptomatic will likely not get tested.Thus, simply exposed does not seem like the description that describes the majority of the population in these 20 studies.Be consistent in the terms of infected or exposed.For example, the title of Table 2 is "Characteristics of research on mothers with COVID-19 and antibody status in breast milk" but on Page 4 under the section Data abstraction and synthesis, "  2 to "Characteristics of research on mothers infected with COVID-19 and antibody status in breast milk".Furthermore, I would not refer to Table 2 in the text as mothers who had been exposed.

2.
Recommend removing or modifying certain points that are not necessary to the flow of the manuscript.)."This is too strong of a statement since the cohort was retrospectively reviewed in a secondary analysis and used "Any breastfeeding" vs. exclusive formula feeding and these were in children up to 13 years of age.The conclusion section uses the word suggest that there is protection with breastfeeding.Thus, I recommend changing the work "can" reduce to "may" reduce.

4.
Important point to consider -Mechanism of antibody transfer in the breast milk.I am uncertain what the meaning of this statement is.As per World Health Organization, the presence of IgA in breast milk is one of the ways in which breastfeeding protects infants against infection and death.This appears to be a big part of the conclusion section for future direction but it is not clear that it is based on the results presented.

5.
Tables Table 1: The question of How precise are the results?Is not a yes/ no question.Per CASP checklist for cohort study the included HINT was Look for range of confidence interval.I would also include p values if given.If not available, list "no data available" for that particular study.This is important for the statistical analysis portion.After you can compare  It would be informative to see an addition of antibody breakdown by type added to Table 3.

Figures
Figure 1 is the Flow chart of the study selection but Figure 1 is mentioned under Results subheading Time duration of antibodies appearing and is described as showing "Duration of antibody time associated with PCR confirmation or symptoms, breast milk testing, and vaccine administration.Was this supposed to be labeled as Figure 2. If so, I didn't see a Figure 2.
Figure 1 Flowchart is confusing because the numbers do not add up as they are explained.It appears that from the 1465, 500 were eliminated to leave 965.Then another 935 were eliminated to leave 30.Next 10 were eliminated to ultimately leave 20 studies.Each elimination should be explained clearly and the numbers should match up.Minor point: From 30 to 20 in the last 2 boxes in the flowchart reports excluded explained 11 that were eliminated but 30-11=19, not 20.Please revise this flowchart using an example from another systematic review.

Small revision points:
In methods on keyword search, it seems that "breast milk" and "COVID-19" were typed twice as an error.Please fix this.These are details of the methods and analysis to allow replication by others.
What is meant by "unclear information was included in the exclusion criteria"?Is this for a few of the studies that were not included or is this describing the majority of the 20 studies that were included?
What is a funnel plot?If you mention it, you may want to explain it.
Page 11, Results section, Heading Antibody status in breast milk from vaccinated, second to last sentence, Serum levels in infants appears to only be included in one study, so please provide the reference for that study as a superscript and also mention it was only one of the 20 studies.Also, how many infants had serum levels?Not many mothers would want you to poke their babies to get a blood draw.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
= Critical Appraisal Skills Programme, Y = yes, C = can't tell, N = No, N/A = not applicable, M = moderate overall quality, L = low overall quality.

6 .24 of 27 F1000Research 2024, 11 :
Page 785 Last updated:15 JUL 2024      and contrast studies within each group of vaccinated or infected mothers.

Table 1 .
The results of the critical appraisal using CASP checklist.

Table 3 .
Characteristics of research on vaccinated breastfeeding mothers and antibody status in breast milk.

Table 3 .
Continued antibody status in breast milk and is written in a systematic table including (1) authors, year, country, (2) research time, (3) study type and sample timing, (4) vaccine type, (5) number of vaccinated women and the dose given, (6) antibody test, (7) type of antibody, (8) finding, and (9) duration time.Heterogeneity in the data was explored including duration of baseline antibody measurement and sample characteristics.
Note: COVID-19 = coronavirus disease 2019, IgA = immunoglobulin A, IgG = immunoglobulin G, IgM = immunoglobulin M.and Introduction section with confusing sentences (page numbers refer to the pdf version of the manuscript): Page 1, 1 st paragraph, First line: …and caused changes in the phases of life.In what ways?Page 1, 1 st paragraph, 3 rd sentence: Consider changing to New mothers and their infants are more susceptible to infection compared to the general population.Page 1, 2 nd paragraph, "Breast milk is the only nutrient needed in the first 6 months of life."Did you mean only nutrition?Page 1, 2 nd paragraph, "Breast milk can be an opportunity and hope for babies to achieve protection".Would take out hope to make it more scientific.Page 11, 2 nd discussion paragraph, "IgA is the most essential glass of Ig provided by breast milk…produced by infants is still in development."What is still in development?"The presence of IgM in some samples (breast milk) suggests the possibility that breast milk may have a protective effect on the newborn."In what way in the context of this systematic review?Page 11, 3 rd paragraph, 3 rd sentence, "It gives the baby strong IgA and IgG antibodies and may increase immunity comparted to natural infection."What is "it"?mRNA vaccines?If so, say these vaccines or mRNA vaccines.Page 12 of discussion first sentence, "Needs to be studied further because different studies can find different results."This sentence is too vague.Do you mean that studies report conflicting results?If so, say that.Page 12, 3 rd paragraph , Dashdorj et al. 2021, which antibody?
Table 2 is antibody contact in mothers exposed to COVID-19" but later on Page 10 under Antibody status in breast milk from infected other."Table 2 disuses antibody status in mothers who had been infected with COVID-19.Based on the majority of the studies confirming COVID-19 with PCR (with the exception of 1 study by Bauerl et al. 2021 which had CPR and/or after COVID-19 infection), I would advise changing the title of Table " and also cite WHO, CDC and AAP recommendations for precautions.Page 12, 4 th paragraph, I would update the sentence "The need to encourage breastfeeding may be justified at least during the first 6 moths of life, when the infant's secretory IgA production is insignificant."There are clear and updated recommendations on the duration of breastfeeding from American Academy of Pediatrics, UNICEF and WHO: which all say exclusive breastfeeding for first 6 months and then up to 2 years of age.You could then highlight the importance of the first 6 month based on infant's low production of sIgA after discussion the general breastfeeding duration recommendations.
Page 12, 4 th paragraph, I would remove the sentence "Therefore, women with confirmed COVID-19 must adhere to standard precautions for contact with breastfeeding."Contact precautions were not previously discussed and may be confusing or send unclear messages.If it is strongly desired to keep this sentence, would change must to "should be 3. advised

Table 2 :
To make it more consistent for no. of infected mothers, just include the numerical value, (two are in words).

Table 2 :
Bauerl et al. sampling time is listed as before and after.How many days or weeks?Also how is infection defined in this study, if not by PCR?

Table 2 :
last entry Demers-Mathieu et al lists sampling time as 16-84 days.What is that in relation to?Table 2 Heading Duration time: What is this the duration of?Follow up with patients?Table 3 Heading Time: of what?Timing after vaccination when antibody levels are measured?

Table 3 :
Perl et al: which antibodies under "Antibody status"?

Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes Are sufficient details of the methods and analysis provided to allow replication by others? Partly Is the statistical analysis and its interpretation appropriate? No Are the conclusions drawn adequately supported by the results presented in the review? Partly If this is a Living Systematic Review, is the 'living' method appropriate and is the search schedule clearly defined and justified? ('Living Systematic Review' or a variation of this term should be included in the title.) Partly Competing Interests:
Page 11, Results section, heading time duration of antibodies appearing, Last sentence is an interpretation of results thus, belongs in discussion section (which you already included on page 12, 2 nd paragraph), so you can delete this sentence.Limitations and recommendations section: instead of the last sentence, "…because it included various types of academic papers."Consider saying lack of homogeneity.Why do Pace et al. and Fox et al. not fit with other available evidence?This should be explained in the discussion section.Personally, from Table 2 the Demers-Mathieu et all paper has all 26 patients with all antibodies in milk including IgA, IgG and IgM.I don't believe those results.Do you? References 1. Meek J, Noble L, Section on Breastfeeding: Policy Statement: Breastfeeding and the Use of Human Milk.Pediatrics.2022; 150 (1).Publisher Full Text No competing interests were disclosed.