Profile of pro-inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age.

Gestations at the extremes of reproductive age are characterized as high-risk pregnancies, conditions that might influence colostrum composition. This first milk secretion contains nutrients necessary for the development and immunity of the newborn; therefore, this study aims to compare adolescent and advanced maternal age mothers regarding sociodemographic, gestational, and perinatal characteristics and the colostrum levels of pro-inflammatory cytokines in these groups of study. This cross-sectional study has compared sociodemographic, gestational and perinatal data from adolescent mothers (between 10 and 24 years old) (n = 117), advanced maternal age mothers (over 35 years of age) (n = 39) and mothers considered a control group (25 to 34 years old) (n = 58). Additionally, colostrum samples were obtained from the studied and control group subjects by manual milking, between 48 and 72 hours postpartum, and the samples were analyzed for cytokine concentrations by enzyme-linked immunosorbent assay (ELISA). The majority of the studied mothers reported living a stable union, and 81.2% of the adolescent mothers did not carry out any paid activity. Mothers with advanced maternal age mainly delivered by cesarean section and presented a higher body mass index (BMI). Neonatal weight and Apgar score were not different between the groups. The concentrations of interleukin (IL)-1β and IL-6 were higher in the colostrum of mothers with advanced age compared to adolescent mothers, but did not differ from the control group. The concentrations of IL-8 and tumor necrosis factor alpha did not differ between the three groups. Therefore, our data demonstrated that maternal age influenced the sociodemographic and gestational characteristics as well as the composition of colostrum cytokines.

Introduction

Gestations at the extremes of reproductive age are have become more frequent in the last decade, and such conditions are characterized as high-risk pregnancies, defined as "those in which the life or health of the mother and/or the fetus and/or the newborn are more likely to be affected than the mean population considered” [1]. In this context, adolescence pregnancies present higher rates of obstetric complications, and are also considered a public health problem in some developing countries, as they carry social and biological outcomes, for example dropping out of school, social isolation, temporary or permanent education interruption, emotional instability and a stable union with a partner at an early stage of life [2,3,4].

Furthermore, the perinatal results from adolescent pregnant women can be adverse, mainly due to their biological immaturity regarding growth and development. Adversities include a greater number of low birth weight newborns (LBW, <2500g), higher risks of intrauterine growth restriction (IUGR), Apgar scores below seven in the fifth minute and, in particular, a higher preterm birth rate (<37 gestational weeks) [3].

On the other hand, pregnancies after the age of 35 years are considered late gestation, which has become increasingly frequent due to aspects such as birth control improvement, advances in assisted reproductive technology, late marriages, increased rates of divorce followed by new unions, women with a higher level of education, and advances in health care [5,6]. However, obstetric complications are observed in the pregnancies of advanced maternal age mothers, including antepartum hemorrhage, pregnancy-induced hypertension, diabetes, IUGR, anomalous presentation, macrosomia, dystocia, prolonged gestation, oligohydramnios, polyhydramnios, and preterm labor [5-7].

The main World Health Organization (WHO) strategy to reduce child mortality is the promotion of breastfeeding around the world [8]. Breastfeeding is considered an age-old practice, with numerous recognized nutritional, immunological, cognitive, economic, and social benefits [9]. Colostrum is the first produced milk secretion, which ordinarily occurs between the second and third postpartum day; the main colostrum components are cytokines, fats, immunoglobulins, proteins, carbohydrates, vitamins, leukocytes, lactoperoxidase, lactoferrin, and lysozyme, as well as hormones and growth-promoting peptides [10-12]. Among these components, the presence of some cytokines is suggested to compensate for the delay in development of the newborn immune system, although the specific role of cytokines is still controversial [13].

Cytokines are soluble glycoproteins that signal intercellular communication, and share several functions with each other. Their presence in human breast milk results in a cascade of effects involved in the regulation of inflammatory response and in the function of the newborn’s immune system, such as their role in maturating the infant intestine [14]. The frequently studied cytokines in breast milk include interleukins (IL)-1β, IL-6, IL-8, IL-10 and IL-12 and tumor necrosis factor (TNF)-α, which are all found in the colostrum and mature milk secretion [13,15-17].

Primarily, IL-1β is an inflammatory mediator, involved in cell proliferation, differentiation, and apoptosis. The concentration of IL-1β in colostrum seems to be constant regardless of the timing of birth; however, in mature milk, its concentration is higher in preterm than in term deliveries. The action of IL-1β is not well known, but it appears to be involved in mammary gland defense mechanisms, including the production of immunoglobulin A and the cytokines, such as IL-6, IL-8, TNF-α, and IL-12 [15-17]. IL-6 is an important pro-inflammatory and anti-inflammatory cytokine produced by keratinocytes, fibroblasts, macrophages, endothelial cells, and T-cells. It is secreted by macrophages and T cells in response to trauma, such as burns or tissue inflammation, with a potent pyrogenic action. IL-6 stimulates the acute phase response, B-cell differentiation, and antibody production by the same pathways in colostrum [12,17].

IL-8 is produced by phagocytes and epithelial cells and is capable of recruiting neutrophils. It is an important component of innate immunity and inflammatory response, has a physiological role in the development and maturation of the gut, and may be involved in the anti-infective protection mechanisms of breast milk. Another role of IL-8 in breast milk is related to newborn protection due to its strong chemotactic activity and activation of the cells involved in allergic diseases [18]. TNF-α is secreted by mammary epithelium and macrophages of breast milk, has a higher concentration in colostrum than in mature milk and participates in the mechanism of defense against infections and trauma [16-17].

Considering that maternal breast milk is the gold standard in feeding all newborns and that the effect of maternal age on breast milk composition is still poorly understood, the objectives of this study were to compare adolescent and advanced maternal age mothers regarding sociodemographic, gestational, and perinatal characteristics and the colostrum levels of pro-inflammatory cytokines in these groups.

Materials and methods

A cross-sectional study was conducted with adolescent mothers and those of advanced maternal age and their newborns who attended the maternity unit of the Regional Hospital of Presidente Prudente, SP, Brazil, from March 2017 to July 2018. The work was approved by the Research Ethics Committee of the Universidade do Oeste Paulista, UNOESTE, Presidente Prudente, SP, Brazil, CAAE 67772617.8.0000.5515. The participants signed the informed consent form and the person responsible for the nursing mother under the age of 18 authorized the participation in the research by signing the Informed Consent Form and the Free and Informed Consent Form according to the Ethics in Research with Human Beings criteria as per Resolution no. 466/2012 of the National Health Council.

Subjects

Nursing mothers and their newborns were recruited using the following inclusion criteria: gestational age at birth between 37 and 41 weeks and negative serological reactions for hepatitis, human immunodeficiency virus (HIV) and syphilis. Twin pregnancies, fetal malformations or loss of data regarding delivery and the neonatal period were the exclusion and/or discontinuity criteria.

Subjects were divided in two groups of study: adolescent mothers (between 10 and 24 years old) [2] and advanced maternal age (over 35 years of age). Mothers aged between 25 and 34 years old were considered the control group.

The weight classification of the newborns was small for gestational age (SGA), appropriate for gestational age (AGA), and large for gestational age (LGA) (weight/gestational age ratio) according to the service protocol.

Colostrum collection

The colostrum was collected by manual expressing, always in the morning (from 8 to 10 am) and in the interval between two feedings, considering the period between 48 and 72 hours after delivery. Half of the samples from each study group and the control group were collected at 48 h postpartum and half at 72 h postpartum in order to avoid variations in the cytokine concentrations over time. A maximum colostrum volume of 10 mL was collected into sterile plastic tubes, immediately transported to the laboratory and then frozen and stored at -80°C until analysis.

Determination of IL-1β, IL-6, IL-8, and TNF-α in colostrum by ELISA assay

Specimens were thawed at 4°C, and a 2 mL aliquot of collected colostrum was centrifuged at 160 g for 10 minutes at 4°C. This allowed the separation of the sample into three phases: cell pellet, an intermediate aqueous phase, and the supernatant containing the fats, which was discarded; the aqueous supernatant was used for the dosage of the cytokines of interest [20]. Commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to quantify IL-1β, IL-6, IL-8 and TNF-α levels in the studied samples. All assays were performed according to the manufacturer’s instructions. Samples in which cytokine levels were estimated to be below the sensitivity of the assay were set as equal to the sensitivity of the assay and those with concentrations at levels above standard curve were diluted and re-assayed. The assays readers were performed in an ELISA automatic reader (Epoch-BioTek, Winooski, VT, USA), at a wavelength of 492 nm. The concentrations of cytokines in the colostrum were calculated on the standard curve obtained with different concentrations of the recombinant human cytokines of interest.

Statistical analysis

Data on the sociodemographic and gestational variables were submitted to the X² or Fisher's exact test, or to the Kruskal-Wallis non-parametric test for comparison between the three study groups, after analysis of data normality by the Kolmogorov-Smirnov test. The concentration of cytokines in colostrum was analyzed by the Kruskal-Wallis test, followed by the Dunn test. Statistical analysis was performed using Graph Pad Prism software, version 6.0 (San Diego, CA), and the level of significance adopted for all tests was 5%.

Results

Sociodemographic and gestational characteristics of the mothers

The sample size was composed of 158 nursing mothers in the control group, 117 in the group of adolescents, and 39 in the advanced maternal age group. The reduced number in the latter group was justified by a smaller number of women who had recently given birth with advanced maternal age in the study period. Regarding pregnancy-related complications, 5 mothers in the adolescent group, 7 in the control group and 4 in the advanced maternal age group presented complications such as hypertension or gestational diabetes.

Table 1 depicts the sociodemographic characteristics of the studied mothers during the period considered. As expected, the median maternal age was statistically lower in the adolescent group (p<0.0001). The predominant marital status was the stable union in all groups (p<0.0001). The unpaid profession was predominant among the adolescent nursing group and paid profession in the advanced maternal age group (p = 0.0002). The variables ethnicity, smoking, alcohol consumption (up to 7 weekly doses), living with smokers, and practicing physical activity (3 or more times a week) did not differ between the groups (p>0.05).

Table 1

Sociodemographic characteristics of adolescent, control, and advanced maternal age nursing mothers included in the study.

Characteristics	Adolescents (n = 117)	Controls (n = 158)	AMA (n = 39)	p
Maternal characteristics
Age (years)#	20 (12–23)a0	29 (24–35)b0	37 (36–46)c0	<0.0001*
Ethnicity ɣ
White	50 (42.7)	76 (48.1)	12 (30.8)	0.14
Non White	67 (57.3)	82 (51.9)	27 (69.2)
Marital status ɣ
Single	41 (35.0)	17 (10.7)	6 (15.4)	<0.0001*
Stable union	76 (65.0)	141 (89.3)	33 (84.6)
Profession ɣ
Paid	22 (18.8)	62 (39.3)	21 (53.8)	0.0002*
Unpaid	95 (81.2)	96 (60.7)	18 (46.2)
Smoking habit ɣ
Yes	6 (5.1)	16 (10.1)	7 (17.9)	0.13
Ex-smoker	18 (15.4)	17 (10.7)	4 (10.2)
Mother living with smokers ɣ
Yes	45 (38.4)	48 (30.4)	14 (35.9)	0.36
Consumed Alcohol ɣ
Yes	3 (2.6)	1 (0.6)	1 (2.6)	0.49
Physical activity practice ɣ
Yes	11 (9.4)	8 (5.1)	2 (5.1)	0.64

# Kruskal-Wallis test, followed by Dunn test for comparison between groups: median (minimum—maximum);

ɣ X2 Test or Fisher's exact test, n (%);

* statistically significant (p<0.05); In lines, values followed by the same letters and the same index do not statistically differ.

Table 2 presents the gestational and obstetric characteristics of the nursing mothers included in the study. Body mass index (BMI) at the beginning and end of gestation was statistically lower in adolescents (p<0.0001); however, the weight gain was higher in this group (p = 0.0004). A higher number of multiparous women was observed in the advanced maternal age group (p<0.0001), which also presented predominant cesarean deliveries rates (p = 0.0001). The number of prenatal visits did not differ between groups (p>0.05).

Table 2

Gestational and obstetric characteristics of adolescent, control and advanced maternal age nursing mothers included in the study.

Characteristics	Adolescents (n = 117)	Controls (n = 158)	AMA (n = 39)	p
Gestational and Obstetric Characteristics
Body mass index (BMI) #
At the beginning of gestation	21.8 (14.3–38.2) a1	25.8 (16.4–49.9) b1	28.7 (19.5–43.0) b1	<0.0001*
At the end of gestation	26.6(19.2–40.50)a2	30.0 (20.4–63.9)b2	31.4 (23.3–51.4) b2	<0.0001*
Weight gain (kg)#	11.9 (-4.3–23.3) a3	9.5 (-6.3–70.0) b3	7.0 (-2.0–32.0) b3	0.0004*
Parity ɣ
Primigravida	80 (68.4)	32 (20.3)	2 (5.1)	<0.0001*
Secundigravida	26 (22.2)	50 (31.6)	6 (15.4)
Multigravida	11 (9.4)	76 (48.1)	31 (79.5)
Number of prenatal consultations ɣ
≤7 consultations	30 (25.6)	47 (29.7)	12 (30.8)	0.70
> 7 consultations	87 (74.4)	111 (70.3)	27 (69.2)
Type of birth ɣ
Vaginal	81 (69.2)	97 (61.4)	12 (30.8)	0.0001*
Cesarean	36 (30.8)	61 (38.6)	27 (69.2)

# Kruskal-Wallis test, followed by Dunn test for comparison between groups: median (minimum—maximum);

X2 Test or Fisher's exact test, n (%);

* statistically significant (p<0.05); In lines, values followed by the same letters and the same index do not statistically differ.

Perinatal outcomes of the newborns

The perinatal results of the nursing mothers’ newborns are demonstrated in Table 3. The New Ballard perinatal characteristics birth weight, weight classification, Apgar score at 1 and 5 minutes of life, and days of hospitalization were analyzed. The perinatal outcomes of the nursing mothers at the extremes of reproductive ages were not statistically different.

Table 3

Perinatal results of newborns of adolescent, control, and advanced maternal age nursing mothers included in the study.

Characteristics	Adolescents (n = 117)	Controls (n = 158)	AMA (n = 39)	p*
New Ballard (weeks, days) #	39w (35w1d – 41w)	38w3d (34w2d – 41w)	38w3d (37w – 40w3d)	0.18
Birth weight (Kg) ¥	3.252 ± 0.431	3.284 ± 0.483	3.297 ± 0.443	0.80
Weight classification ɣ
PIG	7 (6.0)	11 (7.0)	2 (5.1)	0.58
AIG	104 (88.9)	136 (86.0)	32 (82.1)
GIG	6 (5.1)	11 (7.0)	5 (12.8)
Apgar at 1 minute ɣ
≥7	110 (94.0)	154 (97.5)	35 (89.7)	0.09
<7	7 (6.0)	4 (2.5)	4 (10.3)
Apgar at 5 minutes ɣ
≥7	115 (98.3)	158 (100.0)	39 (100.0)	0.18
<7	2 (1.7)	0	0
Days of hospitalization #	2 (2–6)	2 (2–8)	2 (2–3)	0.09

# Kruskal-Wallis test, followed by Dunn test for comparison among groups: median (minimum—maximum);

¥ ANOVA test, followed by comparative test, mean ± SD;

x2 test or Fisher’s exact test, n (%);

* statistically significant (p<0.05).

Colostrum cytokine concentrations

The sample size used for cytokine concentration analysis was 40 in the control group and adolescents group, and 39 in the group of mothers with advanced maternal age, as this was the minimal number of samples needed to detected differences in cytokine levels. Intra-assay coefficient of variation, detection limit and median values of cytokine detection are presented in Table 4. Approximately 10% of the samples presented levels below the limit of detection and the levels of IL-1β and IL-6 were significantly increased in colostrum samples from the advanced age mothers compared to adolescent mothers (p<0.05), but did not differ in relation to the control group. The concentrations of IL-8 and TNF-α did not present differences between the three studied groups (Table 4).

Table 4

Concentration of the evaluated cytokines in colostrum of adolescent, control and advanced maternal age nursing mothers included in the study.

Cytokine (pg/mL) #	Adolescents (n = 40)	Control (n = 40)	AMA (n = 39)	p
IL-1β	18.99 (2.37–96.55) a4	26.28 (0.99–261.0) a4,b4	31.32 (7.15–250.0) b4	0.019*
IL-6	20.73 (2.68–90.87) a5	24.94 (1.62–97.99) a5,b5	42.82 (0.17–195.5) b5	0.04*
IL-8	1276.0 (51.71–1921.0)	1621.0 (70.6–1927.0)	1602.0 (484.4–2080.0)	0.14
TNF-α	79.97 (14.79–2600.0)	84.76 (3.66–3369.0)	109.8 (22.79–5113.0)	0.22

#Kruskal-Wallis test, followed by Dunn test for comparison between groups: median (minimum—maximum). In lines, values followed by the same letters and the same index do not differ;

* statistically significant (p<0.05).

Discussion

The present study has compared the sociodemographic, gestational, and perinatal characteristics, as well as the colostrum cytokine concentrations from nursing mothers at the extremes of reproductive age with full-term gestation.

Brazilian demographic characteristics are heterogeneous and, although changes in family structures currently trend towards an increase in single parents, mainly single women, our findings were different as they showed the predominance of a stable union in both groups. This might reflect the regional habits regarding the preference of couples to form a stable union by dividing responsibilities in relation to financial expenses and child care, as observed in 65–70% of the Brazilian population [19,20]. When analyzing the variable profession, it was observed that unpaid activity prevailed in the adolescent group, while paid activity was predominant in the advanced maternal age group. We suggest that the results of our study may refer to the demands of the labor market, with a preference for professionals with experience and/or to the lack of interest that adolescents demonstrate in finding a job.

Parity tends to increase with the age, as described in a multicenter study involving Brazilian population [19]. The authors reported that women between the ages of 15 and 19 have 0.18 children per woman on average, while those at the end of their reproductive period (between the ages of 45 and 49) have a mean parity of 3.14. In our study, although the number of children was not evaluated, we similarly observed almost 80% of mothers over 35 years presenting three or more pregnancies. However, the mode of delivery was predominantly cesarean section in the advanced age women compared to adolescents; previous data suggest that maternal conditions linked to fetal impairment are more frequent and more severe in this age group [5,6,21]. In this context, Domingues et al. [22] reported the reasons for cesarean deliveries in Brazilian hospitals, including women with complications in pregnancy, which, in turn, is more frequent in advanced age women. Besides an occasional pregnancy disease or complication, the authors showed that the main motivation for cesarean choice was the fear of pain, which increases this mode of delivery in Brazilian hospitals, regardless of the diagnosis of complications or the sector (private or public) in which women work.

Several factors may influence the composition of maternal milk, such as demographic data, age, ethnicity, and gestational characteristics, as well as BMI, parity, and gestational period [23, 24]. Sinanoglou et al. [24] evaluated the possible impact of sociodemographic factors in colostrum composition in Greek nursing mothers, and found that the fatty acid profile and colostrum fat mainly depend on nationality and age rather than on the mode of delivery or maternal pregnancy BMI. In the present study, we found higher BMI in the advanced maternal age group, which is higher than the mean from postpartum Brazilian women (23.6±4.6), as described by de Castro et al. [20]. Conversely, these authors demonstrated that women of older age were more likely to adhere to a healthy dietary pattern, which suggests a regional disparity; however, diet was not evaluated in this study.

It is important to mention that prematurity has been implicated as a factor influencing the cytokine concentration in breast milk. Trend et al. [25] showed that maternal milk from mothers who are breastfeeding preterm newborns contains significantly higher concentrations of certain immune system factors (TGF-β2 and antimicrobials, such as beta-defensin 1) compared to breast milk from full-term newborns. Moreover, Gila-Diaz et al. [26] reviewed the literature and reported that the highest levels of bioactive factors, including cytokines, are found in the colostrum; these decrease throughout the lactation period, being found at higher levels in preterm compared to full-term milk [25]. However, these studies compared gestational age at birth categorized as preterm and term and did not consider adolescent and advanced maternal age ranges.

Breast milk has a protective role against neonatal sepsis, probably mediated by antimicrobial components and modulation of the neonatal immune system. Chemokines in human milk, such as IL-8, and their receptors CXC chemokine receptors (CXCR)-1 and CXCR-2, can activate the immune system of the neonate against invading pathogens [18,26,27]. In our study, when comparing the concentration of IL-8 in colostrum at the extremes of maternal age, we observed that there were no significant differences between the nursing mothers. Similarly, previous data showed no differences regarding IL-8 levels between term and preterm mother's milk [27]. Additionally, our results may reflect those from Munblit et al. [13], who reported that cytokines are present at much lower levels than growth factors in breast milk, and that the cytokine decline over time was less consistent, suggesting that the role of IL-8 is constant during breastfeeding.

Some obstetric conditions may also influence cytokine levels, and although these were not addressed in the present study, they are important for understanding the dynamics of these mediators found in biological fluids. When considering adverse conditions such as preeclampsia, Freitas et al. [28] showed that this condition alters the levels of pro-inflammatory cytokines in breast milk, given that there is an increase in IL-1β and IL-6 levels in the colostrum of mothers with preeclampsia. Although the study did not consider reproductive age, the risk group for preeclampsia was maternal age over 35 years [29], which may also influence cytokines. This was observed in our study, which demonstrated higher IL-1β and IL-6 concentrations in the colostrum from advanced maternal age mothers compared to adolescent mothers. In preeclampsia, higher levels of cytokines in human milk are related to immunological signals for the host defense in high-risk neonates [30]. Therefore, the increased levels of IL-1β and IL-6 in the colostrum of mothers with advanced maternal age, evidenced in the present study, may be beneficial to the newborn.

Therefore, our findings have added knowledge regarding the colostrum composition of cytokines at the extremes of reproductive age, as there is a scarcity of data to date. Maternal age influenced colostrum composition, likely due to the biological immaturity of adolescent mothers. However, further studies should be carried out to better understand the inflammatory environment in pregnancy, to provide additional incentives for breastfeeding and avoid premature weaning and to improve the quality of life for both mothers and their children, as well as stimulating the mother/newborn bond.

Limitations of the study

There are some limitations to the present study that may have influenced the results or understanding and analysis of the results, such as the absence of concentrations of cytokines in the milk during the different periods of lactation, the lack of evaluation of the presence or absence of infection and allergic diseases in nursing mothers, and, finally, our analysis covered only a few cytokines, so the results may not be generalizable for all immunological factors.

28 Jan 2020

PONE-D-19-35580

Profile of inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age. A cross-sectional study.

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Reviewer #1: Although the examination of the profile of inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age seems to be very interesting, a number of points need clarifying and certain statements require further justification. These are given below.

1. In this manuscript, the authors measured concentrations of IL-1β, IL-6, IL-8 and TNF-α in colostrum of adolescent, control, and advanced maternal age nursing mothers (Table 4). The numbers of adolescents, controls, and advanced maternal age nursing mothers were 40, 40, and 39, respectively. In contrast, the numbers of socio-demographic characteristics (Table 1), gestational and obstetric characteristics (Table 2), and perinatal results of newborns (Table 3) of adolescents, controls, and advanced maternal age nursing mothers were 117, 158, and 39, respectively. They (adolescent and control) are different groups. Therefore, the tables 1-3 should be changed, including 40 adolescents and 40 controls that are identical to table 4.

2. How ELISA were performed especially what ELISA kits were used should be described in “Materials and methods”.

4. In references, Refs. 1-4, 6-7, 9, 11, 13, 17, and 23 were not English references. Most of readers are unable to understand whether they are reasonable/appropriate. The authors should change the references to English references.

5. The manuscript contains multiple errors in English writing (e.g. lines 33, 59, 105, 132, 134, 147, 165, etc.); therefore, it should be checked by native English speakers/scientists before submission.

Reviewer #2: Review: Manuscript number: PONE-D-19-35580

General comments:

The manuscript describes the profile of cytokines in mothers at early (10-24 years) vis-à-vis those at late gestational age (≥35 years) relative to a control group (25-34).The study is of interest considering that both populations are within the high risk group for complications for mother and infant. Colostrum is notably for defense and protection of the baby, rather than as a nutrient so assessment of cytokine profiles is relevant.

The article can be improved with some help in the style of writing and in the grammar. Details on specific sections are presented below.

Title: The title could benefit from a minor modification: cytokines are either pro- or anti-inflammatory or both so “selected” could replace “inflammatory” in the title.

Keywords: I suggest you replace “extremes age” with “ age extremes”

Abstract:

Line 39: the age classification is incorrect in the abstract

Methods: The methods section of the abstract needs significant editing to improve the sentence structure and grammar. The results section also need to be improved.

Introduction:

Line 89: …glycoproteins that signal…….

Line 92: ….and function of the newborn’s immune……………

Line 105 : reference numbering has an error

Line 106-107: Please improve the sentence.

Methods

Lines 124-125: Please improve the sentence

Line 133-137: Please revise

Lines 138-139: The sentence is too long. Break it up appropriately.

Lines 142-147: Please revise

Lines 151-152: The dependent variables are not determination of the concentrations……..

Lines 154-155: Please write out the full meaning of the abbreviated letters

Lines 159 -163: Please be specific on the exact times when samples were collected. What time in the morning? How many samples were collected within 48 hours and how many within 72 hours. These are important because there are established variations in the cytokine concentrations with time.

Line 156: This should be under the results section

Discussion

Line 232-233: Please modify

Line 281-286: IL-6 is proinflammatory in the acute phase and involved in immune regulation of the inflammatory response. Its secretion is augmented by TNF-α and IL-1 β so it is not surprising that both are elevated although TNF-α is not. Considering that these are elevated in pre-eclampsia and sickle cell disease in pregnancy, it seems that the role of IL-1 β in augment IL-6 secretion in pregnancy is highly significant compared to TNF-α. Incidentally the risk group for preeclampsia is maternal age >35 years. This is quite interesting in understanding the inflammatory environment in pregnancy.

Conclusion:

The data is relevant and the article is publishable after the corrections are made.

Reviewer #3: The manuscript titled “Profile of inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age” investigated the effect of maternal reproductive age (including adolescents group, control group (25-34 years), advanced maternal age group) on socio-demographic, gestational and perinatal parameters and in particular on the composition of pro-inflammatory cytokines in colostrum. Although it is of interest how the maternal reproductive age influences the different parameters that were investigated within this study, the reviewer does not understand the rationale behind this investigation.

Already the abstract should clearly point out why the authors aimed to study the composition of pro-inflammatory cytokines in the colostrum of the different groups. A first explanation is provided in the last paragraph of the introduction section.

Following several main questions remain unanswered:

What was the reason to present data on the marital status and profession of the study subjects? Was there any association expected to the composition of the colostrum cytokines? The reviewer would rather expect differences in the composition of the colostrum cytokines if there were any pregnancy-related complications (infections, preeclampsia, IUGR, ect) caused by the age of the mother; however this was either not investigated or there were no differences between the groups. It would be helpful to include analyses that are more detailed.

It would be of interest to analyze the socio-demographic, gestational and perinatal parameters only in those study subjects where cytokine data are available.

Do the authors have any information about the development of the neonates, for instance the infection rate during the first year of life? This could be correlated to the composition of the colostrum cytokines.

Very often, it is not clear between which groups a significant difference was detected.

Did the authors include those samples where cytokines could not be detected at all in their analysis?

What can the authors tell about changes in the colostrum cytokine composition within the first 72h after birth? These changes may influence their results as samples were taken between 48 and 72h after delivery.

Reviewer #4: The current study compared the concentrations of IL-1β, IL-6, IL-8, and TNF-α cytokines in the colostrum of adolescent mothers and those with advanced maternal age and to describe sociodemographic, gestational, and perinatal characteristics. The large sample size and cross sectional nature of the study is a significant strength of the study. The main findings of the study are that concentrations of IL-1β and IL-6 were higher in the colostrum of mothers with advanced age compared to adolescent mothers, but did not differ from the control group. Although the findings are modest, the study is carefully conducted, dataset is rich with regards to prenatal and postnatal observations in the mothers and offspring, limitations are acknowledged and the manuscript is well written. I have some minor comments that need to be addressed below.

• What ELISA platform (multiplex, single plex, company) was used for the cytokine analysis?

**********

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Reviewer #1: No

Reviewer #2: Yes: Isaac K. Quaye

Reviewer #3: No

Reviewer #4: No

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Submitted filename: PLOS_rev_colos.docx

Click here for additional data file.

17 Mar 2020

Review: Manuscript number PONE-D-19-35580

Response to reviewers

All answers are detailed below each reviewers’ comments, as well highlighted in the revised version of the manuscript.

REVIEW COMMENTS TO THE AUTHOR

Reviewer #1: Although the examination of the profile of inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age seems to be very interesting, a number of points need clarifying and certain statements require further justification. These are given below.

1) In this manuscript, the authors measured concentrations of IL-1β, IL-6, IL-8 and TNF-α in colostrum of adolescent, control, and advanced maternal age nursing mothers (Table 4). The numbers of adolescents, controls, and advanced maternal age nursing mothers were 40, 40, and 39, respectively. In contrast, the numbers of socio-demographic characteristics (Table 1), gestational and obstetric characteristics (Table 2), and perinatal results of newborns (Table 3) of adolescents, controls, and advanced maternal age nursing mothers were 117, 158, and 39, respectively. They (adolescent and control) are different groups. Therefore, the tables 1-3 should be changed, including 40 adolescents and 40 controls that are identical to table 4.

Answer: We thanks the reviewer for this observation, and we justify that a higher number of sociodemographic data was included in order to achieve one of our objectives regarding the comparison between adolescent and advanced maternal age mothers’ characteristics, besides cytokines colostrum levels. We’ve re-written the abstract and the final paragraph of introduction to make this purpose clearer. We agree the matched numbers would be ideal, however, 40 samples were the minimal number of samples needed to detected cytokines level differences. Therefore, due to the high cost of the technique, we decided to maintain the descriptive data from all studied patients in order to characterize the regional population during the studied period, and perform the Elisa assays in the minimal number of samples. Moreover, we took care of collecting half of the samples in the period of 48h postpartum, and half of them after 72h, which difficult increasing the number of colostrum collected in each group (please see answer to reviewer #2, question #7). In order to reply reviewer’s concern about the number of samples, we performed a statistical analysis for sociodemographic data considering only the same samples tested for cytokines, and we’ve obtained the same trend of results, therefore, the population characteristics are better represented by a larger number of samples, which also represents the reality in the Service during the studied period.

2. How ELISA were performed especially what ELISA kits were used should be described in “Materials and methods”.

Answer: We provided the detailed description of the assays in the revised version of the manuscript (highlighted in blue), as well as the technique protocol was sent for assigned its own identifier (DOI), as suggested by Plos One.

DOI number: dx.doi.org/10.17504/protocols.io.bdggi3tw

Lines 158-165: “Commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to quantify individually the IL-1β, IL-6, IL-8 e TNF-α levels in the studied samples. All assays were performed according to the manufacturer’s instructions. Samples in which cytokine levels were estimated to be below the sensitivity of the assay were set equal to the sensitivity of the assay and those with concentrations at levels above standard curve were diluted and re-assayed. The assays readers were performed in an ELISA automatic reader (Epoch-BioTek, Winooski, VT, USA), at a wavelength of 492 nm.”

3. In references, Refs. 1-4, 6-7, 9, 11, 13, 17, and 23 were not English references. Most of readers are unable to understand whether they are reasonable/appropriate. The authors should change the references to English references.

Answer: We appreciate this cautious observation and we clarify all references were reviewed and changed to English references, and replaced when appropriate.

4. The manuscript contains multiple errors in English writing (e.g. lines 33, 59, 105, 132, 134, 147, 165, etc.); therefore, it should be checked by native English speakers/scientists before submission.

Answer: We clarify the text has been professionally proofread for improving writing and grammar (please find attached the certificate at the end of this letter).

Reviewer #2:

General comments:

The manuscript describes the profile of cytokines in mothers at early (10-24 years) vis-à-vis those at late gestational age (≥35 years) relative to a control group (25-34).The study is of interest considering that both populations are within the high risk group for complications for mother and infant. Colostrum is notably for defense and protection of the baby, rather than as a nutrient so assessment of cytokine profiles is relevant.

The article can be improved with some help in the style of writing and in the grammar. Details on specific sections are presented below.

Answer: We thank your review and we clarify the text has been professionally proofread for improving writing and grammar (please find attached the certificate at the end of this letter).

1) Title: The title could benefit from a minor modification: cytokines are either pro- or anti-inflammatory or both so “selected” could replace “inflammatory” in the title.

Answer: We´ve agreed with reviewer´s suggestion and the change to “pro-inflammatory cytokines” was done in the title, as highlighted in blue in the revised version of the manuscript.

2) Keywords: I suggest you replace “extremes age” with “ age extremes”

Answer: The suggested change was performed.

3) Abstract:

Line 39: the age classification is incorrect in the abstract

Answer: Thanks for the observation, we made the description of adolescent mother consistent with material and methods section content. We’ve considered the adolescence as 10-24 years following the description by Sawyer et al. (1), as the studied population present similar characteristics regarding financial and emotional dependence in this period. We’ve included this reference in the manuscript (ref.#2).

(1) Sawyer S, Azzopardi P, Wickremarathne D, Patton G. The age of adolescence. The Lancet Child & Adolescent Health 2018;2:223–8.

4) Methods: The methods section of the abstract needs significant editing to improve the sentence structure and grammar. The results section also need to be improved.

Answer: The suggested changes were performed, as well as the structure and grammar were reviewed by a professional service as clarified above.

5) Introduction:

Answer: We appreciate your careful review, and the suggested changes were performed, as highlighted in blue in the revised version of the manuscript in the respective lines named below. Line 89: …glycoproteins that signal……. (line 89)

Line 92: ….and function of the newborn’s immune…………… (line 91-92)

Line 105 : reference numbering has an error (line 106)

Line 106-107: Please improve the sentence. (lines 107-108)

6) Methods

Answer: We appreciate your careful review, and the suggested changes were performed, as highlighted in blue in the revised version of the manuscript. We have expanded the abbreviations as suggested.

Lines 124-125: Please improve the sentence (lines 123-125)

Line 133-137: Please revise (lines 135-138)

Lines 138-139: The sentence is too long. Break it up appropriately.

The suggested changes were performed.

Lines 142-147: Please revise

The information regarding total sample size was removed under results section, as we’ve characterized the population considering the period of the study (please also see the answer to question #1, reviewer #1)

Lines 151-152: The dependent variables are not determination of the concentrations.

The suggested changes were performed.

Lines 154-155: Please write out the full meaning of the abbreviated letters (lines 142-143)

7) Lines 159 -163: Please be specific on the exact times when samples were collected. What time in the morning? How many samples were collected within 48 hours and how many within 72 hours. These are important because there are established variations in the cytokine concentrations with time.

Answer: We thank the reviewer for this important observation, and a better description was included in Methods section (lines 146-152).

We clarify that we took care of collecting half of the samples in the period of 48h postpartum, and half of them after 72h, which difficult increasing the number of colostrum collected in each group.

8) Line 156: This should be under the results section

Answer: The suggested changes were performed, as highlighted in blue in the revised version of the manuscript (lines 182-185).

9) Discussion

Line 232-233: Please modify

Answer: The suggested changes were performed, as highlighted in blue in the revised version of the manuscript (lines 231-232)

10) IL-6 is proinflammatory in the acute phase and involved in immune regulation of the inflammatory response. Its secretion is augmented by TNF-α and IL-1 β so it is not surprising that both are elevated although TNF-α is not. Considering that these are elevated in pre-eclampsia and sickle cell disease in pregnancy, it seems that the role of IL-1 β in augment IL-6 secretion in pregnancy is highly significant compared to TNF-α. Incidentally the risk group for preeclampsia is maternal age >35 years. This is quite interesting in understanding the inflammatory environment in pregnancy.

Answer: We thanks the reviewer for this interesting correlation, thus we added some discussion regarding cytokines dynamics in order to improve the interpretation of our data (lines 274-287).

Conclusion:

The data is relevant and the article is publishable after the corrections are made.

Reviewer #3: The manuscript titled “Profile of inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age” investigated the effect of maternal reproductive age (including adolescents group, control group (25-34 years), advanced maternal age group) on socio-demographic, gestational and perinatal parameters and in particular on the composition of pro-inflammatory cytokines in colostrum. Although it is of interest how the maternal reproductive age influences the different parameters that were investigated within this study, the reviewer does not understand the rationale behind this investigation. Already the abstract should clearly point out why the authors aimed to study the composition of pro-inflammatory cytokines in the colostrum of the different groups. A first explanation is provided in the last paragraph of the introduction section.

Answer: We agree with the reviewer, and the abstract was re-written for a better consistence between the context and the objective of this study. We hope we’ve clarified the reasonability of our work:

Abstract: Gestations at the extremes of reproductive age are characterized as high-risk pregnancies, conditions that might influence colostrum composition. This first milk secretion contains the nutrients necessary for the development and immunity of the newborn, therefore, this study aims to compare adolescent and advanced maternal age mothers regarding sociodemographic, gestational, and perinatal characteristics and regarding colostrum levels of pro-inflammatory cytokines in these groups of study.

1) Following several main questions remain unanswered: What was the reason to present data on the marital status and profession of the study subjects?

Answer: As mentioned in the previous reviewer’s question, one of the objectives of this study was to characterize the population regarding sociodemographic, gestational, and perinatal characteristics, as the such data are scarce considering the extremes of reproductive age population.

2) Was there any association expected to the composition of the colostrum cytokines?

Answer: Considering the scarcity of data, the cytokine concentration may reflect either immaturity of adolescent mothers or a compensation for high-risk in advanced maternal age group. Considering other complications that affects this last group, we suggest increase of colostrum cytokines may be beneficial to the newborn, and some discussion regarding this point was added to the text (please see lines 274-287).

3) The reviewer would rather expect differences in the composition of the colostrum cytokines if there were any pregnancy-related complications (infections, preeclampsia, IUGR, ect) caused by the age of the mother; however this was either not investigated or there were no differences between the groups. It would be helpful to include analyses that are more detailed.

Answer: That’s an important point, we specify here that 5 mothers in adolescent group, 7 in control group and 4 in advanced maternal age group presented complications as hypertension or gestational diabetes (others were not analyzed). Although the studied groups are high-risk ones, we considered the low number of pregnancy-related complications are due to the inclusion of term births, as such complications would likely lead to preterm birth. In this view, we did not perform a secondary analysis of cytokines levels as number of samples in complicated groups is too small. We included this information in Results section (lines 182-185).

4) It would be of interest to analyze the socio-demographic, gestational and perinatal parameters only in those study subjects where cytokine data are available. Do the authors have any information about the development of the neonates, for instance the infection rate during the first year of life? This could be correlated to the composition of the colostrum cytokines.

Answer: We agree with reviewer concern and clarify that we’ve characterized the population considering the period of the study, therefore, all patients were included, and the analysis of cytokines were restricted due to the assays - please see the answer to question #1, reviewer #1.

Although neonatal information would add valuable mean to the study, the main objective was to perform a cross-sectional study, to characterize the population and bring data regarding cytokines in colostrum at extremes of reproductive age, as this data are scarce. Moreover, we don’t have such data as this work was performed during a Master Course, with maximum of 2 years for collection, analysis and publication, so, unfortunately, the follow up was not possible.

5) Very often, it is not clear between which groups a significant difference was detected. Did the authors include those samples where cytokines could not be detected at all in their analysis?

Answer: We thanks for this observation, thus, changes were made along the text and in the Tables’ legends to make clear the significant differences. Moreover, we’ve included the description how samples were analyzed regarding the detection limit (lines 158-163).

What can the authors tell about changes in the colostrum cytokine composition within the first 72h after birth? These changes may influence their results as samples were taken between 48 and 72h after delivery.

Answer: We understand reviewer’s concern and we justify the interval 48-72h as colostrum secretion based on the literature (1-4). Although with some variation in time definition, mainly milk secretion consists of 3 phases: the first is colostrum produced in small quantities in the first five days after delivery (colostrum overload occurs 2 to 3 days after delivery) and is rich in cytokines, biochemical and immunological components; the second phase is the transition milk from the sixth day until the end of the second week after delivery, still with colostrum characteristics and the third stage is considered the mature milk that occurs from 15 days postpartum, with the highest composition of water, around 70% (1-4). Additionally, we took care of collecting half of the samples in the period of 48h postpartum, and half of them after 72h to avoid variations in the cytokines concentrations with time (we’ve included this detail in Material and Methods section, lines 141-143). Therefore, we believe time-collection had no influence in our results.

1- “colostrum (once in the first 6 days of life) and mature human milk (once at 4–6 weeks postpartum” Munblit D, Treneva M, Peroni DG, Colicino S, Chow L, Dissanayeke S, et al. Colostrum and mature human milk of women from London, Moscow, and Verona: determinants of immune composition. Nutrients. 2016; 8(11). pii: E695. doi:10.3390/nu8110695.

2- “colostrum (1st–6th day postpartum), transitional (7th–15th day postpartum) and mature milk (from the 16th day onwards)”

Collado MC, Santaella M, Mira-Pascual L, Martínez-Arias E, Khodayar-Pardo P, Ros G, et al. Longitudinal study of cytokine expression, lipid profile and neuronal growth factors in human breast milk from term and preterm deliveries. Nutrients 2015;7(10):8577-91. doi: 10.3390/nu7105415.

3- Pang WW, Hartmann PE. Initiation of human lactation: secretory differentiation and secretory activation. J Mammary Gland Biol Neoplasia. 2007;12: 211–21.

4- “colostrum was collected from 24-72 hours postpartum, and mature milk was collected at the end of the first month”

Freitas NA, Santiago LTC, Kurokawa CS, Meira Júnior JD, Corrente JE, Rugolo LMSS. Effect of preeclampsia on human milk cytokine levels. J Matern Fetal Neonatal Med.2018;25:1-5. doi: 10.1080/14767058.2018.1429395.

Reviewer #4: The current study compared the concentrations of IL-1β, IL-6, IL-8, and TNF-α cytokines in the colostrum of adolescent mothers and those with advanced maternal age and to describe sociodemographic, gestational, and perinatal characteristics. The large sample size and cross sectional nature of the study is a significant strength of the study. The main findings of the study are that concentrations of IL-1β and IL-6 were higher in the colostrum of mothers with advanced age compared to adolescent mothers, but did not differ from the control group. Although the findings are modest, the study is carefully conducted, dataset is rich with regards to prenatal and postnatal observations in the mothers and offspring, limitations are acknowledged and the manuscript is well written. I have some minor comments that need to be addressed below.

• What ELISA platform (multiplex, single plex, company) was used for the cytokine analysis?

Answer: We provided the detailed description of the assays in the revised version of the manuscript, as well as the technique protocol was sent for assigned its own identifier (DOI), as suggested by Plos One.

Lines 158-165: “Commercially available ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to quantify individually the IL-1β, IL-6, IL-8 e TNF-α levels in the studied samples. All assays were performed according to the manufacturer’s instructions. Samples in which cytokine levels were estimated to be below the sensitivity of the assay were set equal to the sensitivity of the assay and those with concentrations at levels above standard curve were diluted and re-assayed. The assays readers were performed in an ELISA automatic reader (Epoch-BioTek, Winooski, VT, USA), at a wavelength of 492 nm.”

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

24 Mar 2020

PONE-D-19-35580R1

Profile of pro-inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age

PLOS ONE

Dear Dr Morceli,

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PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #4: Yes

**********

6. Review Comments to the Author

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

Reviewer #1: Judged by the responses from the authors, most of the problems are resolved in the revised version. However, there remains some points for correction. These are given below.

1. Lines 41-42: “immunoenzymatic assay (ELISA)” should be changed to “enzyme-linked immunosorbent assay (ELISA)”.

2. Line 149: “72h” should be changed to “72 h”.

4. Line 154: “2mL” should be changed to “2 mL”.

5. Line 155: “160g” should be changed to “160 g”.

6. Line 189: “IL-8 e TNF-α” should be changed to “IL-8, and TNF-α”.

7. Lines 173-174: “Graph Pad Prism software, version 6.0” should be changed “Graph Pad Prism software, version 6.0 (San Diego, CA)”.

Reviewer #2: The question raised previously have been well addressed. There are a few typographic errors as indicated below:

Abstract: Line 41: ..attended at the.....should be replaced with: who attended the maternity-----

line 139:... should be punctuated as: advanced maternal age. This...

line 153: should be punctuated as: give birth. (included in the study group : should be deleted.)

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

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

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

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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

30 Mar 2020

March 30 2020

Review: Manuscript number PONE-D-19-35580

Editorial Board, PLOS ONE

Thank you for the careful review of our work and for providing us the opportunity to submit a revised version of the manuscript that addresses the points below.

All answers are detailed below each reviewers’ comments, as well highlighted in the revised version of the manuscript.

REVIEW COMMENTS TO THE AUTHOR

Reviewer #1: Judged by the responses from the authors, most of the problems are resolved in the revised version. However, there remains some points for correction.

Answer: We appreciate your careful review, and the suggested changes were performed, as highlighted in blue in the revised version of the manuscript in the respective lines named below.

1. Lines 41-42: “immunoenzymatic assay (ELISA)” should be changed to “enzyme-linked immunosorbent assay (ELISA)” (Line 41-42).

2. Line 149: “72h” should be changed to “72 h” (Line 149).

4. Line 154: “2mL” should be changed to “2 mL” (Line 154).

5. Line 155: “160g” should be changed to “160 g” (Line 155).

6. Line 189: “IL-8 e TNF-α” should be changed to “IL-8, and TNF-α” (Line 159).

7. Lines 173-174: “Graph Pad Prism software, version 6.0” should be changed “Graph Pad Prism software, version 6.0 (San Diego, CA)” (Line 173-174).

Reviewer #2: The question raised previously have been well addressed. There are a few typographic errors as indicated below:

Answer: We appreciate your careful review, and the suggested changes were performed, as highlighted in blue in the revised version of the manuscript in the respective lines named below.

1. Abstract: Line 41: attended at the.....should be replaced with: who attended the maternity—The study’s location had been suppressed from the abstract in the first revision, we include the suggestion grammar in the Methods section (Line 124)

2. line 139:... should be punctuated as: advanced maternal age. This...(Line 140)

3. line 153: should be punctuated as: give birth. (included in the study group : should be deleted.)

We thank the reviewer and clarify that paragraph was rephrased in the revised version (Lines 139-141)

Hope these changes are satisfactory and will consider our paper for publication in PLoS ONE.

Sincerely,

Morceli, G

Submitted filename: Response to Reviewers_2.docx

Click here for additional data file.

3 Apr 2020

Profile of pro-inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age

PONE-D-19-35580R2

Dear Dr. Morceli,

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

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Frank T. Spradley

Academic Editor

PLOS ONE

4 Jun 2020

PONE-D-19-35580R2

Profile of pro-inflammatory cytokines in colostrum of nursing mothers at the extremes of reproductive age

Dear Dr. Morceli:

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