Association of Zn and Cu Levels in Cord Blood and Maternal Milk with Pregnancy Outcomes among the Slovenian Population

Round 1

Reviewer 1 Report

In the present manuscript, the authors measured Zn and Cu in cord blood and maternal milk with ICP-MS, and assessed the correlations of Zn, Cu, Zn/Cu ratio with maternal characteristics and birth outcomes. The manuscript is written fluently, it presented valuable data for further policy formulation and scientific research. However, I have several comments and suggestions to improve the overall quality of it.

 

1.     The current study collected rich data of maternal characteristics, but the authors didn’t apply multiple variable models in statistical analysis. Since the authors and I both concerned the confounding effects, I strongly suggest using multiple variable models to claim the correlation between maternal characteristics and the elements. Moreover, using the maternal characteristics as covariates in assessments between elements and birth outcomes will also improve statistical power of the results. If the authors follow this suggestion, the relative discussion should also be modified.

2.     Nutrients in both cord blood and maternal milk were from mother, the authors should claim the scientific significance of each comparing findings, especially between levels in maternal milk and birth outcomes.

3.     Line 44-49. This section should be moved after the introduction of Zn and Cu to make it more readable for readers unfamiliar with these elements.

4.     Line 59. More evidence is needed to support “and have significant prominence particularly during embryonic and foetal development”

5.     Line 122-146. This section contains too much information, which should be respectively moved to sections of Material and Method or Discussion.

6.     Line 170. The actual values of SGA and AGA cut points should be provided in this section to make the result more comparable.

7.     Line 261-268 & 374-384, I highly recommended the authors to use one or two table to display these data.

Author Response

Dear Reviewer #1,

Thank you for the comments.

Please see the attachment.

Sincerely,

Ksenija Gersak

Author Response File: Author Response.pdf

Reviewer 2 Report

This study assessed the levels of Zn and Cu in cord blood 17 and maternal milk, and analyzed their association with maternal and infant characteristics and 18 pregnancy outcomes in a Slovenian study population. The most important result showed that a decreased Zn/Cu ratio in cord blood was associated with lower gestational age and lower birth weight.

However, authors should rewrite some points before publications.

1. Authors should edit spell and English sentences because there are some spell miss and too short sentences.

2. The excesses and deficiencies in Table 1 are too broad. Concentration range should be stated.

3. Table 2 and Table 3 are difficult to read, so it is better to divide them into columns for each item.

4. Authors discussed that "This indicates the overall positive effects of Zn  and negative effects of Cu on pregnancy outcomes".  The conclusion is also too broad.　Zn/Cu  should be an optimal concentration range. 

Author Response

Dear Reviewer #2,

Thank you for the comments.

Please see the attachment.

Sincerely,

Ksenija Gersak

Author Response File: Author Response.pdf

Reviewer 3 Report

Dear Editor：

Thank you for inviting me to review the article“Association of Zn and Cu levels in cord blood and maternal milk with pregnancy outcomes among the Slovenian population”. In this article, the authors investigated the levels of Zn and Cu in cord blood and maternal milk that may be associated with socio-demographic and health status, and lifestyle habits. The manuscript found that low Zn levels in cord blood were associated with lower gestational age and birth weight. Maternal smoking influenced the Cu levels in both cord blood and maternal milk. Overall, the article is well organized and its presentation is good. Here are the comments on this article:

Major Comments#1：

The manuscript found that low Zn levels in cord blood were associated with lower gestational age and birth weight. Maternal smoking influenced the Cu levels in both cord blood and maternal milk. However, the manuscript lacks nutritional habits in the main text. Maybe dietary habits play the most important role in the levels of Zn and Cu.

Major Comments#2：

For the statistical methods, maybe multivariate logistic regression can better find the factors that affect the levels of Zn and Cu and the role of each factor in them.

From the above, I suggest rejecting the manuscript.

Author Response

The authors are appreciating you and the reviewers for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them. We hope the manuscript after careful revisions meets your high standards. In the revised version of the manuscript, all changes are marked in green. Please let us know if you still have any questions or concerns about the manuscript. We will be happy to address them.
Yours sincerely,
K. Geršak and J. Osredkar

Reviewer #3:
Dear Editor:
Thank you for inviting me to review the article “Association of Zn and Cu levels in cord blood and maternal milk with pregnancy outcomes among the Slovenian population”. In this article, the authors investigated the levels of Zn and Cu in cord blood and maternal milk that may be associated with socio-demographic and health status, and lifestyle habits. The manuscript found that low Zn levels in cord blood were associated with lower gestational age and birth weight. Maternal smoking influenced the Cu levels in both cord blood and maternal milk. Overall, the article is well organized and its presentation is good. Here are the comments on this article:
Major Comments#1：
The manuscript found that low Zn levels in cord blood were associated with lower gestational age and birth weight. Maternal smoking influenced the Cu levels in both cord blood and maternal milk. However, the manuscript lacks nutritional habits in the main text. Maybe dietary habits play the most important role in the levels of Zn and Cu.

Thank you for the comments.
We added (in green fonts, lines 516-531):
»According to this, we could not confirm a positive association between Zn levels or Zn/Cu ratio in cord blood and birth weight, however, we demonstrated that mothers of SGA infants had a higher Zn/Cu ratio in maternal milk than mothers of AGA and LGA infants, which we are the first to report, to the best of our knowledge.

This is an important finding; particularly as preterm and SGA infants have higher essential micronutrient requirements due to their rapid postnatal growth and development and the limited capacity for storage of these elements [93]. Therefore, these infants are at an increased risk of developing nutritional deficiencies. Ustundag et al. reported significantly lower Zn levels in milk from mothers of pre-term versus fullterm babies [94], however, our study showed the opposite, indicating that higher
micronutrient requirements of infants born with low birth weight for gestational age might be compensated through higher Zn levels in maternal milk in comparison to infants who were born with normal or higher weight according to gestational age.

To date, benefits of zinc nutriture during pregnancy have not been demonstrated and as zinc deficiency is likely a reflection of poor diet, strategies to improve overall maternal nutrition are likely to yield more tangible health benefits than the use of zinc supplements alone [95].«

Major Comments#2:
For the statistical methods, maybe multivariate logistic regression can better find the factors that affect the levels of Zn and Cu and the role of each factor in them.

We did the required calculations using multiple regression and multinominal regression models (depending on the type of the outcome variable). We added the required information to the Results section, subsection 3.3. Association of infant characteristics and pregnancy outcomes with Cu and Zn levels in cord blood and maternal milk (in green fonts, lines 342-344, 354-356, 361-363, 365-369, 372-375 and 383-386).
We described the used statistical methods in the Methods section (in green fonts, lines 260-365).
The significance of the results and their interpretation after the adjustment for the confounding variables in multiple regression and multinominal regression models did not change in majority of cases, so we made only minor adjustments to the Discussion section to match the changes made to the Results (in green fonts, lines 498-501). 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Generally, I am satisfied with the current edition of this article. But there are still several minor concerns as follows.

1.     Line 264. The full name of VIF should be here with the first appearance of this abbreviation.

2.     The format of Table 3 is not standard.

3.     Figure 1 looks confusing; I recommend using percent bar chart.

4.     In Figure 3, ‘term’ maybe means ‘normal’? and only part (D) had an asterisk.

Moreover, the description of multiple regression should be in Lines 260-265, which made a mistake in the authors’ reply.

Author Response

The authors thank the reviewer for further comments. We considered all the reviewer’s concerns and accordingly revised the manuscript. In the revised version of the manuscript (nutrients-1874247_07sep2022.docx), all changes are marked in green and deleted fragments are marked in red. Please let us know if you still have any questions or concerns about the manuscript. We will be happy to address them.

Yours sincerely,

K. Geršak and J. Osredkar

 

Reviewer 1:

Generally, I am satisfied with the current edition of this article. But there are still several minor concerns as follows.

1. Line 264. The full name of VIF should be here with the first appearance of this abbreviation.

Reply: The full name variance inflation factor (VIF) was added in green font (line 263).

2. The format of Table 3 is not standard.

Reply: Table 3 was corrected according to the reviewer`s request.

3. Figure 1 looks confusing; I recommend using percent bar chart.

Reply: Figure 1 is now represented as a percent bar chart.

4. In Figure 3, ‘term’ maybe means ‘normal’? and only part (D) had an asterisk.

Reply: term designates the delivery between 37 and 42 weeks of gestation, which is considered the normal duration of pregnancy. Pre-term birth is considered before 37 weeks of pregnancy and post-term after 42 weeks. This is described in Methods section, lines 185-186.

The asterisk beside the p value in Figure 3D represents the extreme outlier and not statistical significance. Therefore, the p was removed away from the asterisk to avoid the conclusion.

5. Moreover, the description of multiple regression should be in Lines 260-265, which made a mistake in the authors’ reply.

Reply: We apologize for this mistake and confirm that description of multiple regression is in the lines 259-264.

6. English language and style are fine/minor spell check required.

Reply: the spellcheck of the paper was performed and text was corrected where needed.

Author Response File: Author Response.docx