Oral magnesium supplementation for leg cramps in pregnancy-An observational controlled trial.

BACKGROUND: Oral magnesium for leg cramps treatment in pregnancy is a controversial issue according to recent Cochrane systematic review. This study aims to evaluate the efficacy of Mg++ supplementation in leg cramps treatment in pregnancy.
METHODS: This observational clinical trial studied 132 pregnant women with leg cramps in the first trimester of pregnancy. At baseline, 74 (56.3%) had two leg cramps episodes per week, 28 (21.1%) three episodes, 13 (9.8%) four episodes and 9 (6.8%) five or more episodes. They were randomized 1:1 to 300 mg/day of oral Mg++ citrate (n = 66) or placebo (n = 66). The primary outcome was the frequency of leg cramps episodes per week reported by pregnant women. Secondary outcomes were the ocurrence of leg cramps and oral magnesium side effects.
RESULTS: 130 pregnant women completed the study and the two groups were comparable according to some sociodemographic and clinical characteristics. After 4 weeks of intervention it was observed a 28.4% (39/132) (CI 95%: 20.9-37.0) reduction of leg cramps in all participants and no difference between the two groups was found; reduction of 27.2% (18/66) (CI 95%: 17.0-39.6) in Mg++ group and 32.8% (21/66) (CI 95%: 21.6-45.7) in the placebo group. The OR of leg cramps was 1.3 (CI 95%: 0.6-2.9), p = 0.527, taking the placebo group as reference. Among pregnant women who remained with leg cramps the mean of leg cramps episodes per week showed no significance difference between the Mg++ and placebo groups; t-student test: p = 0.408. Four pregnant women showed gastrointestinal side effects; 2 in each group had nauseas and diarrhoea.
CONCLUSION: Oral magnesium supplementation during pregnancy did not reduce the ocurrence and frequency of episodes of leg cramps.

RCT Entities:   Population Interventions Outcomes

Introduction

Leg cramps are involuntary painful skeletal muscle contractions lasting from seconds to minutes, often nocturnal, and frequently involve the gastrocnemius [1,2]. About 30–50% of pregnant women experience leg cramps at least twice a week during the third trimester [2,3]. The etiology of cramps during pregnancy is unclear, but is believed to be due to overload of the ankle plantar flexors, excessive exercise, metabolic disorders, circulatory problems, underlying medical conditions, nutritional deficiencies (vitamins E and D) or electrolyte imbalances (eg. magnesium, calcium and sodium) [1,2,4].

Many therapies for leg cramps have been tried as gabapentin, pycnogenol, electrolytes and vitamins (magnesium, calcium, sodium, and vitamin E and vitamin D), massage, muscle stretching, relaxation, heat therapy and dorsiflexion of the foot [2]. However, there are still no consistent conclusions for treating leg cramps in pregnancy. [2]. Magnesium plays an important role in many metabolic reactions, neuronal excitability and muscle function [5]. Magnesium deficiency enhances neuromuscular transmission. Magnesium therapy has been shown to be effective in eclampsia-related seizures [6]. For this reason some studies have suggested a beneficial role of magnesium in leg cramps. Even more because magnesium requirements increase during pregnancy [7]. Magnesium supplementation has been indicated as a therapy capable of minimizing fetal growth restrictions, reducing the risk of preeclampsia and favoring newborn weight gain, although there is no strong evidence of these benefits [8]. Besides insufficient magnesium intake is common, especially in low-income regions [9].

Some systematic reviews showed that is unclear whether oral magnesium provides an effective treatment for leg cramps and large well-conducted randomised controlled trials are needed to answer the question of leg cramps treatment [2, 10, 11, 12]. This is even more important for low-income regions where insufficient magnesium intake is common [9]. This study aims to evaluate the efficacy of oral magnesium supplementation in the treatment of cramps during pregnancy in a low-income region.

Methods

Study design

This observational blinded controlled trial investigated the effect of oral magnesium citrate supplementation for leg cramps in pregnant women. This trial tests the hypothesis that oral magnesium supplementaton during pregnancy may reduce the frequency od leg cramps episodes. The intervention lasted 4 weeks and was completed between November 2015 and January 2018. This study was part of the Brazil MAGnesium trial [13] and registered at ClinicalTrials.gov (Identifier NCT02032186).

Setting and participants

The study took place at Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Brazil. IMIP registers about 6,000 deliveries per year. Pregnant women who attended the antenatal care clinic at the Department of Obstetrics, IMIP, were invited to join this study. Inclusion criteria were pregnant women aging between 18–45 years, gestational age between 12 and 20 weeks, a single gestation and currently residents of the city of Recife. Gestation age was based on the last menstrual period among women with a regular menstrual cycle or by first-trimester pregnancy dating ultrasound. Exclusion criteria were uncontrolled known hyperthyroidism, any type of known active parathyroid disease, chronic diarrheal disease, chronic kidney disease, defined by an estimated glomerular filtration rate below 60 mL / min / 1.73 m2, as determined by the initial assessment or by known history, Mg ++ serum concentration at baseline > 2.6 mg / dL.

Randomization and intervention

Randomization was performed in a 1:1 ratio using a table of random numbers, prepared by a researcher who did not participate in the data collection. These numbers were generated in a computer by Random Allocation Software 2.0 program. Allocation concealment was ensured, as the referred researcher did not release the randomization code until the participants were recruited into the trial after all baseline measurements were completed.

Consenting pregnant women received a 4 weeks magnesium citrate capsule (300 mg elemental magnesium citrate per capsule) or a daily placebo capsule identical in colour and shape. Both capsules were manufactured by IMIP’s Department of Pharmacology. The study medication packages were supplied with sequential numbers. Code break envelopes were supplied to the lead pharmacist but not available for the investigation team. Each pack was individually prescribed for each participant. Compliance, adverse events, and clinical intercurrences were monitored by the research team at routine prenatal visit during the intervention. Adherence to treatment was defined as the ingestion of at least 80% of the prescribed dose for 30 days.

The criteria for discontinuation of the study were: symptoms reported by the patient or clinical signals due to the intake of the magnesium capsules or the cancellation of prenatal care at IMIP.

Outcomes

The primary outcome was the frequency of leg cramps which was defined as painful, involuntary contraction of muscles occurring at rest, mostly at night, and causing a palpable knot in the muscle, recorded at least twice a week. The frequency was measured as the number of leg cramps per week. Secondary outcomes were the presence of leg cramps episodes and oral magnesium side effects. All this informations were taken from the research diaries previously given to participants.

Ethical considerations

All participants were informed about the data confidentiality, and were informed about their capacity to withdraw from the study. All pregnant women provided written informed consent. The study was approved by the IMIP’s Committee on Research (document number 4033), and was registered in the ClinicaTrials.gov (NCT 02032186).

Data analysis

The sample size calculation was calculated based on Dahle et al (12) and Supakatisant C & Phupong (13) trials to compare 50% reduction of leg cramps between intervention and placebo groups. It was assumed a 5% bilateral alpha error, a 80% power and an expected 35% percentage of cramps in the intervention group. With adjustments for a withdrawal rate of 10%, a minimum of 66 women in each group were required. Stata version 12.1 was used for statistical analysis. Chi-squared test for categorical variables and independent t-test for continuous variables were used when appropriate. It was considered a p < 0.05. Intent-to-treat analysis was performed.

Results

A total of 394 pregnant women were screened and 132 enrolled according to the inclusion criteria. At baseline, 74 (56.0%) had two leg cramps episodes per week, 28 (21.2%) three episodes, 7 (9.8%) four episodes and 23 (13.0%) five or more episodes; 66 pregnant women were assigned to the Mg++ group (300 mg per day) and 66 were assigned to the placebo (Fig 1). Two pregnant women of placebo group were lost to follow-up but 132 participants were included in the intention-to-treat analysis.

Fig 1

CONSORT flow diagram.

The groups showed no significant diferences with respect to age, years of study, employment, income, parity, body mass index, gestational age, number of leg cramps episodes per week and serum magnesium level (Table 1).

Table 1

Some sociodemographic and clinical characteristics of the participants.

	Participants(132)	Mg++ group(66)	Placebo group(66)	p-value
Age (years)	26.6± 5.5	26.2± 4.9	27.0.± 6.0	0.407
Years of study	4.8± 1.1	5.0± 1.2	4.7± 1.0	0.204
Currently employed	68 (51.5%)	36 (54.5%)	32 (48.4%)	0.486
Income per capita monthly (US$)	144 ± 65	153 ±40	135 ±65	0.107
Primipara	51 (38.6%)	30 (45.4%)	21 (31.8%)	0.568
Body Mass Index (BMI)	25.9±5.2	26.5± 5.8	25.2±4.4	0.173
Gestational age (weeks)	15.0±3.5	15.0± 3.2	15.1± 3.9	0.576
Number of cramps per week	4.8±2.8	4.1±2.9	5.5±3.0	0.300
Serum magnesium level < 1.8 mg/dl	66 (50.0%)	32 (48.4%)	34 (51.5%)	0.431

After 4 weeks of intervention it was observed a 28.4% (39/132) (CI 95%: 20.9–37.0) reduction of leg cramps in all participants and no difference between the two groups was found; reduction of 27.2% (18/66) (CI 95%: 17.0–39.6) in Mg++ group and a reduction of 32.8% (21/66) (CI 95%: 21.6–45.7) in the placebo group. The OR of leg cramps was 1.3 (CI 95%: 0.6–2.9), p = 0.527, taking the placebo group as reference. Among pregnant women who remained with leg cramps the number of leg cramps episodes per week showed no significance difference between the Mg++ and placebo groups (t-Student test: p = 0.408) (Table 2). The mean magnesium serum level was 1.84 mg/dL (CI 95%: 1.80–1.87) in the Mg++ group and 1.84 mg/dL (CI 95%: CI 1.80–1.87) in placebo group (t-Student test: p = 0.872).

Table 2

Leg cramps after 4 weeks of intervention.

	Mg++ (66)	Placebo (66)	P
Leg cramps
Yes	48 (73.8%)	45 (68.2%)	0.352
No	18 (27.2%)	21 (31.8%)
Number of episodes per week (95% CI)	4.1 (2.4–7.1)	4.8 (3.7–10.0)	0.300

Four pregnant women showed gastrointestinal side effects; 2 in each group had nauseas and diarrhoea.

Discussion

This observational controlled trial showed no efficacy of oral magnesium supplementation in the treatment of leg cramps during pregnancy. Oral magnesium treatment for leg cramps during pregnancy is still a controversial issue and only a few randomized controlled trials have accessed this intervention. Recently a Cochrane systematic review concluded that magnesium supplements did not consistently reduce how often women experienced leg cramps when compared with placebo [2].

Our results were similar with Nygaard et al [14]. This randomized controlled trial assessed the effect of two weeks of oral magnesium (360 mg) on leg cramps in 38 pregnant women. Leg cramp frequency and intensity were not influenced by oral magnesium supplementation.

However other studies showed different results. Dahle et al studied 73 women with pregnancy-related leg cramps in a randomized trial and oral magnesium for 3 weeks decreased leg cramp distress [15]. Supakatisant & Phupong in a RCT studied 80 pregnant women for 4 weeks [16] and observed a fifty per cent reduction of cramp frequency and intensity in the oral magnesium bisglycinate chelate group (300mg/day). Zarean & Tarjan verified that pregnant women with low magnesium level supllemented with magnesium (200 mg) had less leg cramps during pregnancy [17].

These trials have different measurements. Pregnant women with different gestational ages were studied, interventions for variable periods of time (2 to 4 weeks) were used, different oral magnesium doses and oral formulations were administered. In addition, the definition of leg cramps was not well stablished in some studies. All this may explain different findings.

Gastrointestinal side effects were observed in a few women and there were no differences between the magnesium and placebo groups. The Cochrane systematic review concluded that there was no difference in the experience of side effects, such as nausea and diarrhoea [2].

The pregnant women in this study had a magnesium serum level in the minimum limit of normality (1.8 mg/dl) and around half of participants had hypomagnesemia. This seems to indicate that the pregnant women studied were at nutritional risk. The 300 mg daily dose of magnesium citrate used herein approximated that recommended in pregnancy [7]. Although magnesium deficiency has been implicated with an increased risk for gestational and adverse perinatal outcomes, there is not enough high-quality evidence to show that dietary magnesium supplementation during pregnancy is beneficial. [2, 7, 9].

Our study has strengths and limitation. Our strengths include: 1) A large number of participants and a very low rate of dropout; 2) Magnesium serum level was assessed; 3) Intention-to-treat analysis.

The current sample size might not be sufficient for detecting the differences in the primary outcome, since they calculated it according to the frequency of leg cramp. Additionally, the 50% reduction in frequency of leg cramp in treatment group might be too positive. All these above might be the reasons why this study had a negative result. Other limitations are as follows: 1) The intensity of leg cramps pain was not evaluated. However this evaluation includes a high degreee of subjectivity; 2) Magnesium serum level was not assessed after intervention; 3) Pregnancy women were studied in the first trimester of pregnancy but leg cramps is a more common problem in the third trimester; 4) Because this study was observational, it could be prone to biases.

In conclusion, oral magnesium supplementation during pregnancy did not reduce the ocurrence and frequency of episodes of leg cramps.

Original clinical protocol in Portuguese.

(TIF)

Click here for additional data file.

CONSORT 2010 checklist.

(TIF)

Click here for additional data file.

Protocol English.

(TIF)

Click here for additional data file.

(DTA)

Click here for additional data file.

24 Jul 2019

PONE-D-19-16639

Oral magnesium supplementation for leg cramps in pregnancy – a randomized controlled trial

PLOS ONE

Dear Dr. Alves,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised by all the reviewers.

We would appreciate receiving your revised manuscript by Sep 07 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Yiqing Song, MD, ScD

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially identifying or sensitive patient information) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. Please see http://www.bmj.com/content/340/bmj.c181.long for guidelines on how to de-identify and prepare clinical data for publication. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

**********

3. 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: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. 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: No

Reviewer #3: Yes

Reviewer #4: No

**********

5. 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: I will focus on methods and reporting

Major

1) the introduction is very short for a research paper. Much more information is needed in terms of the background to the study.

2) I don't understand how the sizes of the groups differ so much following an 1 to 1 allocation process

3) the methods section is very poor. Power analyses are incomplete. What is the baseline level for cramps? power and numbers needed are very different between a very prevalent and a rare outcome. a 50% reduction is a a very large effect. What is the odds ratio? the analysis plan is pretty poor. Even if perfectly balanced why did they not analyse in the context of a logistic regression, where the effects could be quantified in ORs?

Minor

1) Abstract. 1 to 1 matching yes 50 vs 63 in the placebo group. that's not 1 to 1?

2) information on the blinding is needed in title and/or abstract. single? double?

3) abstract says the 2 groups were comparable. some elaboration is needed

4) no information on analyses methods in the abstract

5) no information on baseline levels in the abstract - only the reduction. we need some measure of the absolute, not only relative, reduction.

6) Abstract: report effects and their CIs rather than p-values

Reviewer #2: This is a randomized controlled trial to evaluate the effect of oral magnesium supplementation on leg cramps in pregnant women. There are many critical design flaws for this study. The data provided in the manuscript was not consistent in whole manuscript. Also, many writing issues and language questions presented in the manuscript.

I will list several important design flaws:

1. The ratio for randomization was 1:1, while the number of women in Mg supplementation group was 57 and 64 in the placebo group. The number of participants in two groups were imbalanced. And the author did not provide the detailed allocation concealment for randomization. This is very important in a RCT study. Without allocation concealment, randomization would be unsuccessful.

2. The sample size calculation was based on the estimation of 50% reduction in frequency of leg cramp in Mg treatment group. The primary outcome was the presence of the leg cramps. The current sample size might not be sufficient for detecting the differences in the primary outcome, since they calculated it according to the frequency of leg cramp. Additionally, the 50% reduction in frequency of leg cramp in treatment group might be too positive. All these above might be the reasons why this study had a negative result.

3. The method for missing values were not mentioned in the study.

Reviewer #3: 1. The hypothesis testing, power and type 1 error should be included in the study design section. Please also be clear whether the type 1 error is one-sided or two sided.

2. The screening failure rate is very high. Any reasons for this?

3. Could the authors perform some additional subgroup analyses to explore the effect of intervention?

Reviewer #4: In this RCT, oral magnesium did not reduce the occurrence nor frequency of leg cramp episodes in the second trimester of pregnancy. This is an interesting topic, but some details are missing from this manuscript.

1.) The rationale for the trial requires more explanation in the introduction to highlight any gaps in knowledge. For example, the Garrison et al. (2012) systematic reviewed showed no evidence that oral Mg is helpful for leg cramps during pregnancy, with low heterogeneity among trials. What will this RCT add?

2.) In the introduction, it would be more relevant to cite magnesium effects on muscle/ mechanistic effects rather than cite general information on the etiology of cramps.

3.) How common are cramps during the second trimester (study population used in the RCT)?

4.) pg. 5: Exclusion of those with Mg at baseline >9.5mmol/dL is not physiologically plausible given the Mg reference range.. this value is a typo?

5.) Was adherence self reported?

6.) How were outcomes ascertained?

7.) Additional statistical rationale for the sample size calculation is needed. Why was a 50% reduction in leg cramps expected?

8.) pg. 7: Lines 3-4: are units in mg/dL? Three serum levels are listed: should one be deleted?

9.) Provide information on whether any stopping rules were instituted (CONSORT missing item)

10.) Who was blinded to the information? (eg. only participants, their doctors, the PI, those assessing outcomes, the statistical analyst, etc)?

11.) Table 1: income values are approximately $50,000 annually? Units need checking

12.) Flowchart (Fig 1): Indicate which arm is the placebo; which is the Mg arm (currently unlabeled)

13.) Can the original Portuguese protocol be translated into English via an online translator or individual? The English summary of the protocol is an abbreviated version of the manuscript and provides no additional detail.

14.) The overall manuscript would benefit from copyediting before resubmission

**********

6. 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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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.

13 Aug 2019

Answers to the Reviewers:

Answer: Thank you very much for reviewing our manuscript. We also greatly appreciate the reviewers for their comments and suggestions. We have carried out all the recommendations suggested by the reviewers. The manuscript has been completely revised and we hope it will be able to be published.

Reviewer #1: I will focus on methods and reporting

Major

1) the introduction is very short for a research paper. Much more information is needed in terms of the background to the study.

Answer: The introduction was complemented. A new paragraph and five news references were added.

2) I don't understand how the sizes of the groups differ so much following an 1 to 1 allocation process

Answer: We apologize for this mistake. All the database was reviewed with a biostatistician and it was observed that eleven 18-year-old pregnant women had not been included in the analysis. These 11 pregnant women (2 in the intervention group and 9 in the control group) were now included in the analysis and the ratio 1:1 restored.

3) the methods section is very poor. Power analyses are incomplete. What is the baseline level for cramps? power and numbers needed are very different between a very prevalent and a rare outcome. a 50% reduction is a a very large effect. What is the odds ratio? the analysis plan is pretty poor. Even if perfectly balanced why did they not analyse in the context of a logistic regression, where the effects could be quantified in ORs?

Answer: Baseline level for cramps was included. The sample size calculation was based upon the 50% reduction in frequency of leg cramps in both groups obtained from Dahle et al (reference number 12) and Supakatisant C & Phupong V (reference number 13) trials. The odds ratio was calculated by logistic regression. The effects were now quantified in ORs.

Minor

1) Abstract. 1 to 1 matching yes 50 vs 63 in the placebo group. that's not 1 to 1?

Answer: It was corrected (66:66)

2) information on the blinding is needed in title and/or abstract. single? double?

Answer: It was provided; double blind.

3) abstract says the 2 groups were comparable. some elaboration is needed

Answer: It was added this information.

4) no information on analyses methods in the abstract

Answer: It was provided.

5) no information on baseline levels in the abstract - only the reduction. we need some measure of the absolute, not only relative, reduction.

Answer: This information was added in the abstract.

6) Abstract: report effects and their CIs rather than p-values

Answer: Effects and their CIs were provided.

Reviewer #2: This is a randomized controlled trial to evaluate the effect of oral magnesium supplementation on leg cramps in pregnant women. There are many critical design flaws for this study. The data provided in the manuscript was not consistent in whole manuscript. Also, many writing issues and language questions presented in the manuscript.

I will list several important design flaws:

1. The ratio for randomization was 1:1, while the number of women in Mg supplementation group was 57 and 64 in the placebo group. The number of participants in two groups were imbalanced. And the author did not provide the detailed allocation concealment for randomization. This is very important in a RCT study. Without allocation concealment, randomization would be unsuccessful.

Answer: We apologize for this mistake. All the database was reviewed with a biostatistician and it was observed that eleven 18-year-old pregnant women had not been included in the analysis. These 11 pregnant women (2 in the intervention group and 9 in the control group) were now included in the analysis and the ratio 1:1 restored. Allocation concealment was ensured, as the service did not release the randomization code until the participants were recruited into the trial after all baseline measurements were completed.

2. The sample size calculation was based on the estimation of 50% reduction in frequency of leg cramp in Mg treatment group. The primary outcome was the presence of the leg cramps. The current sample size might not be sufficient for detecting the differences in the primary outcome, since they calculated it according to the frequency of leg cramp. Additionally, the 50% reduction in frequency of leg cramp in treatment group might be too positive. All these above might be the reasons why this study had a negative result.

Answer: The sample size calculation was based upon the 50% reduction in frequency of leg cramps in both groups obtained from Dahle et al (reference number 12) and Supakatisant C & Phupong V (reference number 13) studies. This limitation was included in the discussion.

3. The method for missing values were not mentioned in the study.

Answer: Thank you for this observation. However as we had no missing values we did not include this in the methods section.

Reviewer #3: 1. The hypothesis testing, power and type 1 error should be included in the study design section. Please also be clear whether the type 1 error is one-sided or two sided.

Answer: The hypothesis testing, power and type error were included in the methods section.

2. The screening failure rate is very high. Any reasons for this?

Answer: The flowchart was corrected. The main reason for the screening failure was absence of leg cramps.

Reviewer #4: In this RCT, oral magnesium did not reduce the occurrence nor frequency of leg cramp episodes in the second trimester of pregnancy. This is an interesting topic, but some details are missing from this manuscript.

1.) The rationale for the trial requires more explanation in the introduction to highlight any gaps in knowledge. For example, the Garrison et al. (2012) systematic reviewed showed no evidence that oral Mg is helpful for leg cramps during pregnancy, with low heterogeneity among trials. What will this RCT add?

Answer: Garrison et al is cited in the introduction (reference number 11) and they final conclusions are that “It is unlikely that magnesium supplementation provides clinically meaningful cramp prophylaxis to older adults experiencing skeletal muscle cramps. In contrast, for those experiencing pregnancy-associated rest cramps the literature is conflicting and further research in this patient population is needed.” We studied pregnancy-associated cramps and our conclusion is the same of Garrison et al, i.e. it is unlikely that magnesium supplementation during pregnancy provides clinically meaningful cramp prophylaxis to leg-cramps.

This systematic review implications for research were: “To resolve the uncertainty surrounding the role of magnesium in pregnant women, parallel‐group blinded placebo‐controlled RCTs of magnesium in that population are needed”. We tried to develop a randomized double-blind controlled trial.

2.) In the introduction, it would be more relevant to cite magnesium effects on muscle/ mechanistic effects rather than cite general information on the etiology of cramps.

Answer: The introduction was complemented. A new paragraph and four news references were added.

3.) How common are cramps during the second trimester (study population used in the RCT)?

Answer: During the second trimester 70.0 % (91/130) of 130 participants that had leg cramps in the first trimester continued presenting leg cramps.

4.) pg. 5: Exclusion of those with Mg at baseline >9.5mmol/dL is not physiologically plausible given the Mg reference range. this value is a typo?

Answer: Thank you for this correction. It was a typo error and it was corrected (2.6 mg/dL)

5.) Was adherence self reported?

Answer: Compliance/adherence, adverse events, and clinical intercurrences were monitored by the research team at each routine prenatal visit until the completion of the treatment. Adherence was defined as the ingestion of at least 80% of the prescribed dose.

6.) How were outcomes ascertained?

Answer: All this informations were taken from the research diaries previously given to participants.

7.) Additional statistical rationale for the sample size calculation is needed. Why was a 50% reduction in leg cramps expected?

Answer: The sample size calculation was based upon the 50% reduction in frequency of leg cramps in both groups obtained from Dahle et al (reference number 12) and Supakatisant C & Phupong V (reference number 13) studies.

8.) pg. 7: Lines 3-4: are units in mg/dL? Three serum levels are listed: should one be deleted?

Answer: The units are in mg/dL. It was provided. The first serum level was deleted.

9.) Provide information on whether any stopping rules were instituted (CONSORT missing item)

Answer: The criteria for discontinuation of the study were: clinical signs or symptoms reported by the patient due to the intake of the capsules or the cancellation of prenatal care at hospital.

10.) Who was blinded to the information? (eg. only participants, their doctors, the PI, those assessing outcomes, the statistical analyst, etc)?

Answer: Participants, their doctors and all investigators. This information was now provided in the method section.

11.) Table 1: income values are approximately $50,000 annually? Units need checking

Answer: Thank you for this observation. It was corrected.

12.) Flowchart (Fig 1): Indicate which arm is the placebo; which is the Mg arm (currently unlabeled)

Answer: It was indicated.

13.) Can the original Portuguese protocol be translated into English via an online translator or individual? The English summary of the protocol is an abbreviated version of the manuscript and provides no additional detail.

Answer: The Portuguese protocol was translated into English.

14.) The overall manuscript would benefit from copyediting before resubmission

Answer: The manuscript was reviewed.

Submitted filename: Answers to the Reviewers (1).docx

Click here for additional data file.

8 Nov 2019

PONE-D-19-16639R1

Oral magnesium supplementation for leg cramps in pregnancy – a randomized double-blind controlled trial

PLOS ONE

Dear Dr. Alves,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Dec 23 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Yiqing Song, MD, ScD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

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 #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #5: (No Response)

**********

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 #2: No

Reviewer #3: Yes

Reviewer #5: No

**********

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

Reviewer #2: No

Reviewer #3: Yes

Reviewer #5: No

**********

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 #2: Yes

Reviewer #3: Yes

Reviewer #5: No

**********

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 #2: No

Reviewer #3: Yes

Reviewer #5: 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 #2: 2. For sample size calculation: You have not answer my question yet. The primary outcome is the presence of leg cramps, while the outcome used for calculating sample size is the secondary outcome. This is a substantial issue for your protocol. That means your current sample size might be only sufficient for analysis of your secondary outcome. Also, please provide your function for sample size calculation. The reference Currently the 20% dropout rate is a little bit large. And I do not find a 50% reduction in your referent papers. Please have a check.

3. For missing value: In your flwo chart, all 66*2 were included into your analysis, however, in your table 2 only 64 in treatment and 64 in control group were included into analyses. While actually, the overall number of placebo is 66. Please re-check your numbers. Many other inconsistences in your manuscript. For example, in tbale 1, 36 in Mg group and 30 in placebo group were currently employed; while in total, 68 were currently employed. In table 2, 27,2 should be 27.2. Please check your manuscript thoroughly.

Reviewer #3: (No Response)

Reviewer #5: The authors aimed to evaluate to evaluate the effect of magnesium supplementation for the prevention of leg cramps in pregnant women.

According to information provided by the authors, the study was part of the Brazil MAGnesium trial and registered at ClinicalTrials.gov (Identifier NCT02032186). The registration refers to the Brazil MAGnesium trial, but neither the current study is referred to nor are the aims of the study defined as secondary outcomes. Therefore, the presented manuscript describes an unplanned secondary analysis of the above mentioned randomized trial Brazil MAGnesium trial. A secondary analysis should be regarded as an observational trial and should be identified explicitly as such.

The CONSORT checklist does not longer correspond to the presented study. The STROBE checklist would be more appropriate and will guide the authors to important aspects, which should be included in the description of the study. E.g. the consideration of potential confounders in the statistical analysis of observational data is necessary and should be added.

One further aspect is, that randomization corresponds to the estimated sample size (2000 assigned to magnesium, 1000 assigned to placebo (with 2:1 allocation ratio) according to protocol on ClinicalTrials.gov) and not to the subsample of 132 patients (with an apparent 1:1 allocation ratio). This means, that the quality characteristics of randomization no longer apply. The study is merely an observational trial.

The sample size of the initial study is based on the primary outcome (perinatal composite outcome). Therefore, the sample size and corresponding power is not adequate for the current outcome (presence of leg cramps) and puts the validity of the analysis and subsequent conclusion into question.

Apart from that, the “new” calculation is based on “50% reduction of leg cramps” and not on the variable defined as the primary endpoint (presence of leg cramps) so that the methodological basis of the study in itself is also questionable.

I am sorry to say, but from my point of view there are unsurmountable methodological deficits.

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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 #2: No

Reviewer #3: No

Reviewer #5: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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.

13 Nov 2019

Rebuttal Letter

Dear Editor,

We appreciate your attention. Thank you very much for the opportunity to address the comments from the Reviewers. We carefully considered all comments offered by the reviewers. The authors hope that the Reviewers will be satisfied with the further amendments which we have made to the manuscript. Please see below the point-by-point responses to the reviewers’ specific comments.

Best regards,

Joao Guilherme Alves

- Corresponding Author -

Reviewer #2: 2. For sample size calculation: You have not answer my question yet. The primary outcome is the presence of leg cramps, while the outcome used for calculating sample size is the secondary outcome. This is a substantial issue for your protocol. That means your current sample size might be only sufficient for analysis of your secondary outcome. Also, please provide your function for sample size calculation. The reference Currently the 20% dropout rate is a little bit large. And I do not find a 50% reduction in your referent papers. Please have a check.

Answer: Thank you for this observation. You are completely rigth. Our sample size was calculated based on the frequency of leg cramps which is the parameter most used in trials assessing leg cramps interventions. Systematic reviews have used frequency of leg cramps as a primary outcomes. Based on this we changed our primary outcome from the presence of leg cramps to frequency of leg cramps.

Function for sample size was determined by the software “Clinical.com” →“Statistics” → “Sample Size Calculator” → “View Power Calculations”:

N1={z1−α/2∗p¯∗q¯∗(1+1k−−−−−−−−−−−√)+z1−β∗p1∗q1+(p2∗q2k−−−−−−−−−−−−−−√)}2/Δ2q1=1−p1q2=1−p2p¯=p1+kp21+Kq¯=1−p¯N1={1.96∗0.57∗0.43∗(1+11−−−−−−−−−−−−−−−√)+0.84∗0.7∗0.3+(0.45∗0.551−−−−−−−−−−−−−−−−−−√)}2/0.252N1=60N2=K∗N1=60N1={z1−α/2∗p¯∗q¯∗(1+1k)+z1−β∗p1∗q1+(p2∗q2k)}2/Δ2q1=1−p1q2=1−p2p¯=p1+kp21+Kq¯=1−p¯N1={1.96∗0.57∗0.43∗(1+11)+0.84∗0.7∗0.3+(0.45∗0.551)}2/0.252N1=60N2=K∗N1=60

p1, p2 = proportion (incidence) of groups #1 and #2

Δ = |p2-p1| = absolute difference between two proportions

n1 = sample size for group #1

n2 = sample size for group #2

α = probability of type I error (usually 0.05)

β = probability of type II error (usually 0.2)

z = critical Z value for a given α or β

K = ratio of sample size for group #2 to group #1

The 20% dropout rate is really a little big. However, we lost to follow-up only two participants and we studied 66 pregnant women in each arm, this allowed us to reduce de drop out to 10% in our sample size calculation (60 + 6 = 66).

A 50% reduction was cited by Supakatisant C, Phupong V, reference number 16 (Oral magnesium for relief in pregnancy-induced leg cramps: a randomised controlled trial. Maternal & Child Nutrition 2015; 11(2):139–145), methods section, “The sample size calculation was based upon the 50% reduction in frequency of leg cramps in both groups obtained from Dahle et al.'s study (Dahle et al. 1995)”.

3.For missing value: In your flwo chart, all 66*2 were included into your analysis, however, in your table 2 only 64 in treatment and 64 in control group were included into analyses. While actually, the overall number of placebo is 66. Please re-check your numbers. Many other inconsistences in your manuscript. For example, in tbale 1, 36 in Mg group and 30 in placebo group were currently employed; while in total, 68 were currently employed. In table 2, 27,2 should be 27.2. Please check your manuscript thoroughly.

Answer: Sorry for this mistake. This number in the table 2 was corrected (Placebo 64 to Placebo 66). Table 1 was also checked and corrected. All the manuscript was completely reviewed.

Reviewer #3: (No Response)

Reviewer #5: The authors aimed to evaluate to evaluate the effect of magnesium supplementation for the prevention of leg cramps in pregnant women.

According to information provided by the authors, the study was part of the Brazil MAGnesium trial and registered at ClinicalTrials.gov (Identifier NCT02032186). The registration refers to the Brazil MAGnesium trial, but neither the current study is referred to nor are the aims of the study defined as secondary outcomes. Therefore, the presented manuscript describes an unplanned secondary analysis of the above mentioned randomized trial Brazil MAGnesium trial. A secondary analysis should be regarded as an observational trial and should be identified explicitly as such.

Answer: The study was now identified as an observational trial.

The CONSORT checklist does not longer correspond to the presented study. The STROBE checklist would be more appropriate and will guide the authors to important aspects, which should be included in the description of the study. E.g. the consideration of potential confounders in the statistical analysis of observational data is necessary and should be added.

Answer: The CONSORT check list was changed to STROBE checklist. This limitation was added in the discussion: 4) Because this study was observational, it could be prone to biases.

One further aspect is, that randomization corresponds to the estimated sample size (2000 assigned to magnesium, 1000 assigned to placebo (with 2:1 allocation ratio) according to protocol on ClinicalTrials.gov) and not to the subsample of 132 patients (with an apparent 1:1 allocation ratio). This means, that the quality characteristics of randomization no longer apply. The study is merely an observational trial.

Answer: Following your orientation the study design was changed to an observational study.

The sample size of the initial study is based on the primary outcome (perinatal composite outcome). Therefore, the sample size and corresponding power is not adequate for the current outcome (presence of leg cramps) and puts the validity of the analysis and subsequent conclusion into question.

Answer: We agree with you but a sub-sample size was calculated based on previous trials with interventions to reduce the frequency of leg cramps. This is supposed to offer robustness to our results.

Apart from that, the “new” calculation is based on “50% reduction of leg cramps” and not on the variable defined as the primary endpoint (presence of leg cramps) so that the methodological basis of the study in itself is also questionable.

Answer: The primary endpoint was changed to the frequency of leg cramps. The sample size was calculated based on 50% reduction of leg cramps. Presence of leg cramps was now considered as a secondary outcome.

I am sorry to say, but from my point of view there are unsurmountable methodological deficits.

Answer: Thank you very much for your analysis. We hope that as the study design was now changed to an observational trial you can review your position.

Submitted filename: Rebuttal Letter.docx

Click here for additional data file.

20 Dec 2019

Oral magnesium supplementation for leg cramps in pregnancy – an  observational  controlled trial

PONE-D-19-16639R2

Dear Dr. Alves,

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.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Yiqing Song, MD, ScD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The critiques by the reviewers have been nicely addressed and the manuscript has been improved significantly.

Reviewers' comments:

27 Dec 2019

PONE-D-19-16639R2

Oral magnesium supplementation for leg cramps in pregnancy – an  observational  controlled trial

Dear Dr. Alves:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Yiqing Song

Academic Editor

PLOS ONE