Study of Concussion in Rugby Union through MicroRNAs (SCRUM): a study protocol of a prospective, observational cohort study

Introduction The diagnosis of mild traumatic brain injury or sports-related concussion is a challenge for all clinicians, players, coaches and parents involved in contact sports. Currently, there is no validated objective biomarker available to assess the presence or severity of concussion in sport, and so it is necessary to rely on subjective measures like self-reporting of symptoms which depend on the cooperation of the athlete. There is a significant health risk associated with repetitive injury if the diagnosis is missed, and so there is great value in an objective biomarker to assist diagnostic and prognostic decisions. Objective To establish a panel of non-invasive MicroRNA biomarkers in urine and saliva for the rapid diagnosis of sports-related concussion and investigate the kinetics and clinical utility of these biomarkers in assisting diagnostic, prognostic and return-to-play decisions. Methods and analysis Observational, prospective, multicentre cohort study recruiting between the 2017–2018 and 2018–2019 Rugby Union seasons. Professional rugby players in the two highest tiers of senior professional domestic rugby competition in England will be recruited prospectively to the study. During the season, three groups will be identified: athletes entering the World Rugby Head Injury Assessment (HIA) protocol, uninjured control athletes and control athletes with musculoskeletal injuries. Saliva and urine will be collected from these athletes at multiple timepoints, coinciding with key times in the HIA protocol and return-to-play process. Ethics and dissemination Ethics approval has been obtained. The compiled and analysed results will be presented at national and international conferences concerning the care of patients with traumatic brain injury. Results will also be submitted for peer review and publication in the subject journals/literature.

In the methods and analysis section, the three groups of players (uninjured, orthopaedic and concussed) could be described more clearly.

Article summary
For the last point of the article summary, I would also include younger athletes as a population that requires further study, as this is an important group that displays differences in concussion symptom presentations. So I would suggest saying "such as female or youth athletes".

Introduction
In the first sentence of the introduction, "sport related concussion" is capitalised, but in the abstract it is in lower caps and then abbreviated. This needs to be kept consistent.
The first sentence reads: "Sport Related Concussion is form of mild traumatic brain injury caused by..", this should be changed to "Sport Related Concussion is a form of mild traumatic brain injury caused by..".
In the second sentence there is a space between "England" and the punctuation.
The third sentence needs to be grammatically revised. Possibly change to: "In the majority of cases, concussion results in a rapid onset, but short-lived, impairment of neurological function that resolves spontaneously." The fourth sentence is somewhat contradictory, as it presents two conflicting pieces of information. It is stated that 80-90% of concussed athletes return to pre-injury levels within two weeks, while 20-30% experience prolonged symptoms. both statements can't be simultaneously true.
The fourth sentence should also read: "pre-injury levels within two weeks" The second paragraph of the introduction is one long sentence. This could be split into two or three sentences.
In paragraph five, "baseline information for athletes" can be changed to "baseline athlete information".
In the first sentence of paragraph 6, the word "process" might not need to be capitalised.
For the description of the HIA process in paragraph six, I would explain where it is currently being used after the first sentence and before going into a description of it. I feel that one needs the context of it being used in professional leagues to understand the description of video replays etc.
In paragraph nine, I would add more biological information on why miRNA's are being suggested as biomarkers. For example, highlighting the temporal expression of miRNA and why/how quantifying these levels may provide an indication of the extent of underlying gene expression and the biological processes initiated by head injury. This may give the reader a better understanding of why miRNA analysis may be a better biomarker candidate in comparison to other gene or protein expression assays.
It might also be worth explaining how miRNA's are distributed in bodily fluids, how they interact with the blood brain barrier and the relevance of these dynamics to concussion.
In general, I think more detail needs to be provided about miRNA's and how they function to give readers without a knowledge of genetics or molecular biology a better understanding of why they are potential biomarkers.

Methods and analysis
In the first sentence of the methods the number of participants is written as "1100", while in the sample size it is written as "1,100".
In the first sentence, I would also add that the two leagues consist of senior professional players. Some international readers may not be familiar with the Premiership and Championship. For example, maybe say "the two highest tiers of senior professional domestic rugby competition", or something similar. It will be important to record the timing of these baseline extractions in relation to preseason training sessions, other injuries, or supplement/medication use. There are so many potential confounding factors that getting a true "baseline" from a single extraction will always be difficult.
In the fourth paragraph, "in match HIA" could be changed to "pitchside HIA" and "36-48 HIA" could be changed to "36h-48h HIA", to be consistent with Table 1.
Paragraphs five and six could be joined into a single paragraph. Both paragraphs consist of a single, slightly long-winded, sentence.
In the first sentence of paragraph seven, shown below, it is not especially clear which athletes are being referred to.
"In the case of players who are diagnosed with concussion at any time point, they will also be asked for a sample of saliva and urine at the point at which they are cleared by their team physician to return to rugby (Stage V) as per the Berlin Consensus Graduated Returnto-Play (GRTP) protocol. [2]" The sentence could be made more succinct, for example: "Players diagnosed with concussion will be asked for a sample of saliva and urine at the point at which they are cleared by their team physician to return to rugby (Stage V) as per the Berlin Consensus Graduated Return-to-Play (GRTP) protocol. [2]" Will the playing position of the uninjured control be taken into account? For example, using a scrum-half as a comparison for a forward position with a high collision profile might not be the best match. I would suggest trying, if possible, to match the playing position and the minutes of game time, as we know each playing position has a different physical demand.
I'm not sure what benefit the orthopaedic controls will provide, unless the concussed player also sustained a musculoskeletal injury. They may provide a good comparison at the post-match time points, as they too will be removed from activity, but the effect of a moderate to severe musculoskeletal injury will overshadow these differences. In my opinion, the uninjured player would provide a better comparison to measure the effects of "sub-clinical" nonneurological injuries and musculoskeletal trauma. However, it would be interesting to see the differences in miRNA profiles between the injury types (concussion vs. musculoskeletal).
The tenth paragraph of the study design section introduces a new aim that was not mentioned in the introduction. Specifically, to investigate the effects of collision sport involvement in baseline miRNA levels. I would suggest adding some rationale for this in the introduction. I would also consider the chance of false positives when comparing single extractions of miRNA. Because of the number of factors that influence miRNA expression, it is highly likely that the two baseline levels will be different. It would be bold to suggest that these differences are due to rugby participation alone. If one wanted to thoroughly investigate the baseline miRNA levels, one would ideally need to collect a number of samples to get an individual's "average range" that can then be compared.
In the final paragraph of the study design section, the number of samples that will be sent to identify promising targets needs to be stated, even if this is a rough estimated number. How confident are the authors that this number of samples is sufficient to ensure that all the potential biomarkers are accurately identified?
At the end of the of the outcome measures and statistical analysis section, it is mentioned that imaging data will be analysed. There is no previous mention of what imaging will be done or what it would be used for. Please clarify this.

VERSION 1 -AUTHOR RESPONSE
3) Pg 6, Ln 18: Complied? compiled? Corrected 4) Page 8, Ln 6: ... is "a" form of Corrected 5) I found it odd that all references are cited after a period or a comma, is it a requirement for this journal?
The authors noticed that in the recently published BMJ Open study protocols. Therefore, we decided to follow the same format. 7) In the methods and analysis section, the three groups of players (uninjured, orthopaedic and concussed) could be described more clearly. Corrected

Article summary
8) For the last point of the article summary, I would also include younger athletes as a population that requires further study, as this is an important group that displays differences in concussion symptom presentations. So I would suggest saying "such as female or youth athletes". Corrected Introduction 9) In the first sentence of the introduction, "sport related concussion" is capitalised, but in the abstract it is in lower caps and then abbreviated. This needs to be kept consistent. Corrected 10) The first sentence reads: "Sport Related Concussion is form of mild traumatic brain injury caused by..", this should be changed to "Sport Related Concussion is aform of mild traumatic brain injury caused by..". Corrected 11) In the second sentence there is a space between "England" and the punctuation. Corrected 12) The third sentence needs to be grammatically revised. Possibly change to: "In the majority of cases, concussion results in a rapid onset, but short-lived, impairment of neurological function that resolves spontaneously." Corrected 13) The fourth sentence is somewhat contradictory, as it presents two conflicting pieces of information. It is stated that 80-90% of concussed athletes return to pre-injury levels within two weeks, while 20-30% experience prolonged symptoms. both statements can't be simultaneously true. Corrected

14)
The fourth sentence should also read: "pre-injury levels within two weeks" Corrected 15) The second paragraph of the introduction is one long sentence. This could be split into two or three sentences. Corrected 16) In paragraph five, "baseline information for athletes" can be changed to "baseline athlete information". Corrected 17) In the first sentence of paragraph 6, the word "process" might not need to be capitalised. Corrected 18) For the description of the HIA process in paragraph six, I would explain where it is currently being used after the first sentence and before going into a description of it. I feel that one needs the context of it being used in professional leagues to understand the description of video replays etc. Corrected 19) In paragraph nine, I would add more biological information on why miRNA's are being suggested as biomarkers. For example, highlighting the temporal expression of miRNA and why/how quantifying these levels may provide an indication of the extent of underlying gene expression and the biological processes initiated by head injury. This may give the reader a better understanding of why miRNA analysis may be a better biomarker candidate in comparison to other gene or protein expression assays.
20) It might also be worth explaining how miRNA's are distributed in bodily fluids, how they interact with the blood brain barrier and the relevance of these dynamics to concussion. In general, I think more detail needs to be provided about miRNA's and how they function to give readers without a knowledge of genetics or molecular biology a better understanding of why they are potential biomarkers. 19, 20) The authors think that would be beyond the scope of this study protocol and it would be better to refer the readers to our newly published paper "MicroRNA Signature of Traumatic Brain Injury:

21)
In the first sentence of the methods the number of participants is written as "1100", while in the sample size it is written as "1,100". Corrected 22) In the first sentence, I would also add that the two leagues consist of senior professional players. Some international readers may not be familiar with the Premiership and Championship. For example, maybe say "the two highest tiers of senior professional domestic rugby competition", or something similar. The investigators do record the timing of these baseline extractions in relation to preseason training sessions and other injuries. But, we do not collect information about supplement/medication use. The reason being that we have concerns we may not get reliable information from the players due to concerns about their confidentiality etc. 25) In the fourth paragraph, "in match HIA" could be changed to "pitch-side HIA" and "36-48 HIA" could be changed to "36h-48h HIA", to be consistent with Table 1. Corrected 26) Paragraphs five and six could be joined into a single paragraph. Both paragraphs consist of a single, slightly long-winded, sentence. Corrected 27) In the first sentence of paragraph seven, shown below, it is not especially clear which athletes are being referred to.
"In the case of players who are diagnosed with concussion at any time point, they will also be asked for a sample of saliva and urine at the point at which they are cleared by their team physician to return to rugby (Stage V) as per the Berlin Consensus Graduated Return-to-Play (GRTP) protocol. [2]" The sentence could be made more succinct, for example: "Players diagnosed with concussion will be asked for a sample of saliva and urine at the point at which they are cleared by their team physician to return to rugby (Stage V) as per the Berlin Consensus Graduated Return-to-Play (GRTP) protocol.
[2]" Corrected 28) Will the playing position of the uninjured control be taken into account? For example, using a scrum-half as a comparison for a forward position with a high collision profile might not be the best match. I would suggest trying, if possible, to match the playing position and the minutes of game time, as we know each playing position has a different physical demand. We have not asked clubs to match to playing position, just that they try and match a roughly similar number of played minutes in the game. 29) I'm not sure what benefit the orthopaedic controls will provide, unless the concussed player also sustained a musculoskeletal injury. They may provide a good comparison at the post-match time points, as they too will be removed from activity, but the effect of a moderate to severe musculoskeletal injury will overshadow these differences. In my opinion, the uninjured player would provide a better comparison to measure the effects of "sub-clinical" non-neurological injuries and musculoskeletal trauma. However, it would be interesting to see the differences in miRNA profiles between the injury types (concussion vs. musculoskeletal).
To rule out contamination from non-neurological injuries and musculoskeletal trauma (extra-cranial injuries) that would affect the sensitivity and specificity of miRNAs as reliable concussion biomarkers in polytrauma patients.
30) The tenth paragraph of the study design section introduces a new aim that was not mentioned in the introduction. Specifically, to investigate the effects of collision sport involvement in baseline miRNA levels. I would suggest adding some rationale for this in the introduction. I would also consider the chance of false positives when comparing single extractions of miRNA. Because of the number of factors that influence miRNA expression, it is highly likely that the two baseline levels will be different. It would be bold to suggest that these differences are due to rugby participation alone. If one wanted to thoroughly investigate the baseline miRNA levels, one would ideally need to collect a number of samples to get an individual's "average range" that can then be compared. Corrected 31) In the final paragraph of the study design section, the number of samples that will be sent to identify promising targets needs to be stated, even if this is a rough estimated number. How confident are the authors that this number of samples is sufficient to ensure that all the potential biomarkers are accurately identified? Corrected 32) At the end of the of the outcome measures and statistical analysis section, it is mentioned that imaging data will be analysed. There is no previous mention of what imaging will be done or what it would be used for. Please clarify this. Corrected

GENERAL COMMENTS
Overall, I believe the manuscript meets the requirements for the journal and details an important area of research.
I noticed a few small things that could be addressed: In the first paragraph of the introduction, "short term" should be written as "short-term".
When referring to Table 1, be consistent in using capital letters (" Table 1" vs "table 1").
The "University of Birmingham" abbreviation is described twice, once in the methods (paragraph 8) and once in the ethics and dissemination section.
The abbreviation "SCRUM" should be defined in Table 1.