EMRSHN 2: Exploring the Modulatory Role of Sex Hormones Along the Neuromechanical Axis in Females
Conditions: Role of Sex Hormones Along the Neuromechanical Axis
Sponsor: University of Texas Southwestern Medical Center

STUDY PROTOCOL AND STATISTICAL ANALYSIS PLAN 
 
 
 
Official title:  EMRSHN 2: Exploring the Modulatory Role of 
Sex Hormones Along the Neuromechanical Axis in Females 
 
NCT number: NCT03947684 
IRB Approved date: 05-08-2020 

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PROTOCOL FORM / RESEARCH DESCRIPTION 
 
If an item does not apply to your research project, indicate that the question is "not applicable” – do not leave sections blank 
 
Click once on the highlighted entry in each box to provide your response.  Click the item number/letter or word, if hyperlinked, 
for detailed instructions for that question.  If your response requires inserting a table, picture, etc, you may need to first delete the 
box that surrounds the answer and then insert your table or other special document. 
 
 
1.  Purpose and objectives.           List the purpose and objectives: 
 
OBJECTIVES:  
The goal of this project is to test our central hypothesis that changes in sex hormone concentration result in 
changes to the basic elements of motor control – at multiple levels, from the musculotendinous unit to 
motor control circuitry.  Under Aim 1 we will determine the influence of sex hormone fluctuations on the 
muscle stretch reflex during active and passive states, and the time lag between hormone concentration 
changes and the reflex response.  We will use a technically simple assessment that could be implemented 
in the field.  Under Aim 2 we will determine the influence of sex hormone fluctuations on spinal motor 
neuron excitability using H-reflex as a probe and the simultaneous change in the muscle mechanics using 
muscle twitch response. Aims 1 & 2 will include a focus on the differential role of oral contraceptives.  In 
Aim 3 we will use paired-pulse transcranial magnetic stimulation during active contraction to determine 
the influence of sex hormone fluctuations on cortical excitability in naturally cycling women. In the basic 
elements of motor control explained in Aim 2 and Aim 3 is that the sex hormones modulate the synaptic 
transmission and plasticity of neuron and muscles. The modulation of the synaptic transmission and 
plasticity of neuron and muscles might be caused by the changes of the neurotransmitter or muscle fiber in 
the transcriptional level, translational level, or combination of both. In Aim 4, we want to explore these 
changes through sequencing of whole exome and microRNA in addition to evaluating the expression level 
of mRNA and proteins related to synaptic transmission and muscle. Our findings have the potential to 
guide future research in injury prevention strategies, including targeted neuromuscular training.  This 
knowledge will enable clinicians to educate female patients on neuromuscular effects of sex hormones 
during times of high fluctuations, including puberty, pregnancy, and menopause. 
 
Abbreviations:  
nOC = non- users of oral contraceptives 
OC =   oral contraceptive users 
 
 
 
 
2.  Background. 
• 
Describe past experimental and/or clinical findings leading to the formulation of your study.   
• 
For research involving investigational drugs, describe the previously conducted animal and human studies.   
• 
For research that involves FDA approved drugs or devices, describe the FDA approved uses of this drug/device in 
relation to your protocol.   
• 
Attach a copy of the approved labeling as a product package insert or from the Physician’s Desk Reference.   
You may reference sponsor’s full protocol or grant application (section number and/or title) or if none, ensure background 
includes references. 
 
Please respond to all components of this item, or clearly indicate which components are not applicable. 

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a. 
Background 
BACKGROUND: 
The National Institutes of Health has called for the study of sex differences in all areas of biomedical 
research. One of the most striking sex disparities in musculoskeletal medicine is the increased risk of 
anterior cruciate ligament (ACL) injuries in female athletes. Female athletes are up to 9 times more 
likely to experience ACL injuries than males competing in the same sports. These injuries cause 
significant morbidity, including time lost from school or work, psychological distress, and post-
traumatic knee osteoarthritis rates (10-15 years after injury) up to 80% higher than in the uninjured 
knee. 
 
Joint stability depends upon articular congruence, passive stiffness of connective tissues, and reflexive 
and voluntary muscle contractions. Therefore, changes in muscle mechanics could compromise the 
force- generating capacity of skeletal muscle and lead to decreased joint stability. Impaired stability 
increases the risk of musculoskeletal injury, particularly to the ACL. Such injuries are linked to 
impaired neuromuscular control or abnormal patterns of muscle activation. Specifically, the female 
pattern of landing with decreased hip and knee flexion, increased knee valgus, and an increased 
quadriceps-to-hamstring activation ratio is linked to higher rates of injury. Prevention focused on 
neuromuscular training has reported success, but the sex disparity in ACL injury persists. 
 
While injury rates, hormones, and some motor control patterns diverge around puberty, it is unclear 
how such changes increase the risk of ligament injury. Neuromuscular control is influenced by 
fluctuations in estradiol and progesterone during the menstrual cycle, but the mechanism of this 
interaction remains unknown. Post-pubertal females have shown variable muscle activation patterns of 
the gluteus maximus and semitendinosus during a landing task that correlate with cyclic variations in 
estradiol. Neuromuscular control during hopping also fluctuates with hormone changes, but not in 
women with more stable levels of estradiol and progesterone induced by monophasic oral 
contraceptives. To fill the current research gap, we propose to determine the hormonal regulation of 
different constituents of neuromuscular control. 
 
Our project will increase understanding in four realms. First, we propose a paradigm shift, arguing that 
increased rates of injury in females is not due to direct changes in cycle specific connective tissue 
properties, but are secondary to hormone mediated changes in neuromuscular control. Second, we will 
determine if our muscle stretch reflex protocol can identify hormone-mediated alterations in joint 
control. If so, this protocol can be employed to determine whether this process causes joint injury. Due 
to its technical simplicity, the protocol and the associated equipment (hammer/load cell/wireless EMG 
unit and a tablet) can be used in clinical settings and at athletic training sites, facilitating large-scale 
examination of the neuromuscular underpinnings of joint injuries. Importantly, our pilot data showed a 
significant change in the reflexes (on average, up to 2.5 folds reductions) from the luteal to the 
ovulatory phase, highlighting the robustness of the test in detecting changes in reflex excitability, which 
may be applicable to multiple clinical settings. Third, our findings should allow clinicians to time 
neuromuscular retraining programs to certain points during maturation or the menstrual cycle when 
females might be most receptive to reprogramming neuromuscular control strategies. Similarly, 
identifying a cortical level locus of sex hormone mediated changes in neuromuscular control would 
inform the implementation and timing of motor learning specific strategies. Fourth, by including 

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women taking hormonal contraceptives, we will determine if, and to what extent, endogenous and 
exogenous estradiol and progesterone have different effects on knee neuromechanics. 
 
It has been suggested that oral contraceptives might prevent musculoskeletal injury in female athletes, 
but there is a paucity of literature to guide clinicians in this area. As 40-70% of female collegiate 
basketball and soccer players [more than 10 million women in the United States, and 100 million 
women worldwide] report taking oral contraceptives, understanding their effects on the 
neuromusculoskeletal system is critical. Answering these questions has the potential to reduce the risk 
and rates of ACL injury and mitigate the long- term knee pain in future generations of female athletes. 
While our focus is on basic neuromuscular changes due to natural hormone fluctuations, we expect that 
this knowledge will illuminate other effects of sex hormones on the neuromusculoskeletal system, such 
as in osteoarthritis in perimenopausal women or lumbopelvic control in peripartum females.   
 
Most studies have focused on hormonal modulation of the passive mechanics of connective tissues, 
measured in humans at the aggregate joint scale. Such studies have yielded conflicting results. Instead, 
we argue that sex hormones may have a more significant effect on active properties of the 
neuromuscular system. We suggest the injury sex disparity is likely due to changes in neuromuscular 
control rather than in aggregate joint stiffness. To the best of our knowledge, this study will be the first 
to examine, simultaneously, hormonal effects in the musculotendinous unit (Aim 1), motor neuron 
(Aim 2), and cortical motor neuronal networks (Aim 3), which are key contributors to movement 
control. This project builds a foundation for the study of sex differences in neurophysiologic properties 
of cortical networks. 
 
 
b. 
Current practice 
N/A  
 
 
3.  Study Design.  
Describe the study design (e.g., single/double blind, parallel, crossover, etc.)  Consider inserting a scheme to visually present the 
study design. 
 
Same as #4 below. 
 
 
4.  Research Plan / Description of the Research Methods:   
4.a.  Provide a comprehensive narrative describing the research methods.   
     1) Provide the order in which tests/procedures will be performed,  
     2) Provide the setting for these events and a description of the methods used to protect privacy during the study.   
     3) Provide the plan for data analysis (include as applicable the sample size calculation) 
 
Please respond to all components of this item, or clearly indicate which components are not applicable. 
 
 
STUDY TIMELINE: 
 

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Year 1 
Year 2 
Year 3 
Year 4 
Year 5 
month 
1-3 
4-11 
12 
1-5 
6 7-11 
12 
1-5 
6 7-11 
12 
1-5 
6 7-11 
12 
1-5 
6 7-11 
12 
Set-up, hiring 
Recruitment 
Testing 
Project Meeting 
Data Analysis 
Conferences 
Manuscripts 
 
PROCEDURES INVOLVED: 
The sequence of experiments is shown in Fig. 1. During month one, all eumenorrheic subjects (nOCs), 
will provide blood samples every other day to track their hormones across the menstrual cycle (they will 
track menstrual cycle for 2-3 months prior). Post hoc analyses will compute subject-specific hormonal 
fluctuations throughout the month using a nonlinear mixed-effect model.  nOCs will use urinary ovulation 
kits to verify that they have ovulatory cycles and identify the approximate day of ovulation. nOCs and 
male controls will have a brain MRI to establish consistent coil placement across visits in Aim 3, where 
TMS will be used to assess cortical excitability. 
In month 2, all female subjects, both 
OCs and nOCs, will undergo every 
other day testing as described in Aims 
1 and 2 (described in detail below) and 
provide every other day blood samples. 
nOC subjects will use urinary 
ovulation kits daily as described above. 
Hormone concentrations of each nOC 
subject will be compared to the 
subject-specific mixed-effect model 
from month one to determine where 
she is in her menstrual cycle. This 
estimation will enable TMS testing at 
six key points during the cycle (Aim 
3). For example, when the subject’s 
daily estradiol concentrations are rising 
and the model indicates that her peak 
estradiol should occur the next day, she will 
be instructed to report to the lab for TMS 
testing in the next 24 hours. On these days, 
she will complete the protocols for Aims 1-
3. Subjects will be randomized to start 
testing in the follicular, peri-ovulatory, or luteal phase where Day 1 is defined as the first day of menses. 
The time required for each experiment, including setup, is approximately 2½ to 3 hours for Aims 1 and 2 
combined and 2 - 3 hours for Aim 3. The investigator will be blinded to menstrual cycle phase and 
contraceptive use. The comparative groups for Aims 1 and 2 will be women on monophasic oral 
contraceptives containing progestins of similar potency and androgenicity,as justified by our published and 
pilot data. The control group for all Aims will be age-matched healthy males. Our choice of males as 
Figure 1. Sequence of experiments. In month one, non-users of 
oral contraceptives will have their blood drawn to determine 
their hormonal signature. In month two, subjects will undergo 
daily blood draws as well as testing for Aims 1 and 2. Subjects will 
complete tests for Aim 3 on six occasions defined by key 
hormonal fluctuations. 

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controls for Aim 3 is motivated by the lack of data to guide interpretation of the effects of exogenous 
hormones on cortical circuitry. 
 
Below is the specific experimental protocol for each AIM: 
 
AIM 1: Determine the influence of sex hormone fluctuations on muscle stretch reflex at relaxed and active 
states.  
 
Venipuncture will be performed at UT Southwestern Medical Center or UT Southwestern Lab. 
Venipuncture will be completed every other day during a full menstrual cycle, for a total of 2 cycles. This 
equates to 28 to 40 blood draws total (not necessarily consecutively), up to 3.04 teaspoons of blood per 
draw, for a maximum of 85.12 to 121.6 teaspoons (425.6 ml to 608 ml) over the course of study 
participation (2 – 3 months, equivalent to 2 menstrual cycles). To minimize the effect of diurnal 
fluctuations of hormone levels, testing will be done at a consistent time of day for each test session across 
all Aims. Furthermore, OCs will be asked to ingest their birth control pill at a consistent time each every 
day of the testing.  
 
At each session, subjects will be seated in an experimental chair with their right hip and knee positioned in 
90o of flexion. The ankle joint will be secured in a coupling ring, which will fix the limb to a load cell, via 
a rigid beam. Pre-amplified surface electromyography will be placed along the lower limb muscles. In an 
effort to ensure the same electrode placement at subsequent testing sessions, each electrode site will be 
marked with a permanent marker and subjects will be encouraged not to wash off the markings between 
sessions. Three maximum voluntary contractions (MVCs) will be recorded with a minimum of 10 seconds 
of rest between trials. The muscle reflex will be evoked by tapping the patellar tendon with a hammer 
instrumented with a load cell. The load cell instrumented hammer will allow us to quantify the reflex gain 
by normalizing the reflex EMG by the tap force. The hammer will be fixed on a pendulum to standardize 
the tap force and tap rate used to elicit the reflexes. For each pre-activation level (0%, 5%, 10%, and 20% 
knee extension torque at MVC), up to 40 total taps will be delivered during each testing session. Taps will 
be randomly spaced (varying from 10-20 seconds between taps) to limit the subjects’ ability to anticipate 
the timing of the next tap.  In an attempt to further minimize supraspinal modulation of the observed tap 
induced muscle activity at rest (0% MVC), subjects will be instructed to relax and maintain a standardized 
position. Finally, to assess the joint’s connective tissue response at varying points in the cycle, anterior 
knee laxity (AKL) will be measured with the knee arthrometer. 
AIM 2: Determine the influence of sex hormone fluctuations on spinal motor neuron excitability 
Subjects will be placed in a prone position on an examination table with the right knee in full extension 
(the targeted side for all participants). An AirCast® boot will secure the ankle joint. The boot will be placed 
within a coupling ring, which will fix the limb to a six degrees-of-freedom load cell (JR3®). This testing 
protocol will be employed in an attempt to standardize the testing environment, behavioral state, degree of 
muscle activation, and ankle joint angle across testing sessions as changes in these factors can influence 
spinal reflex amplitude and twitch force (i.e., initial muscle length).  Two protocols will be employed in 
order to assess the influence of hormone concentrations on both mono- and poly-synaptic spinal circuits. 
For the monosynaptic H-reflex testing, a stimulating bar bipolar electrode (Digitimer Ltd, Hertfordshire, 
England) will be placed over the tibial nerve in the popliteal fossa with the anode positioned distally. The 

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H-reflex and M-Wave will be recorded from the soleus muscle using surface EMG electrodes (see Aim 1 
methodology) placed over the muscle belly at the myotendinous junction. Stimulus intensity will be 
controlled manually via a digitimer (DS&A constant current stimulator) and waveforms will be visualized 
on an oscilloscope (Tektronix TDS 2014). One- millisecond stimuli will be delivered at 10-20 second 
intervals to prevent post-activation depression. The H- reflex and M-wave recruitment curves will be 
mapped by increasing the stimulus intensity in 1mA increments starting from 2mA, noting the peak of the 
H-reflex and the plateau of the M-wave. To maximize the accuracy of these measurements, additional 
measurements using incremental increases in stimulation intensity (0.2mA) will be taken around the peak 
H-reflex. Once we identify the stimulation intensity necessary to yield the peak H-reflex, we will obtain 5 
measurements at 10-20 second intervals, as well as 5 measurements of the maximum M-wave at 10-20 
second intervals. To compute the associated changes in the contractile properties of the MT, single 
electrical pulses (1 ms in duration) at intensity 20% higher than the intensity at the maximal M-wave will 
be delivered to the soleus muscle. Five supramaximal stimulations will be elicited, and the corresponding 
M-waves and twitch torques will be stored and later averaged. For polysynaptic reflex testing, stimulation 
will be applied behind the medial malleolus of the ankle to the distal tibial nerve using a Grass Instruments 
S48 Stimulator, a Grass SIU5 Stimulus Isolation Unit and a Grass Constant Current Unit (model CCU1) 
(Grass Instument Company, W. Warwick, RI). The constant current stimulator will allow a constant safe 
current to be used throughout the experiment. The isolation unit is standard when using the Grass stimulator 
in human research to ensure maximum safety for the subject. The stimulation consists of a train of eight 
biphasic rectangular pulses at a frequency of 200 Hz and a pulse duration of 2 ms. Subjects will start with 
2 mA stimulation intensity. The current will gradually increase by 2mA until the first visible contraction 
of the abductor halluces muscle is found. The data collection will occur at double the current of the motor 
threshold. In the experiment described by Hubli et al (2011) the average stimulation intensity was ~16.0 
mA (sd 8.1 mA). Stimulation at this intensity has been shown to be non-noxious and safe in healthy subjects 
(Dietz et al, 2009). EMG recordings will be taken from the tibialis anterior muscle using self-adhesive 
surface electrodes. Data collection will include 20 series of 8 train pulses. 
AIM 3: Determine the influence of sex hormone fluctuations on cortical excitability using transcranial 
magnetic stimulation (TMS).  
Surface EMG will be recorded from muscles in the right leg. The paired-pulse paradigm isolates any 
modulatory influence of hormone concentration at cortex. With the subject seated with hip, knee, and ankle 
flexed at 90 degrees, TMS will be delivered to the leg area of the  left motor cortex through using one of 
our  TMS systems (Magstim  (Magstim 200 Bistim2, Whitland, UK), Nexstim (eXimia NBS/T, Nexstim, 
Helsinki, Finland), or MagVenture (MagPro X100, Denmark)). The TMS coil will be positioned over the 
intersection of the central sulcus with the mid-saggital plane using the subject’s own anatomical MRI using 
an optical navigation system ( Localite GmbH, Brainsight, or Nexstim eXimia ). MRI images will be 
acquired prior to the first TMS laboratory visit at the Shirley Ryan AbilityLab Center or the Center for 
Translational Imaging, located across the street from the Shirley Ryan AbilityLab. Optimal coil position 
for producing a motor evoked potential (MEP) in the right leg muscle will be marked on the 3D-rendered 
cortical surface, ensuring consistent coil placement throughout and across sessions. Active MEP threshold 
will be determined as the lowest stimulus intensity producing MEPs just distinguishable above background 
in 5 of 10 consecutive trials during tonic contractions at ~10% of MVC. The conditioning stimulus, first 
pulse, will be set to 5% less than the active MEP threshold, an intensity below the level required to produce 
a recordable descending volley in the corticospinal tract. To ensure this stimulus level is sub-threshold, we 

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will analyze  a set of 10 stimuli online for evidence of evoked potentials. Conditioning stimulus strength 
will be lowered in 1% increments until no evidence is identified. The test stimulation intensity, second 
pulse, will be set to  produce MEPs of 500 - 1500µV in amplitude. While the subject maintains a tonic 
contraction at ~10% MVC  ten to fifteen trials of paired-pulse TMS will be delivered, while an additional 
20-25 test pulse trials (without a preceding conditioning pulse) will be randomly interspersed for the 
control. 
Strength-dexterity (SD) test 
The SD test requires that participants use their thumb and index fingers to compress a spring as 
far as possible before buckling. This test involves dynamic control of finger motions and force 
generation. The SD test consists of a custom spring (Century Springs Corp., Los Angeles, CA, 
USA) with two compression load cells (ELB4–10, Measurement Specialties, Hampton, VA, 
USA). The load cells are connected to a signal-conditioning box and USB-DAQ (National 
Instruments, Austin, TX, USA), sampled at 2000 Hz using custom Matlab (The Mathworks, 
Natick, MA, USA) software. Participants will be asked to compress the spring at their own pace 
to the point of maximal instability they can sustain (i.e., beyond which they feel it will slip out of 
their hand), and maintain that compression at a steady level for at least 5 s. After familiarization, 
10 trials will be performed by the dominant hand and the compression force, defined as the 
mean of the three maximal trials, will be used as the outcome measure. Participants will be 
allowed as many practice trials as needed to obtain steady state compression for the minimum 
required compression time of 5 s.  
 
AIM 4: The modulation of the synaptic transmission and plasticity of neuron and muscles. 
Small portions of the blood samples taken in AIM 1 will be used for analysis in AIM 4. 
The modulation of the synaptic transmission and plasticity of neuron and muscles might be caused by the 
changes of the neurotransmitter or muscle fiber in the transcriptional level, translational level, or 
combination of both.  We want to explore these changes through sequencing of whole exome and 
microRNA in addition to evaluating the expression level of mRNA and proteins related to synaptic 
transmission and muscle. 
This research study includes genetic testing.  Human blood contains genes that determine many of a 
person’s physical characteristics, such as the color of eyes and hair.  In some cases, genetic testing of 
blood can be used to indicate a risk for the development of certain diseases.  Genetic information is unique 
to each individual and could potentially be used to discover possible changes in a person’s future health 
status or life expectancy, or that of his/her children and family members.  
 
Releasing this information to the subject could cause psychological distress, anxiety or family problems.  
Releasing this information to others, such as including it in their medical record, may pose a possible risk 
of discrimination, or increase difficulty in obtaining or maintaining disability, long-term care, or life 
insurance.   

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These risks would occur if the subject’s information is released by mistake.  The measures being taken to 
protect the subject’s privacy are discussed below and make this possibility unlikely. 
 
Even though the results of genetic testing may not be linked to the subject, it is possible that people of 
their ethnic background may be found to be at more risk for certain diseases based on future genetic 
research and this information might harm the subject in the future as a member of the group. Also, there 
may be unknown risks of genetic testing in the future. 
 
DATA AND SPECIMEN BANKING AND MANAGEMENT: 
Paper questionnaires and signed consents will be kept in a locked cabinet at UT Southwestern Medical 
Center or UT Southwestern Lab (CS6.102A and H9.104, respectively), during data collection and analysis. 
Only listed key personnel specifically designated and authorized by the Principal Investigators shall have 
access to these documents. All personnel will be properly trained and supervised regarding the 
management and handling of confidential materials. All data (paper and electronic) will be destroyed 
within 5 years of data analysis completion. Serum samples will be collected UT Southwestern Medical 
Center or UT Southwestern Lab, which will then be analyzed at UT Southwestern Medical Center or UT 
Southwestern Lab. Samples will be labeled with subject number and date and time of testing. They will be 
held in temporary storage at UT Southwestern Medical Center or UT Southwestern Lab. Once the data 
collection is complete, the de-identified frozen samples at UT Southwestern Medical Center/Lab will be 
shipped to Dr. Ellen Casey to be analyzed for additional hormones, such as relaxin and cortisol, and their 
relationship to estradiol and progesterone. All blood samples will be destroyed 5 years after the 
completion of the study. Only members of the study team and those individuals responsible for the 
analysis will have access to the specimen samples. 
 
All necessary data pertaining to the study from SRAlab will be transferred to UT Southwestern Medical 
Center. Data (except for blood samples) will be maintained for future use, for indefinite amount of time. 
All data would be de-identified and stored in a protected database. Researchers only with IRB approval 
and approval from authorized personnel of this study, would have access to the controlled database of 
data, to be used to answer questions related specifically to their research study. 
 
SAMPLE SIZE JUSTIFICATION 
AIMS 1 and 2:  We wish to rule out an effect of hormonal state on motor neuronal excitability. To do this, 
specified the minimum effect size that we considered meaningful along with high statistical power that 
minimized the chances of a Type-I error. We conducted statistical simulations to evaluate this scenario. 
Pooling N = 30 OCs and N = 30 nOCs that provide daily H-refelxes from soleus muscle (Aim 1) and MSR 
from 3 quadricepts muscles (VM, VL, RF) over one menstrual cycle provided power = .95 to detect effects 
as small as an average within-individual correlation of  r = -0.075 (R2 = .005) between a single hormone 
and reflex responses across a range of situations. Smaller differences are considered as pragmatically 
meaningless. This degree of power holds even if menstrual cycles vary from 22 to 32 days, and 
participants miss an average of 25% (see above) of their lab visits (and assumes the data are missing at 
random). 
AIM 3: To test hypothesis 3, that estradiol will produce a targeted facilitatory influence on lower limb 
muscles, we used a GEE model that views the mean conditioned MEPs at each ISI as correlated outcomes 
before separate post-hoc tests for each inter-pulse interval (ISI = 7, 10, 15, 20, 25, 30 ms) and each subject 
between peak estradiol and baseline hormone concentration (Fig. 7). This synthesis establishes a within-

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subject control. Based on preliminary data of four subjects, at each of the 6 ISIs >7 ms (see Fig. 6), a 
sample size of 20 subjects achieved over 90% power to detect a main effect for a GEE model that pools 
the ISIs as correlated outcomes. Additionally, we have sufficient power (.80) to follow up a main effect, 
for each of the six ISIs, to detect differences of 40% or larger from the null hypothesis, with an estimated 
standard deviation of 37% and with a significance level (alpha) of a Bonferroni-corrected 0.05/6 = 0.008. 
To allow for attrition we will aim to enroll 30 subjects in each female cohort.  To ensure that our within-
subject changes can be attributable to changes in the hormonal milieu, we will enroll N = 15 males and test 
them at multiple occasions. 
 
 
4.b.  List of the study intervention(s) being tested or evaluated under this protocol 
 
N/A -  this study does not test or evaluate an intervention.    Skip to item 4.d. 
# 
Study intervention(s) being tested or evaluated under the protocol 
 
 
 
Add or delete rows as needed 
 
Affiliate 
 
Place a check next to 
institution(s) where the  
intervention will be 
performed 
Local Standard 
Practice?  
Indicate whether the 
intervention is 
considered 
acceptable practice 
locally for applicable 
institutions 
1 
Insert study intervention 1 here 
☐ UTSW 
☐ Yes 
☐ PHHS 
☐ Yes 
☐CMC 
☐ Yes 
☐THR 
☐ Yes 
☐TSRH 
☐ Yes 
☐ Other: _____ 
☐ Yes 
2 
Insert study intervention 2 here 
☐ UTSW 
☐ Yes 
☐ PHHS 
☐ Yes 
☐CMC 
☐ Yes 
☐THR 
☐ Yes 
☐TSRH 
☐ Yes 
☐ Other: _____ 
☐ Yes 
 
 
4.c.  Risk:Benefit Analysis of study interventions being tested or evaluated under this protocol 
For each study intervention identified in section 6b above, complete a risk:benefit analysis table. 
 
(Two tables are provided, copy & paste additional tables as needed or delete both tables if this study does not test an 
intervention) 
 
 

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4.c. 
Study Intervention #1 
Insert name used in 4.b. 
List each group exposed to this 
intervention on a separate line. 
(e.g., experimental, control, Arm A, Arm B, 
etc) 
Or state All Groups/Subjects 
For each group, list the benefits of this intervention.  (Benefits can be directly from 
the intervention or from a monitoring procedure likely to contribute to the subject’s 
well being).  If there are no benefits, state “none”. 
 
 
 
 
If you are requesting a Waiver of Informed Consent, complete the table below. 
 
If you have a consent form, list the reasonably foreseeable risks in the consent form (and do not complete this section).   
 
List the risks according to the probability (likely, less likely or rare) and magnitude (serious or not serious). 
(include: 1) expected adverse events; 2) rare and serious adverse events; 3) all other psychological, social, legal harms) 
Do not delete frequency.  Frequency must be estimated because it will assist you with determining which adverse events will require 
prompt reporting.   
 
Not serious 
Serious  
Likely 
These risks are expected to occur in 
more than 20 out of 100 subjects. 
• 
 
• 
 
 
Not serious 
Serious 
Less likely  
These risks are expected to occur in 5-
20 subjects or less out of 100 subjects. 
• 
 
• 
 
 
 
Serious 
Rare  
These risks are expected to occur in 
less than 5 subjects out of 100  
 
• 
 
 

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4.c. 
Study Intervention #1 
Insert name used in 4.b. 
List each group exposed to this 
intervention on a separate line. 
(e.g., experimental, control, Arm A, Arm B, 
etc) 
Or state All Groups/Subjects 
For each group, list the benefits of this intervention.  (Benefits can be directly from 
the intervention or from a monitoring procedure likely to contribute to the subject’s 
well being).  If there are no benefits, state “none”. 
 
 
 
 
If you are requesting a Waiver of Informed Consent, complete the table below. 
 
If you have a consent form, list the reasonably foreseeable risks in the consent form (and do not complete this section).   
 
List the risks according to the probability (likely, less likely or rare) and magnitude (serious or not serious). 
(include: 1) expected adverse events; 2) rare and serious adverse events; 3) all other psychological, social, legal harms) 
Do not delete frequency.  Frequency must be estimated because it will assist you with determining which adverse events will require 
prompt reporting.   
 
Not serious 
Serious  
Likely 
These risks are expected to occur in 
more than 20 out of 100 subjects. 
• 
 
• 
 
 
Not serious 
Serious 
Less likely  
These risks are expected to occur in 5-
20 subjects or less out of 100 subjects. 
• 
 
• 
 
 
 
Serious 
Rare  
These risks are expected to occur in 
less than 5 subjects out of 100  
 
• 
 
 

Form A 
IRB 
Velos 
STU-2018-0192 
26783 
 
STU2018-0192, Dhaher, FormA-ResearchProtocol, Mod_13, 05-08-20 
 
12 
vMar2018 
 
4.d. List ALL other research procedures or components not listed in table 4.b. 
        The combination of Tables 4b and 4d should account for all of the research 
procedures that will take place during this study. 
 
Consider grouping similar procedures under a single component (e.g., blood work, CT = safety 
assessments) 
 
 
 
# 
Research component 
• 
individual 
procedures 
 
example: 
Eligibility Assessments 
• 
History and 
physical 
• 
Questionnaire 
• 
Laboratory tests 
Add or delete rows as needed 
Column A 
Local Standard 
Practice Indicate the 
number of times each 
procedure will be 
performed as stipulated 
in the research plan that 
would be performed if 
the participant were not 
participating in the 
study. 
Column B 
Research Only  
Indicate the number of times 
each procedure will be 
performed solely for research 
purposes (meaning that the 
participant would not undergo 
the same number of 
procedures or would not 
undergo the procedure(s) at 
the same frequency if they 
were not participating in the 
study) 
Column D 
Risks 
If you are requesting a Waiver of Informed 
Consent, complete the table below. 
 
List the reasonably expected risks for each procedure 
or group of procedures under the following categories 
as appropriate: 
• Serious and likely; 
• Serious and less likely; 
• Serious and rare; 
• Not serious and likely; 
• Not serious and less likely 
1 
Aim 1 
N/A 
Every other day, for a 
menstrual cycle 
N/A 
 
Blood draw for Estradiol, 
Progesterone, 
andTestosterone 
 
 
 
 
Reflex testing 
 
 
 
 
Anterior Knee Laxity 
Measurement 
 
 
 
2 
Aim 2 
N/A 
Every other day, for a 
menstrual cycle 
N/A 
 
Blood draw for Estradiol, 
Progesterone, and 
Testosterone 
 
 
 
 
EMG/Nerve conduction 
Testing 
 
 
 
3 
Aim 3 
 
 
 
 
TMS 
N/A 
5 – 6 times in a 
menstrual cycle 
N/A 
 
MRI 
N/A 
once 
N/A 
 
Strength-dexterity test 
N/A 
5 – 6 times 
N/A 
4 
Aim 4 
N/A 
Every day for a 
menstrual cycle, until 
ovulation 
N/A 
 
Blood analysis on 
microRNA,  
N/A 
2 
N/A 
 
Blood analysis for DNA 
sequencing 
N/A 
2 
N/A 
 
Blood analysis on mRNA 
N/A 
2 
N/A 
5 
Ovulation testing 
 
 
 
 
Urine Ovulation Kit  
 
1. 
 
 
 
 

Form A 
IRB 
Velos 
STU-2018-0192 
26783 
 
STU2018-0192, Dhaher, FormA-ResearchProtocol, Mod_13, 05-08-20 
 
13 
vMar2018 
 
5.  Safety Precautions.  (Describe safeguards to address the serious risks listed above.) 
a.  Describe the procedures for protecting against or minimizing any potential risks for each of the more than minimal risk 
research procedures listed above. 
1. 
Skin irritation – cleaning the skin with alcohol prior to placing surface electrodes and tape will help minimize the skin 
irritation associated with the use of surface electrodes 
2. 
Muscle twitching and discomfort – taking breaks during sessions will lessen the discomfort; subjects also have the 
option to stop the testing 
3. 
MRI – there are no known serious risks associated with MRI for subjects without contraindications to the test. 
However, some may become claustrophobic, anxious, or fatigued. Subjects will be in constant communication with 
the study staff/technician throughout the procedure and they have the option to stop the MRI at any time;  
4. 
Noise (from the TMS/MRI procedures) – noise-cancelling ear buds will be provided 
b.  Where appropriate, discuss provisions for ensuring necessary medical or professional intervention in the event of adverse 
events, or unanticipated problems involving subjects. 
The laboratory at H9.104 is adjacent to the James W. Aston Ambulatory Care Center, which is fully equipped to handle the 
very rare probability of adverse events or unanticipated problems involving subjects. 
c.  Will the safeguards be different between/among groups?   
Yes 
 
No