Alternating Cycle / Direct Confirmation Redox Project (AC/DC)
Conditions: Pregnancy Related, Gender
Sponsor: University of Michigan

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Title of Study: Alternating Urine Redox Status of Consecutive 
Menstrual Cycles [Uro-001] 
Sponsor: Urobiologics LLC, 31628 Glendale St., Livonia MI 48150 
Date of Final Protocol: October 16, 2019 
 
Approved by: 
For the Sponsor: 
[Signature on File] 
  
 10/16/19 
Kuldeep Verma, PhD 
President, Urobiologics, LLC 
 
 
Principal Investigator: 
[Signature on File] 
  
 10/16/19 
Dr. Daniel S. McConnell, PhD 
Associate Research Scientist, Department of Epidemiology 
University of Michigan School of Public Health 

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Title of study: 
Alternating Urine Redox Status of Consecutive Menstrual Cycles [Uro-001] 
 
Name of Sponsor: 
UROBIOLOGICS, LLC 
 
Population: 
40 Female subjects 
 
Estimated duration of study: 
November 2019 – October 2020 
 
Objective: 
The Sponsor believes there is variability in the relative redox status in women’s urine across 
menstrual cycles. This validation study will involve the collection and analysis of urine samples 
from non-pregnant women to determine the existence of alternating redox status between two 
consecutive menstrual cycles. If the hypothesis is proved, future research could examine if there 
are related fertility issues. 
 
Study design synopsis: 
Approximately 40 women with normal reported menstrual cycles will be included in this validation 
study. 
All subjects will receive an explanation of the study and what is asked of them. They will sign 
informed consent and then undergo screening for study eligibility. If subjects fail the screening 
evaluation for reasons related to incidental transitory conditions yet wish to participate, they may 
subsequently be re-screened again after providing informed consent. 
Subjects who meet the entry criteria (along with providing informed consent) will receive written 
instructions for participation in the study; an information sheet for logging their period dates, 
tracking medications and fevers, and instructions for returning the samples; and all supplies needed 
for collecting the urine samples (collection kits). 
Subjects will collect urine over two days at the mid-point of their menstrual cycles (see Appendix 
1) until three separate pairs of two consecutive cycles are obtained (requiring a minimum of 6 
months of participation in the study). Subjects may receive telephone calls, texts, or other 
reminders to obtain the urine, as long as the reminder method is acceptable to the subject. 
Urine specimens will be delivered to the University of Michigan CLASS Laboratory or to a second 
location for transfer to the CLASS Laboratory; if necessary, specimens may be sent through postal 
or carrier services. In the CLASS Laboratory, aliquots of urine will be drawn from the specimens, 
randomized and blinded in consecutive cycle pairs, and sent to Urobiologics for analysis. 
Urobiologics will send the reports of results to the CLASS Laboratory. 
When all samples have been collected and analyzed, the blind will be broken, the data coded into 
spreadsheets, and statistical analysis will be performed. The principal hypothesis is that women 
have menstrual cycles that alternate between opposite redox states. 

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Inclusion/Exclusion Criteria: 
Inclusion criteria: 
1. Female subject, between the years 18-30, at the time of consent. 
2. Subject reports “regular” menstrual cycles (i.e., cycle is 25 to 35 days and subject can typically 
predict onset of menses within ± 3 days). 
3. Subject may be sexually abstinent or sexually active; if sexually active, she should be trying to 
prevent pregnancy using non-hormonal methods such as condoms. 
4. Subject is willing to participate and is capable of giving informed consent. (Note: Consent 
must be obtained prior to any study-related procedures.) 
5. Subject must be willing to comply with all study procedures. 
6. Subject agrees to avoid alcohol, unapproved medications (prescription, OTC, or other), 
marijuana products, and illicit substances for 3 days prior to each urine collection. If 
medications must be taken, the collection will be discarded and another opportunity offered. 
Exclusion criteria: 
1. Subject is pregnant, or trying to or planning to become pregnant during the study. 
2. Subject was pregnant within 1 year of beginning study, regardless of the outcome of 
pregnancy. 
3. Subject who is breastfeeding. 
4. Subject who is not menstruating for any reason or plans to undergo or change circumstances 
that might affect menstruation (e.g., certain athletic training regimens, significant dietary 
changes). 
5. History of recent or current irregular menstrual cycles (as self-reported by the subject). 
6. Subject reports she has been told by a medical professional that she is not ovulating (i.e., has 
anovulatory periods) at any time during the previous 12 months. 
7. Subject is using or planning to use any method to regulate her periods. 
8. Subject smokes (includes tobacco and marijuana) or vapes. 
9. By history, subject has medical problems that might influence the results of the study, such as 
endocrinologic, renal, or hormonal problems. 
10. Known polycystic ovarian syndrome (by history). 
11. Subject is taking or planning to take hormones or hormone- enhancing/manipulating 
pharmaceuticals (including “morning after pill” [levonorgestrel], birth control pills, Clomid 
[clomiphene citrate], Femara [letrozole], Cialis [tadalafil]*, Viagra [sildenafil]* and similar 
medications) and/or agents to influence hormonal status such as herbal products or nutritional 
supplements. (*Not approved for use and should be avoided by women.) 
12. Subject is using or planning to use hormonal contraceptives of any type, including oral 
contraceptives and intrauterine devices. 
13. Subject is using or planning to use hormonal therapies for acne vulgaris or other conditions, 
including spironolactone, flutamide, metformin, or oral contraceptives used as acne therapies, 
including Ortho Tri-Cyclen (combination of norgestimate and ethinyl estradiol [EE]), 
Estrostep (combination of norethindrone acetate and EE), and Yaz/Yasmin (combination of 
drospirenone and EE). 

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14. Subject is using or planning to use estrogen products, including topicals/creams, for any 
purpose. 
15. Subject is using or planning to use androgens or anabolic steroids (e.g., stanazolol/danazol) for 
any reason, including for training or athletic purposes.. 
16. Subject has attempted or taken IVF or IUI treatment within the prior six months. 
17. Subject has uncontrolled diabetes or other significant medical disorder that might, in the 
investigator’s opinion, preclude completion of the study or suggest that an exclusion criterion 
will ensue. 
18. Subject reports difficulty handling biologic fluids, specifically urine, or gives other reasons 
why she is hesitant to participate. 
 
Methods for subjects to collect urine samples: 
See Appendix 1 for instructions to subjects and methodology. 
 
Delivery of samples to UM research laboratory: 
See Appendix 1. 
 
Processing of samples in UM research laboratory: 
Preparation of samples: 
1. Upon receipt of the sample collection kit from each subject, check to ensure that the bag label 
has been transferred to the Sample Collection Form. Verify that the urine barcode numbers 
listed on the sample sheet match the numbers on the urine vial. Assure that two separate 30 
mL vials are present from the same subject, one labeled “A” or “#1” and one labeled “B” or 
“#2”, AND that they contain dates consistent with being obtained during two consecutive 
menstrual cycles (i.e., about 25-35 days difference), AND that the subject so states on the form 
filled out by the subject. A difference in days outside the range of 25 to 35 days could 
legitimately occur, but if the gap is not within 25-35 days, ask Dr McConnell to investigate. 
2. Indicate date of receipt of samples on the Sample Collection Form. 
3. Obtain a ACDC Aliquot Kit. Transfer the LG Bag Label to the Sample Collection Form in the 
space below the Subject ID, Date, and Time. 
4. Invert repeatedly to ensure homogeneity. 
5. Carefully harvest the urine from the 30 mL “A or #1” vial and pipet 10 mL into one yellow- 
top polypropylene 10 mL tube labeled with the predetermined randomization number. Tube 
will also be labeled “Biologic research specimen. Human urine.” 
6. Cap and place the 10 mL vial in its corresponding numbered box. 
7. Repeat steps 4-6 for the 30 mL “B or #2” vial. 
8. Write the date, time and the technologist’s initials on the Sample Collection Form. Also include 
the tube numbers, volume and number of tubes. 

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9. Place the box containing the 30 mL vial and the randomized yellow-top tubes into the room 
temperature ACDC storage cabinet. 
10. File the Sample Collection Form in the ACDC sample binder. 
 
Quality assurance (QA): 
Under the direction of the study statistician, at other times, samples may be aliquoted from the 30 
mL vials and given randomization numbers (or facsimiles thereof) by following steps 4-6 above. 
Such specimens will come from subjects’ consecutive cycles but may not necessarily be paired in 
the same way as the principal samples. For example, if previous randomized samples were from 
Subject X in months 1&2 and 3&4, then QA could include sending samples from Subject X’s 
months 2&3 and/or repeat samples from the original months such as 3&4. Such QA may be 
performed and analyzed at interim stages of the protocol only if it does not break the blind of the 
principal study. Repeated failure to be in conformation with the hypothesis during QA may result 
in early termination of the study, if the statistician and principal investigator agree. 
 
Shipping of samples: 
When sufficient samples are obtained, the 10 mL yellow-top tubes labeled “Biologic research 
specimen. Human urine.” with random numbers affixed AND NO OTHER INFORMATION 
AFFIXED (specifically excluded is any information that could in any way identify the 
participating subject by name, birthdate, or any other identifying information) will be sent (by mail 
or other approved delivery method) for processing to Urobiologics LLC, 31628 Glendale St., 
Livonia MI 48150. THE GREEN-TOP VIALS THAT SUBJECTS USED FOR THEIR 
COLLECTION OF URINE MUST REMAIN LOCKED IN THE C.L.A.S.S. LABORATORY 
AND MAY NOT BE SHIPPED OUT (because they may contain identifying information about 
the subjects or might otherwise “break the blind”). 
 
WHEN THE SUBJECT BELIEVES THAT SHE WILL LIKELY SHIP HER SPECIMENS BACK AND 
NOT RETURN SAMPLES IN PERSON, THE STUDY WILL PROVIDE THE APPROPRIATE 
MATERIALS TO SHIP THE SPECIMEN THAT COMPLY WITH ALL DOT REGULATIONS. THE 
STUDY WILL PROVIDE THE TRIPLE PACKAGING (1- VIAL, 2 SEALED BAG, 3- ENVELOPE) 
RETURN ENVELOPES WITH PREPAID RETURN SHIPPING LABEL. THESE URINE SAMPLES 
ARE CONSIDERED INFECTIOUS SUBSTANCES CATEGORY B (SEE NOTE BELOW), 
THEREFORE TO COMPLY WITH DOT REGULATIONS THE SAMPLE MUST CONTAIN THE TEXT: 
“EXEMPT HUMAN SPECIMEN” WHICH MUST BE VISIBLE ON OUTSIDE OF ENVELOPE. THIS 
EXEMPT STATUS IS FOR SPECIMEN SHIPPING ONLY AND DOES NOT IMPLY ANY IRB 
EXEMPTION. 
URINE IS CONSIDERED UNDER INFECTIOUS SUBSTANCES CATEGORY B 
 
MATERIALS THAT ARE INFECTIOUS BUT DO NOT MEET THE CRITERIA FOR INCLUSION 
IN CATEGORY A 
ASSIGNED TO UN 3373 (CATEGORY B, INFECTIOUS SUBSTANCES) 
HUMAN OR ANIMAL MATERIAL TRANSPORTED FOR RESEARCH, DIAGNOSIS, DISEASE 
TREATMENT, ETC. 
 
PATIENT SPECIMENS FOR WHICH THERE IS MINIMAL LIKELIHOOD THAT 
PATHOGENS ARE PRESENT AND ARE TRANSPORTED IN A PACKAGING WHICH 

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WILL PREVENT ANY LEAKAGE ARE CONSIDERED “EXEMPT HUMAN SPECIMEN” 
OR “EXEMPT ANIMAL SPECIMEN”.  
 
THEY MUST BE LABELED AS “EXEMPT HUMAN/ANIMAL SPECIMEN”.  
THEY MUST USE TRIPLE PACKAGING SYSTEM. 
 
 
Processing of samples in Urobiologics laboratory: 
Urine samples are processed to remove non-hormonal redox compounds and then reacted with a 
redox indicator to evaluate each sample in the pair of urines as an oxidative or a reductive reaction. 
Results are reported to the UM C.L.A.S.S. Laboratory according to the randomized numbers 
provided with the samples. 
 
At the conclusion of the study the residual de-identified urine will be shipped to the Sponsor.  Upon completion of 
processing, the sponsor will destroy all remaining materials and verify the destruction and disposition to the class 
laboratory. 
 
Statistical power analysis and planned stat analysis: 
From each experimental subject, we will have three non-overlapping pairs of consecutive urine 
samples taken. For each sample pair a blinded determination will be made by Urobiologics as to 
which sample in the pair was reductive. For the sake of simplicity, consider the case where these 
samples are collected in 6 consecutive months. Let “A” be the event that the reductive sample in 
the pair corresponds to the earlier cycle and “B” its converse. Then, the hypothesis of alternating 
reductive cycles is supported if the sequences AAA or BBB are over-represented relative to others. 
To analyze this, we will map whether the 2nd and 3rd pairs are successfully determined conditional 

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on the first. Let “Y” denote success and “N” denote failure. Each subject then has one of the four 
patterns: YY, YN, NY, NN representing whether the 2nd and 3rd paired cycles are correctly 
predicted by the alternating cycle hypothesis. The hypothesis is supported by over-representation 
of YY relative to chance if predictions were made under the null of P(Y) = 0.5 with successive 
pairs called independently. 
Consequently, we will first use a chi-squared goodness of fit test to evaluate the hypothesis below 
at the α=0.01 level: 
H0: p = 0.5 
and P(YY) = p*p; P(YN) = P(NY) = p*(1-p); P(NN) = (1-p)*(1-p). 
Under the alternative that p = 0.75, 37 subjects are needed to give 90% power to reject the null 
hypothesis at a significance level of 0.01. Assuming that <10% of enrolled subjects will be lost to 
attrition, we need to enroll a minimum of 40 subjects to achieve the target sample size. 
Subsequently, we will estimate “p” in the model above using the total proportion of “Y” and form 
a confidence interval for this estimate using a bootstrap procedure to account for the pairing of 
samples within women. As a secondary analysis, we will use a likelihood ratio test to evaluate the 
validity of the independence assumption by comparing the one-parameter independence model to 
the four-parameter model with unique cell probabilities. Finally, for exploratory analyses to 
generate future hypotheses we will fit a mixed logistic regression in which the log odds of outcome 
“Y” are expressed as a linear function of any covariates to be explored and a random intercept 
inducing correlation between repeated measures within each woman. 

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Study Flow Chart 
 
Study 
Procedure 
Screening 
Cycle 
1 
Cycle 
2 
Cycle 
3 
Cycle 
4 
Cycle 
5 
Cycle 
6 
All 
samples 
processed 
Informed Consent 
X 
Inclusion/Exclusion form 
X 
Medical History/ Demographics 
X 
Assign a Subject Number 
X 
Give three urine collection kits to 
subject. 
Each kit will contain two units. The 
two units provide for two collections 
about a month apart for each kit. 
Each unit will have one cup, one 
green-cap vial, one dropper, one 
instruction sheet, and one form to fill 
out with period start date and dates of 
collection of sample along with other 
relevant queries and instructions for 
returning the samples. 
When the subject believes THAT she 
will likely ship her specimens back and 
not return samples in person, THE 
STUDY WILL PROVIDE THE 
APPROPRIATE MATERIALS TO 
SHIP THE SPECIMEN THAT 
COMPLY WITH ALL D.O.T. 
REGULATIONS. THE STUDY WILL 
PROVIDE THE TRIPLE 
PACKAGING (1- VIAL, 2 SEALED 
BAG, 3- ENVELOPE) RETURN 
ENVELOPES WITH PREPAID 
RETURN SHIPPING LABEL. 
THESE URINE SAMPLES ARE 
CONSIDERED INFECTIOUS 
SUBSTANCES CATEGORY B 
(SEE NOTE BELOW), 
THEREFORE TO COMPLY WITH 
D.O.T. REGULATIONS THE 
SAMPLE MUST CONTAIN THE 
TEXT: “EXEMPT HUMAN 
SPECIMEN” WHICH MUST BE 
VISIBLE ON OUTSIDE OF 
ENVELOPE. THIS EXEMPT 
STATUS IS FOR SPECIMEN 
SHIPPING ONLY AND DOES NOT 
IMPLY ANY IRB EXEMPTION. 
X* 
Determine if subject accepts reminders 
X 
Remind subjects at least twice in each 
cycle (when and as agreed by subject) 
X 
X 
X 
X 
X 
X 

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Receive urine samples, process in 
CLASS Lab per protocol. 
X 
X 
X 
Send urine samples to Urobiologics 
IN BATCHES OF SAMPLES 
Code data from Urobiologics 
AS SAMPLES ARE REPORTED 
Quality assurance per protocol 
AS SAMPLES ARE AVAILABLE 
Break blind; statistical analysis 
X 
*At any time, provide more kits if needed. 

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APPENDIX 1: Instructions to subjects: 
 
HOW TO COLLECT URINE SAMPLES 
 
This study may help future women with fertility issues. Because this topic is so important, 
please follow the directions carefully. Call us with any questions at (734) 763-2461. 
 
It is very important that samples be collected over two nights during consecutive periods 
-- otherwise the result may be confusing or misleading. 
 
If you make a mistake, call us and we will see if we can fix it together. If you collect urine 
in one period and forget to collect in the next cycle, please let us know right away. Again, 
we will work with you to get enough pairs of urine collections to complete the study. If you 
don’t tell us, the entire study could be ruined for everyone. 
 
If you develop fever, flu, or a virus at the time of collection, make a note of it on the sheet 
we gave you. If you can get your actual temperature, please write it down. Give us a call 
during business hours and we will advise you if you should reschedule your urine 
collection. 
 
If you are taking some regular medicines, it would be better to stop taking them 2 days 
before and resume them after collecting the overnight samples. Please consult with your 
doctor about this and get permission to stop for two days. If you can avoid unnecessary 
doctor visits during the collection time, it is best because you may receive a treatment 
that affects the research test. Of course, if you need to see a doctor, please do so. Record 
any medications, drugs, over-the-counter products, etc. that you are taking on the sheet 
provided. 
 
To assess our research theory, we need overnight mid-cycle urine samples from two 
nights to be combined into one vial during your period. Then we need this repeated in 
your very next period. In each period, you collect your urine overnight for any two nights 
from the 13th to 17th night after the START of your period. The start of your period is the 
first day of bright red bleeding. Earlier spotting is not considered the first day. 
 
(Example: Suppose your first day of bright red bleeding occurs on Jan 5th. You will collect 
your urine on any two nights from Jan 18, 19, 20, 21, and 22 – that is, 13 to 17 nights 
after the first day. If you collected on the 13th night but forget to collect on the 14th night, 
you can still collect the second night on the 15th, 16th, or 17th night. It is any two nights 
during the 13th to 17th night following the START of your period.) 
 
SUMMARY: 
 
At the mid-point of your period, collect all  urine  over  2  different nights.  Collect all 
urine after you go to bed including the first urine of the  morning.  Put a small  amount  
of each urine collection into the green-top vial. If necessary, top off the green-top vial. 
Repeat in your next period. The two green-top vials are what we need for our research. 

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How do I collect the urine over 2 different nights on days 13 to 17 of my period? 
 
First, set out on the back of your toilet (or nearby) the large plastic collection cup, the 
dropper, and the resealable green-top vial. 
FOR  THESE  FIRST  CYCLE  COLLECTIONS,  THE   GREEN-TOP   
VIAL MUST BE LABELED “#1”. 
We need your urine for the entire night. Before going to bed, pass urine into the toilet. 
Do not collect this -- flush it away. Collect all urine every time you go to the bathroom after 
this, including your urine when you get up in the morning. 
 
The way to do this is to: 
1. Pee (urinate) into the toilet for 2 to 3 seconds 
2. Collect the rest of the urine in the provided plastic collection cup (called “midstream” 
urine). 
3. Then transfer urine from the cup to the resealable green-top vial, using the dropper 
provided. Each time you pee, put some of the urine into the green-top vial as follows: 
 
If you wake up during the night, collect any midstream urine in the collection cup. 
 
4. Take two dropperfuls (total about 6 ml or a bit more than a teaspoon) of the urine 
from the cup and squirt the dropperfuls into the green-top vial provided. You do not 
have to be exact. 
5. Close the vial. 
6. Empty the extra urine left in the collection cup into the toilet and flush the urine away. 
 
When you get up in the morning, collect midstream urine in the collection cup. 
 
7. Again take two dropperfuls from the cup and squirt them into the same green-top 
vial. 
8. If you did not urinate during the night, you will have collected only your morning urine 
in the cup so put FOUR dropperfuls into the green-top vial. 
9. Close the vial. 
10. Empty the extra urine left in the collection cup into the toilet and flush the urine away. 
11. If you wish, rinse the collection cup with tap water and allow it to dry. 
12. Keep the materials at hand for the next night’s collection. 
 
The green-top vial should be about half-full after the first collection night. Make sure 
the cap is on securely and the vial will be leak proof. 
 
Your morning urine is your last collected sample for the day. Do not collect any samples 
during the day. 
 
Repeat during the next night (or, if necessary, another night within the 13 to 17 day 
window of this period). Again, put the materials you need nearby, including the green-top 
vial #1. When you go to bed, flush away the urine you pee. Collect any urine during the 

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night and when you get up in the morning. Add dropperfuls of urine to the SAME green- 
top vial in the same way as above. 
 
With two nights’ collection, you will have approximately 30ml of urine in the vial. The vial 
will be nearly full (near the 30ml mark on the vial). If you have put in less than that amount, 
you can make it up by adding some more urine from the last sample. After that, you may 
throw away the dropper and the collection cup. TIGHTEN THE CAP ON THE GREEN- 
TOP VIAL AND KEEP IT. You will not be adding anything more to it. 
 
Your first menstrual cycle sample is ready! Please be sure you have entered the date of 
your period’s first day and the dates of urine collection on the sheet provided. 
 
Store the green-top vial labelled “#1” at room temperature until after the next collections 
in your next period. You may put a “Do not open” Post-it note on the cap or similar to 
remind you not to add anything else to this vial. Your urine samples won’t go bad at room 
temperature. No preservative is needed. 
 
Please keep your completed green-top vial, these instructions, the sheet you are filling 
out, and the remaining collection items where you will find them during your next period! 
As a reminder, you can find these instructions attached to the Consent Form you signed. 
Call us during business hours at (734) 763-2461 if you need more collection materials or 
if you have ANY questions. 
 
How do I collect the urine over 2 different nights on days 13 to 17 of my next period? 
 
In your next period, repeat the process. Collect your urine in the same way as above. 
BUT THIS TIME PUT THE DROPPERFULS OF URINE in the second green-top vial 
LABELLED “#2”. Finish filling out the information sheet. Now you can hand-deliver or ship 
both green-top vials (“#1” and “#2”) and your information sheet all together to one of our 
University of Michigan campus offices. Instructions are in the sheet provided. 
 
We hope you will participate for a total of 6 menstrual cycles (3 times with 2 consecutive 
periods). Thank you for your help with our research. We appreciate your effort!