Endometriosis and Brown Adipose Tissue Activity

to measure BAT. However, as this test relies on glucose, anatomy doesn’t always parallel activity, especially, apparently, in states of insulin resistance 12. For that reason, experts are now recommending that BAT activity is best assessed by measuring changes following standardized non-shivering cooling as assessed using infrared thermography 26. Another human study of BAT activity in women with PCOS measured supraclavicular (BAT anatomy) skin temperatures versus deltoid (control) skin temperatures and found the greatest differences during sleep 27. A further study used Z-spectral imaging in women with PCOS and showed low BAT activity 28. Animal models of PCOS have also shown lower BAT activity 29. As mentioned, androgenic polycystic ovary syndrome (PCOS) 30 and endometriosis (EndoM) 31 have been proposed to be diametrically opposite reproductive disturbances 32. PCOS is a multidimensional women’s reproductive and metabolic condition characterized by androgen excess, long/irregular menstrual cycles and/or multiple follicular ovarian cysts30 and tends to be associated with obesity and insulin resistance. EndoM is also a common premenopausal reproductive condition in approximately 10% of women; it is characterized by ectopic endometrial-like tissue in the pelvis, ovary or other anatomical sites. EndoM is associated with chronic abdominal/pelvic pain and infertility 33. By contrast to women with PCOS, women with EndoM tend to have lower or normal body weights 34 35. Despite the strong diversities of these two conditions 32, the literature also has documented that those with PCOS have EndoM more commonly than do controls 36,37. In addition, both EndoM38 and PCOS39 are associated with infertility, as well as with ovulatory disturbances and lower-than-normal progesterone levels. Women with PCOS tend to develop insulin resistance, Type 2 Diabetes Mellitus (T2DM) and central or visceral obesity with increased waist circumference (WC). Although, in general, BMI is inversely related to the BAT anatomical area or activity, it is now increasingly clear that insulin resistance is associated with decreased BAT activity 12,40. A recent review asserted that higher BMI and insulin resistance might not change the PET assessment of BAT anatomy but may decrease BAT activity12 (as discussed earlier). BAT, by the new radiographic technique of Z-spectral imaging, showed both smaller area and possibly decreased activity the higher the fat content of the brown adipose tissue 28. That investigation studied Chinese participants; 13 women had PCOS as well as being overweight/obese, 19 women were controls and 17 were men; none had diabetes, but all with PCOS had insulin resistance 28. Results showed greater fat within brown adipose tissue in those with PCOS; that may be related to lower BAT activity 28. The other BAT study in humans with PCOS measured resting (non-stimulated) supraclavicular and upper arm temperatures as a simple way of estimating BAT activity in humans, with the biggest difference being recorded during sleep 27. This cross-sectional study without cycle-phase documentation in 47 women with PCOS (mean BMI 29, waist circumference 101 cm) and 11 women controls (mean BMI 25, WC 77) found that PCOS supraclavicular temperatures, especially at night, were lower than in controls. The lower nighttime supraclavicular than deltoid temperatures in PCOS than in controls had a significant negative relationship with free Version4 H23-03963 September 25, 2024 Page 8 of 22 testosterone levels; however, the temperature difference in the two skin areas was minimally positively related to BMI 27. An important observation, however, was that control, upper arm temperatures did not differ between the three groups. Another human study using PET showed that BAT anatomy had a lower area in women with PCOS than in BMI- and age-matched controls 41. Thus, at least BAT area was negatively related to central adiposity 41. As mentioned previously, one of the anthropomorphic differences between PCOS and EndoM is that the latter tend to be of normal weight 42. Thus, this has increased speculation that PCOS and EndoM may differ in BAT activity. Women with PCOS tend to have more prevalent overweight/obesity, increased WC (that reflects increased visceral as well as subcutaneous fat), and metabolic syndrome. All of these are associated with insulin resistance 43. The key question in support of the divergent relationships of PCOS and EndoM then is: What is the activity of BAT in women with EndoM? The purpose of this research is to document brown adipose tissue cold but non-shivering activity for the first time in women with known endometriosis. In addition, we will determine whether BAT activity in women with EndoM differs from healthy control women who are of similar weight and similarly menstrual cycle characteristics. 1.2 Relevant Literature and Data Endometriosis (EndoM) is a relatively common (9% of a population-based sample of women at age 44 44) condition in which ectopic endometrium-like tissue found outside the uterus is associated with severe menstrual cramps (secondary dysmenorrhea), pain with intercourse (deep dyspareunia), pain with defecation (dyschezia) and urination (dysuria) and also with fatigue and chronic pelvic pain 45. EndoM has been described as an “estrogen-dependent chronic inflammatory process” with a deficiency of progesterone receptors 46. EndoM is understood to be progesterone-resistant, but this assertion is not well-documented. It is also commonly associated with primary infertility 47. Many symptomatic women spend years and see multiple physicians before being diagnosed with EndoM much less, before being effectively treated 48. The pathophysiologic etiology of EndoM is currently unclear but it is associated with early menarche, heavy menstruation, shorter cycles and nulliparity 49; all of these reproductive characteristics are associated with higher estradiol levels. It is currently unclear whether part of the estrogen-progesterone imbalance 50 in EndoM is not only related to higher estradiol values but also to more prevalent subclinical ovulatory disturbances (normal length and clinically normal menstruation but short or insufficient luteal phases, or anovulation [SOD]) 51,52. No prospective studies of ovulatory characteristics in untreated women with documented EndoM are available. Compared with control women, if those with EndoM have higher estradiol exposures we would expect them to have shorter cycles and shorter follicular phases 53. Older studies of women with infertility showed that a late decline in basal temperature variables at the time of flow was associated with endometriosis 54. But without a reliable assessment of ovulation and luteal phase length in these data it is difficult to understand their importance or specificity. Endometriosis is also associated with an overall increased risk for cancers despite adjusting for surgeries that have removed the uterus and ovaries; these increased cancers in a population-based longitudinal study, included breast, ovarian and hematological malignancies (particularly Hodgkin’s lymphoma) 55. Cancer-related mutations have been found in deep endometriosis 56 Version4 H23-03963 September 25, 2024 Page 9 of 22 although a full genetic interpretation of this observation is not yet clear. An endometriosis family history is sometimes positive. Dietary intakes and energy expenditures in endometriosis have not been well-studied but the observation of a lower body weight and lower BMI than in controls is consistent 45,57 . We know of no reports of BAT anatomy or cold-stimulated, non-shivering BAT activity in women with EndoM. With increasing interest in measuring human BAT anatomy and activity, there is evidence that many common characteristics (age 26, sex25) and clinical conditions influence or cause abnormalities in these measurements. The evidence that BAT, teleologically was considered the “hibernating gland”58 raises questions about seasonal variations in BAT cool-stimulated activity. Any increased activity of the sympathetic nervous system (SNS) such as in chronic pain59 or the hypothalamic-pituitary-adrenal (HPA) axis as in depression 60 will increase BAT activity. In particular the phenomenon known as central pain sensitization syndrome 6 is a state of hypervigilance with elevated SNS and HPA stimulation. Those with attention deficit/hyperactivity disorder (ADHD) who are being treated with medications such as Methylphenidate (Ritalin) would have elevated BAT activity. The nicotine in smoking/vaping has been shown to increase BAT activity through the HPA axis 7. Insulin resistance, and Type 2 Diabetes Mellitus will decrease BAT anatomy 12; it is not yet entirely clear that they would decrease BAT activity. The increasingly popular “cold water bathing,” or the more traditional use of saunas followed by cold water or snow exposure, both stimulate BAT activity. Because of the involvement of both SNS and HPA in athletic training and competition, these would also likely increase BAT activity. Thus, in doing a cross-sectional, controlled study it is important to avoid these potential confounders. It is also key, as much as possible, that participants in both experimental (EndoM) and control groups have similar BMI, waist circumference and age variables, as well as regular normal-length menstrual cycles. 2 STUDY OBJECTIVES Primary research question: Is the standardized cool-stimulated, non-shivering activity of BAT by infrared thermography similar in regularly cycling women with EndoM and in regularly cycling, healthy controls? The primary hypothesis is that standardized BAT activity (stimulated by non-shivering cooling) using infrared thermography will be higher in regularly menstruating women with endometriosis compared with healthy, similar-age and BMI-range regularly menstruating women/PORA controls. 2.1 Primary Objective The primary objective of this research is to determine whether the increase in standardized brown adipose tissue (BAT) activity random-ordered in the follicular and luteal phases following non-shivering cooling is the same in regularly cycling women with endometriosis (EndoM) and in healthy, regularly cycling controls. Version4 H23-03963 September 25, 2024 Page 10 of 22 2.2 Secondary Objectives The secondary objectives, that arise from the above literature review and these data, are to determine whether the following outcomes differ between women with EndoM and healthy controls: 1) Indicators of in utero androgen exposure (anogenital distance, or the ratio of Digit 2:4 lengths, or both measures). 2) Menstrual Cycle Diary© experiences (especially feelings of self-worth, feeling of energy, and outside stresses) recorded over two complete menstrual cycles. 3) Menstrual cycle, luteal phase lengths and ovulatory characteristics by QBT© in two complete cycles. 4) Premenstrual/luteal phase levels of, progesterone, testosterone, cortisol, and DHEAS. 5) Self-reported 3-day dietary intakes (i.e., energy and macronutrient intakes) 6) Whether in those with EndoM and controls, there is a systematic difference between random-ordered BAT activity in the mid-follicular versus the mid-luteal phase. In addition, in the entire study cohort we will determine: 1) The accuracy of a screening reproductive questionnaire based on the baseline CaMos questionnaire and SF-36 self-rated health, plus the sensitivity and specificity of each variable for predicting two consecutive, normally ovulatory (≥10-day luteal phase) menstrual cycles. 2) The relationship of cooling-stimulated BAT activity with the proportion of body fat and non- bone lean (muscle) by body dual energy X-ray absorptiometry testing in 20 participants/group 3) Whether cooling-stimulated BAT will vary by season. 4) Whether cooling-stimulated BAT, by cycle phase will differ by whether it is the first or the second test. 3.1 INVESTIGATION PLAN 1) Recruitment—we will use accessible and credible websites ( www.cemcor.ubc.ca; reachbc.ca, etc.), social media, REACH BC, VCHRI, public talks and tear-tab strategic postering (all information ethics approved) as well as contacts through agencies and organizations related to endometriosis health care and/or support. 2) Screening—we invite all people approaching researchers, to tell each about the study and invite them to review the Consent Form. If potential participants verbally say they have read the consent form and agree, we will screen with a screening questionnaire, and the Central Sensitization Inventory 6 (CSI; completed online in REDCap) to determine all inclusion and exclusion criteria and make appropriate decisions about inclusion. Version4 H23-03963 September 25, 2024 Page 11 of 22 3) Those volunteering in the EndoM Group will be asked if they have had surgery to diagnose endometriosis and what month and year that occurred. If yes, and more than 3 months but less than five years previously, they will be eligible. If no, they also will be asked if they have had an ultrasound or an MRI showing that they have endometriosis in an ovary (endometrioma). If yes, they are eligible, but if no, they will be ineligible. Eligible participants in each group will complete the interviewer-administered CaMos questionnaire (adapted from the CaMos instruments, www.camos.org)3 by video conferencing with answers directly entered by the interviewer into REDCap. They will individually complete the generic Health-Related Quality of Life instrument, SF-3661 (directly onto the secure database, REDCap). 4) For those volunteering as Controls, the questionnaires as describe above will also be completed. Variables considered likely associated with ovulation are: gyn-age >12, a normal reproductive history without surgeries or past amenorrhea, lack of androgen excess or infertility, age at menarche between 12 and 13 years, parity >0, normal and stable BMI without weight cycling, no report of anxiety, depression or sleep problems, if they live with other adults rather than alone, and rate their health as good, very good or excellent. Those with very abnormal reproductive histories will thanked. All will be told that if their first cycle is not ovulatory, they will not be continued in the study. 5) The overall plan of the study will be discussed with each volunteer. It involves monitoring Diary and first morning temperature over two complete cycles plus part of a third, and two in-person visits that will each last about 2 hours. Given that the order of the follicular and luteal phase testing for BAT activity will be randomized a. The first in-person visit will be in the luteal/premenstrual phase or the mid- follicular phase of the second cycle when the baseline body measures, and first BAT activity will be assessed followed by fingerpick blood spot collection. b. The second in-person visit will be either in the luteal/premenstrual phase of the second cycle or in the mid-follicular phase of the third cycle during which we will do the second BAT activity assessment and finger prick blood spot collection. On that occasion, the first 20/group of women with complete data will also have a whole body DXA for assessment of bone density and % body fat and muscle (performed at the Hip Health Centre less than a block from the research offices). All will be invited to complete the final questionnaire on REDCap, provided with their honorarium of $100.00, a DivaCup® (menstrual cup, if they wish), and an author-signed copy of the informative, award-winning novel about the menopause transition, Estrogen’s Storm Season— stories of perimenopause. 6) Each participant will receive telephone, and email support to complete the Menstrual Cycle Diary©8 (Diary) forms (4 blank paper forms will be mailed to them), including online video resources http://www.cemcor. ca/resources/daily-menstrual-cycle-diary. They will also be provided with (by mail) a single-batch digital oral thermometer and instructed in collecting their temperature in the morning on first awakening https://www.cemcor.ubc.ca/resources/qualitative-basal-temperature-qbt-method-ovulation- detection. Participants will be asked to record their morning temperature at the bottom of the Diary every day (in the evening before bed as they are completing the Diary). 7) In order for the coordinator to keep track of each participant’s cycles, each will be requested to send each complete Diary with its temperature data by fax or email to the coordinator at the end of each cycle. It will be evaluated for completion and its ovulatory characteristics Version4 H23-03963 September 25, 2024 Page 12 of 22 will be assessed by the twice-validated Quantitative Basal Temperature© (QBT©) method4,5.

will determine if that participant, if in the Control Group, needs to repeat a cycle or can be scheduled for testing in the second cycle. Participants will be provided with their own cycle length and ovulatory information about each cycle. 8) At the first in-person visit, the participants will be instructed in completing the 3-day diet diary and provided with the paper form and instructions. They will each be reminded by a telephone/email about 4 days before their scheduled second-in-person visit to begin the Diet Diary. At the second visit, they will return the completed Diary and the coordinator/research will be able to confirm and clarify any missing or confusing entries with the participant. 9) Cool activation of Brown Adipose Tissue will use the method of Levy 62. (See Methods below) 3.1.1 Duration of Study Participation The study duration per participant will usually be up to 70 days, or over 2.2 menstrual cycles. The total estimated time to participate is about 10-11 hours over about 2.2 months. This research time includes five minutes/day for temperature and Diary (5-6 hours), screening questionnaire (.5 hours), initial questionnaire (~1.2 hours), first in-person study visit, including physical measures, 2:4-digit measures, bloodspot test and BAT (1.5 hours) and final visit (1.5 hours). 3.2 Study Population 3.2.1 Inclusion Criteria for Women with Endometriosis 1) Community-dwelling women or PORA ages 19-40 years 2) Body Mass Index (BMI) 18.5-28 3) Normal-length (21-35 days), predictable menstrual cycles 4) History of endometriosis defined as surgical diagnosis in the last 5 years (but not in the last three months) or current imaging diagnosis of ovarian endometriosis (on ultrasound or MRI). (Person-reported diagnoses of endometriosis are reliable)1. 5) Women can enroll even if using a Kyleena IUD for more than 60 days, or a Mirena IUD for 5 years or more and have regular month apart menstrual cycles 3.2.2 Inclusion Criteria for Control Women 1) Community-dwelling women or PORA ages 19-40 years. 2) Body mass index 18.5-28 3) Normal-length (21-35 days), predictable menstrual cycles 4) Women as controls will be healthy with a normal reproductive history by CaMos baseline questionnaire, with normal-length, predictable and proven ovulatory menstrual cycle in the first cycle by twice-validated Quantitative Basal Temperature methods4,5 although a short luteal phase is accepted. 5) Women can enroll even if using a Kyleena IUD for more than 60 days, or a Mirena IUD for 5 years or more and have regular month apart menstrual cycles Version4 H23-03963 September 25, 2024 Page 13 of 22 3.2.3 Exclusion Criteria for both groups 1) Reporting daily chronic pain > 3/10 on a 11-point numeric rating scale at any of several sites and/or a Central Sensitization Inventory score ≥406. 2) Participants with PCOS, clinical androgen excess, hysterectomy, both ovaries removal or a gynecological diagnosis other than EndoM. 3) Any use of combined hormonal contraceptives (CHC) except Kyleena (>60 days), and Mirena (≥5 years) IUD, ovarian hormonal agonists or antagonists within the last three months (3-mo). 4) Current or ongoing mental health issues, addictions or major situational stress that are likely to interfere with completion of this trial 5) Planning pregnancy in the next two months. 6) Diabetes Mellitus (T1DM or T2DM) or known insulin resistance. 7) Cold-water bathing, use of medications with adrenergic stimulating or blocking activity, or intense physical activity or exercise training (< 300 minutes of moderate or vigorous activity per week over the last three months). 8) Current use of nicotine delivered by any method (smoking, vaping, chewing)7. 9) Unwilling or unable to consistently take and record first morning oral temperatures (for QBT© assessment) and keep the Menstrual Cycle Diary© 8, an 18-item interval scale tool collecting reproductive and daily life experiences and for which normative data related to ovulation are available 9,10. Study Procedures 4 STUDY EVALUATION AND MEASUREMENTS 4.1 Screening and Monitoring Evaluations and Measurements 1) Height and weight will be measured in light clothing, in stocking feet on a balance beam with a stadiometer (with each result being a mean of three repeated measures). 2) Waist circumference will be measured with a non-stretch, cloth tape-measure half way between the lowest rib and iliac crest, while standing with quiet breathing; the final will be the mean of three assessments in cm. Hip circumference will be measured at the widest lateral dimension of the hip, with the same tape, while standing with relaxed breathing. The mean of three measures in cm will be final. 3) Quantitative Basal Temperature© (QBT©) will use the Mean Temperature analysis method 4 to assess whether or not ovulation occurred and the length of the luteal phase (with ≥10 days being normal). It will be recorded using a single-batch set of digital thermometers (tested coefficient of variation vs a mercury standard thermometer = .1°C) that is recorded first thing in the morning. The temperature will be recorded at the bottom of the Diary in the evening. 4) Menstrual Cycle Diary©8 (Diary) a self-report tool about diverse women’s experiences throughout the menstrual cycle will be analyzed as previously reported using Principal Components Analysis for each group and comparison of factor loading by group9. We will retrieve cycle lengths from this tool. This is an 18-item ordinal scaled instrument including two Version4 H23-03963 September 25, 2024 Page 14 of 22 validated flow assessments 63; other experiences such as interest in sex and cramps have previously been reported 9,10. Each participant will complete the Diary in the evening just before sleep. 5) Ad libitum 3-day Diet Diary: Participants will be asked to record their dietary intake for 3 days (2 weekdays and 1 weekend) using a study-specific form (paper and pen). Participants will be instructed on correct methods of recording dietary data (i.e., including information on cooking method, serving size, added oils/sugar, time of meal, etc.) and will be asked to track dietary intake for two weekdays and one weekend day. These data will be entered into and analyzed using the FoodProcessor® Nutrition Analysis software (ESHA Research, Chicago, IL, USA) to obtain information on energy, macronutrient, and micronutrient intake. 6) Appetite: Participants will complete two validated and standard questionnaires to assess appetite ‘traits’ – or aspects of appetite that are not dependent on momentary physical or emotional states. These include the 18-item short form Three-Factor Eating Questionnaire 64 (measures dietary restraint, disinhibition, general hunger) and the 35-item Adult Eating Behavior Questionnaire 65 (measures Food responsiveness, general hunger, emotional overeating, enjoyment of food, satiety responsiveness, food fussiness, emotional undereating, and slowness in eating). 7) Exercise: We have a number of questions about daily activity on the baseline comprehensive (CaMos©) questionnaire. In addition, participants will complete the short form International Physical Activity Questionnaire (IPAQ)66 on RedCap. 8) Assessments of in utero androgen exposure will include: a. Three measurements of the second and fourth digits (D) of the dominant hand and their ratio in all participants. The lengths of 2D and 4D will be measured from the midpoint of each proximal crease to the distal finger-tip using a Helio caliper 67. The difference of 2D to 4D will be obtained by subtracting 4D from 2D. The ratio of 2D:4D will be obtained by dividing 2D by 4D lengths. b. The anogenital distance measurements will be optional (indicated by a check box on the consent form) and performed only in those indicating acceptance, in a secure location, in the supine position by a woman physician following standard procedures 68. 9) Brown Adipose Tissue Activity stimulated by cooling will be measured using standard thermographic techniques 26,62,69 (see Appendix 1 for the detailed Protocol) after ≥4 hours fasting (morning or afternoon—both mid-follicular and luteal phase assessments within-woman at the same time of day) in a quiet environment without windows or heaters. The participant will converse with the coordinator during which she will be queried about strenuous exercise in the past 24 hours, any recent caffeine, and about the quality of the previous night’s sleep. She will be shown the cooling suit and explained about the process of the study including that she must tell us when she feels like she will soon start to shiver. Cooling equipment consists of a shirt and pants through which ice water circulates (BCS4 Cooling Unit W/3 PC Suit M-S from MEG-ENG). The participant will be asked to put it on (over a provided tank top and their own shorts or underpants). At baseline after measures of room temperature and humidity and about 10 minutes of participant relaxation, the infrared thermographic camera (FLIR E54, from ITM Instruments, Teledyne FLIR) will first assess the Version4 H23-03963 September 25, 2024 Page 15 of 22 temperature of the midpoint of the top of the sternum (a control point). Then we will position the focus of the camera at an angle to best visualize the area above the clavicle, one meter from first the Left ( L) and then the Right ( R ) supraclavicular regions with thermal pictures to be taken at five-minute intervals for 15 minutes (see Appendix 2 for the data-collection form). The mean of all values will be considered the baseline measurement at the three sites. Once all baseline measures are complete the cooling suit will be turned on. Every five minutes the participant will have measures of BAT in each supraclavicular fossa and in the upper sternum (control) recorded. The participant will also frequently answer a question about shivering— if responses are negative, all three measurements will be repeated and recorded. This will continue for about 30 minutes unless the participant shivers. In that case the cooling unit pump will be turned off and when she stops shivering the imaging will begin again as above. The mean of the finally selected two temperatures will be used. The heart rate per minute will be recorded at four different times: during the relaxation phase, the baseline phase, the cooling phase of the BAT test, and at the end of the visit. 10) Capillary hormone levels: For participant convenience and to allow flexibility in scheduling, progesterone, testosterone, and other hormones will be measured in dried blood spots (DBS) 70,71. To decrease the potential sympathetic activity associated with this, each collection will follow the BAT activity analysis. The hand to provide the sample will be warmed by external and gentle means prior to the fingerpick. DBS involves a finger-prick of soap and water washed, warm fingers following hand and arm movements, external warming and lowering the hand a few minutes. The puncture will be by standard T2DM-like methods. The fresh blood sample will be collected using the 20 µL collector, and an investigator will immediately allow the sample from the capillary tube to fill the circles on the Whatman #903 filter paper (FDA approved). We will allow the filter paper to dry at room temperature for at least 2 hours. These hormones (progesterone, testosterone, cortisol, and DHEAS) will be analyzed with microsample methods that have been validated. DBS will be processed by following way --the filter paper will have the ID # and date. We will dry the filter paper, place in a sealed zip-lock bag with ID # and date. We will store in the 4°C fridge before shipping. When the study is complete, we will transport the frozen samples to Victoria General Hospital where the Dr. Chen's lab (UBC affiliated site within Island Health) will analyze, the hormones using validated laboratory methods. 11) Central Sensitization Inventory (CSI)6 will be a required screening tool (performed by the interested person in REDCap). Each participant must have a score below 40 in order to be eligible. In addition, on the telephone screening questionnaire each potential participant will be asked if they have daily pain. If they respond “no” they are eligible. If they respond yes, we will ask what is their level of pain on a 0-10 scale. If they say it is > 3 they will become ineligible. Version4 H23-03963 September 25, 2024 Page 16 of 22 5 STATISTICAL CONSIDERATIONS 5.1 Primary Endpoint We will examine the BAT activity data, acquired random-ordered in the mid-follicular and the mid-luteal phase/premenstrual phase from final mean of the two BAT examinations in each participant with EndoM and each control. If these data are normally distributed, and the two groups are not different in BAT relevant variables we will perform a parametric comparison of these data using a non-paired T test. If there are differences of relevance between the two groups we will use regression analysis to assess whether BAT activity differs in participants with endometriosis versus controls. 5.2 The secondary objectives, that arise from the above literature review and these data, are to determine whether the following outcomes differ between women with EndoM and healthy controls: i. Indicators of in utero androgen exposure (anogenital distance, or the ratio of Digit 2:4 lengths, or both measures). ii. Menstrual Cycle Diary© experiences (especially feelings of self-worth, feeling of energy, and outside stresses) recorded over two complete menstrual cycles. iii. Menstrual cycle, luteal phase lengths and ovulatory characteristics by QBT© in two complete cycles. iv. Luteal/Premenstrual phase levels of progesterone, testosterone, cortisol, and DHEAS. v. Self-reported 3-day dietary intakes (i.e., energy and macronutrient intakes) vi. Whether in those with EndoM and controls, there is a systematic difference between random- ordered BAT activity in the mid-follicular versus the mid-luteal phase of ovulatory cycles. 5.2a In addition, in the entire study cohort we will determine: 1) The accuracy of a screening reproductive questionnaire based on the baseline CaMos questionnaire and SF-36 self-rated health, plus the sensitivity and specificity of each variable for predicting two consecutive, normally ovulatory (≥10-day luteal phase) menstrual cycles. 2) The relationship of cooling-stimulated BAT activity with the proportion of body fat and non- bone lean (muscle) by body dual energy X-ray absorptiometry testing in 20 participants/group 3) Whether cooling-stimulated BAT will vary by season. 4) Whether cooling-stimulated BAT, by cycle phase, will differ by whether it is the first or the second test. We will evaluate the sensitivity and specificity of the reproductive questionnaire screening process to determine its accuracy. The association between BAT activity and body fat and muscle will be analyzed through correlation analysis. Depending on the data distribution, the cooling-stimulated BAT activity within subjects will be assessed using the Paired-t test or the Wilcoxon Signed Rank test. Furthermore, multivariate analyses will be conducted to estimate Version4 H23-03963 September 25, 2024 Page 17 of 22 cooling-stimulated BAT activity, adjusting for relevant variables. All statistical analyses will be performed using R and SPSS, with a significance level set at p < 0.05. 6 STUDY ADMINISTRATION 6.1 Data Collection and Management 1. The primary database will be a specially created one in REDCap, a secure online form used by UBC into which participants can upload questionnaires and the results can be entered into a study database and cleaned before providing the final database to the statistician. Confidentiality. All participants will be given an ID number without any relationship to personal information. The master list decoding the ID, personal identity and contact information for each participant will be in a locked office cabinet, in a locked office and a secure building (Diamond Centre, UBC space). All researchers dealing with the data will have their electronic equipment encrypted for security. 2. Security. The data will be online in the K-drive that is backed up every five minutes through the University of British Columbia Med-IT. 6.2 Regulatory and Ethical Considerations We will achieve Clinical Research ethics approval for this protocol through the UBC CREB with harmonization with the Women’s and Children’s and Simon Fraser University ethics groups. 7 PUBLICATION Choices Potential journals to which we will submit are: Journal of Clinical Endocrinology and Metabolism, Human Reproduction, Clinical Endocrinology, Journal of Obstetrics and Gynecology, Fertility and Sterility, Journal of Endometriosis and Pelvic Pain Disorders, Women’s Reproductive Health, Reproductive Biology and Endocrinology, Biology of Reproduction, Women’s Health (Sage publisher), PLOS One or PLOS Medicine or BMJ Sexual and Reproductive Health. 8 REFERENCES 1 Shafrir, A. L. et al. Validity of self-reported endometriosis: a comparison across four cohorts. Hum Reprod 36, 1268-1278 (2021). https://doi.org:10.1093/humrep/deab012 2 Nisenblat, V., Bossuyt, P. M., Farquhar, C., Johnson, N. & Hull, M. L. Imaging modalities for the non-invasive diagnosis of endometriosis. Cochrane Database Syst Rev 2, CD009591 (2016). https://doi.org:10.1002/14651858.CD009591.pub2 3 Kreiger, N. et al. The Canadian Multicentre Osteoporosis Study (CaMos): Background, rationale, methods. Canadian Journal Aging 18, 376-387 (1999). 4 Prior, J. C., Vigna, Y. M., Schulzer, M., Hall, J. E. & Bonen, A. Determination of luteal phase length by quantitative basal temperature methods: validation against the midcycle LH peak. Clinical & Investigative Medicine 13, 123-131 (1990). 5 Bedford, J. L., Prior, J. C., Hitchcock, C. L. & Barr, S. I. Detecting evidence of luteal activity by least-squares quantitative basal temperature analysis against urinary Version4 H23-03963 September 25, 2024 Page 18 of 22 progesterone metabolites and the effect of wake-time variability. 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APPENDICES Appendix I—Brown Adipose Tissue Cold- Activated Protocol—Endometriosis BAT Activity Study Appendix 2— Thermal Imaging Data Collection—Endometriosis and Brown Adipose Activity Study