{"paper_id":"c427c255-3fa3-4452-95e6-61aacf765680","body_text":"Version4                              H23-03963                     September 25, 2024                              Page 1 of 22 \n \n \n                 \n                                                                                                                                                      \nPROTOCOL:  NFRFE-2022-00300 \n \nTitle: Prenatal Programming of Female Reproductive \nPhysiology, Life History and Health \nLay Title Endometriosis and Brown Adipose Tissue Activity  \nProtocol Date: November 30, 2023 \nAmendment 1 Date: January 31, 2024 Amendment 4 Date:  \nAmendment 2 Date: May 23, 2024 Amendment 5 Date: \nAmendment 3 Date: September 25, 2024 Amendment 6 Date: \nStudy Principal Investigator—Bernard Crespi   \nDepartment of Biological Sciences, Simon Fraser University,  \n8888 University Drive, Burnaby, B.C.  \nPhone 604 805 4922 \nemail: crespi@sfu.ca  \nStudy Lead—Jerilynn C. Prior  \n2775 Laurel Street, Suite 4111 \nEndocrinology, University of British Columbia \nVancouver, BC, Canada, V5Z 1M9 \nPhone: 604 875-5927  \nEmail: jerilynn.prior@ubc.ca  \nCo-investigator—Paul Yong  \nWomen’s Hospital, 4500 Oak Street \nVancouver, BC V6H 3N1 \nPhone 604 875-2534  \npaul.yong@vch.ca   \nRole: Endometriosis Expert \nCo-investigator—Sarah Purcell  \nReichwald Health Sciences Centre, Room #319 \n1088 Discovery Avenue \nKelowna, BC V1V 1V7  \n\n\nVersion4                              H23-03963                     September 25, 2024                              Page 2 of 22 \n \nPhone: 604-341-6809 \nEmail: sarah.purcell@ubc.ca  \nRole: Nutrition, Energy-Balance Expert \n \nCollaborator— Michael X. Chen \nMedical Biochemistry, Provincial Laboratory Medicine Services \nMedical Biochemistry, Island Health \nUniversity of British Columbia, Vancouver, BC \nPhone: (1) 250-727-4425 email: Michael.Chen@islandhealth.ca  \n \nResearch Coordinator—Nahid Shirazian  \nCentre for Menstrual Cycle and Ovulation Research—Research Associate  \n2775 Laurel Street, Suite 4111 \nEndocrinology, University of British Columbia \nVancouver, BC, Canada, V5Z 1M9 \nPhone: 604 875-5917 \nemail: nahid.shirazian@ubc.ca \n \nResearch Assistant—Sonia Shirin  \nCentre for Menstrual Cycle and Ovulation Research—Research Associate  \n2775 Laurel Street, Suite 4111 \nEndocrinology, University of British Columbia \nVancouver, BC, Canada, V5Z 1M9 \nPhone: 604 875-5960 \nemail: sonia.shirin@ubc.ca   \n \n \n \n \n \n \n \n \n \n\nVersion4                              H23-03963                     September 25, 2024                              Page 3 of 22 \n \nPROTOCOL SYNOPSIS \nStudy Title:     \nENDOMETRIOSIS and \nBrown Adipose Activity  \n• Our evolutionary biology hypothesis is that endometriosis \n(EndoM) and androgenic polycystic ovary syndrome \n(PCOS) represent common reproductive conditions (each in \nabout 10% of premenopausal women) that are diametrically \nopposite to one another.  \n• PCOS has been causally linked with higher androgen \nexposure in utero. EndoM is hypothesized to relate to lower \nthan usual fetal androgen exposure.  \n• PCOS, in which overweight and obesity are common, has \nrecently been shown to be associated with lower activity of \nbrown adipose tissue (BAT). \n• There are no data on BAT area or activity in women with \nEndoM but they tend to have lower body weights than age- \nand ethnicity similar women.  \n• Our goal is to investigate whether stimulated BAT activity, \nmarkers of in utero androgen exposure, anthropometric, diet \nand exercise, menstrual cycle, ovulatory and hormonal \ncharacteristics differ between:  those diagnosed with EndoM \nand normally cycling and ovulatory controls ages 19-40 and \nBMI 18.5-28.  \nFunder New Frontiers in Research Fund—Exploration NFRFE-2022-00300 \nStudy Rationale Cold-stimulated BAT activity in EndoM has yet to be measured and \ncompared with that in healthy controls with normal length and \nnormally ovulatory menstrual cycles and similar, age and weight \ncategories. We aim to fill this knowledge gap by studying both \ngroups in a standardized prospective manner over two menstrual \nphases with documentation of socioeconomic, reproductive, medical \nand lifestyle histories, body measures, digit 2:4 length ratio or \nanogenital distance measures, or both, and cool, non-shivering \nstimulated BAT activity by thermographic methods random-ordered \nin the follicular and luteal phases.  \nStudy Objective(s) Primary  \n• To determine the 4-hour fasting pre-menstrual/luteal and mid-\nfollicular menstrual cycle phase activity of brown adipose tissue \n(BAT) following non-shivering cold stimulus by thermography \nin 60 regularly cycling women/people with ovaries of \nreproductive age (PORA) who have EndoM and 60 similar aged \nand BMI, normally cycling, and ovulatory controls.  \n• Secondary \n      To determine whether BMI, waist circumference and evidence   \nof fetal androgen exposure, e.g., anogenital distance and/or Digit \n2:4 ratio, dietary intake, exercise and normal ovulation significantly \ndiffer in women with EndoM versus controls and/or are related to \nBAT activity. \n\nVersion4                              H23-03963                     September 25, 2024                              Page 4 of 22 \n \nStudy Design \n \nThis is a cross-sectional comparison of two groups of regularly \nmenstruating, untreated (on no hormonal or combined hormonal \ncontraception, CHC (except for Kyleena (>60 days), and Mirena (≥5 \nyears) IUD) premenopausal women/PORA who differ in a \nreproductive diagnosis. It is also a prospective comparison of \nportions of three menstrual cycles and their ovulation characteristics \nin the two groups: those with EndoM; healthy, normally and \npredictably cycling and normally ovulatory controls. We will \ndetermine whether they also differ in standardized, non-shivering \nBAT activity by thermography as assessed during the luteal/ \npremenstrual phase (≥3 days before next flow) or the mid-follicular \nphases (cycle days 5-9). We hypothesize that BAT activity will be \nincreased in EndoM versus ovulatory controls  \nSubject Population \nkey criteria for \nInclusion and Exclusion: \nInclusion Criteria \n1. Community-dwelling women/PORA ages 19-40 years \n(Women/PORA≥ 35 will be screening for perimenopause) \n2. Body mass index 18.5 to 28  \n3. Menstrual cycles 21-35 days long and predictable \n4. Women with endometriosis (EndoM) defined as self-report \nof surgery for endometriosis diagnosis in the last 5 years \n(but not <3 months ago) or current imaging diagnosis of \novarian endometriosis or endometrioma via ultrasound or \nMRI.  (Person-reported diagnoses of endometriosis are \nreliable)1,2.  \n5. Controls will be healthy individuals with a normal \nreproductive history by the CaMos baseline questionnaire3, \nnormal-length, predictable and a proven ovulatory 1st  \nmenstrual cycle (including short luteal phase <10 days) by \ntwice-validated Quantitative Basal Temperature methods4,5.  \n6.  Women can enroll even if using a Kyleena IUD for more \nthan 60 days, or a Mirena IUD for 5 years or more and have \nregular month apart menstrual cycles.  \nExclusion Criteria \n1) Reporting daily chronic pain >3/10 at any of several sites on an \n11-point numeric rating scale. \n2) Having a Central Sensitization Inventory score ≥40 6. \n3) Participants with PCOS, clinical androgen excess, hysterectomy, \nboth ovaries removal or a non-EndoM gynecological diagnosis. \n4) Any use of combined hormonal contraceptives (CHC), ovarian \nhormonal agonists or antagonists, or hormonal IUDs (except for \nKyleena (>60 days), and Mirena (≥5 years) IUD) or implants \nwithin 3-months.  \n5) Emotional instability, addictions or major situational stressors \n6) Planning pregnancy in the next three months. \n7) Type 1 or Type 2 Diabetes Mellitus or known insulin resistance.  \n\nVersion4                              H23-03963                     September 25, 2024                              Page 5 of 22 \n \n8) Frequent Cold-water bathing, use of medications with \nadrenergic stimulating or blocking activity, or intense physical \nactivity or exercise training. \n9) Current use of nicotine delivered by any method (smoking, \nvaping, chewing)7.  \n10) Unwilling or unable to consistently take and record first \nmorning oral temperatures (for QBT© assessment) and keep the \nMenstrual Cycle Diary© 8, an 18-item interval scale tool \ncollecting reproductive and daily life experiences and for which \nnormative data related to ovulation are available 9,10.  \nNumber of Participants  \n \nTotal of 120  \n60 with Endometriosis, and 60 Ovulatory Controls \nStudy Duration Each volunteer’s participation will last about two months (two and a \nquarter complete ovarian cycles)  \nEfficacy Evaluations Primary evaluation measurement—4- hour fasting, standardized \nassessments of BAT activity by thermographic methods (after \ncooling but not shivering) during cycle days 18-25 (or >3 days \nbefore next flow) and also cycle days 4-9. (Within-woman each of \nthe two BAT activity assessments will be random-ordered and at the \nsame time of day and duration of fasting).  Luteal phase and \nfollicular phase assessment timing will be confirmed by dried blood \nspot hormonal measures. \nStatistical and Analytic \nPlan \nWe will assess the distribution and normality of all collected \nvariables including BAT activity data using appropriate \nparametric/nonparametric statistics. We will analyze both \nunadjusted and BAT-related variables (using results in these data \nand/or from the literature).  First morning oral temperature records \nwill be assessed for presence/absence of ovulation and the luteal \nphase length using validated Quantitative Basal Temperature© \n(QBT©) methods4,5.  Luteal phase lengths of <10 days will be \nconsidered short luteal phases11; those without a clear, sustained (>3 \ndays) rise in QBT© will arbitrarily be given a “luteal length” of 0.1 \nday (for analysis purposes) and be deemed anovulatory.  The \nMenstrual Cycle Diary© data from the two groups will be assessed \nby separate Principal Components Analysis to determine if factors \nand factor loading are similar or differ. In addition, mean Diary data \nwill be analyzed using non-parametric statistics to compare the \nmeans of all data per woman/PORA in the EndoM versus control \ngroups. Using SPSS version 28 and R we will analyze data, \naccepting a P value of <.05 as important.  \n\nVersion4                              H23-03963                     September 25, 2024                              Page 6 of 22 \n \n1 BACKGROUND INFORMATION AND RATIONALE (focused literature review) \n1.1 Introduction \nReproduction and energy balance in human females (women or people with ovaries of \nreproductive age [PORA]) are modulated by the brain and specifically the hypothalamus. In \nwarm-blooded mammals, core temperature is maintained within a physiological range by \nvariable, adaptive energy creation mechanisms. Energy balance relates to core temperature, basal \nmetabolic rate, stores of white fat, energy intake/expenditure and likely brown adipose tissue \n(BAT) activity12. Progesterone, a hormone that increases to high levels after ovulation, raises \ncore body temperature by approximately .2-.3 degrees Celsius13. It may be for that reason that \ndietary energy intakes are increased in ovulatory (with high progesterone) versus anovulatory \nmenstrual cycles in weight-stable premenopausal women 14. Estradiol, the dominant follicular \nphase ovarian hormone, tends to decrease body temperature and inhibit progesterone’s thermal \neffects 15. Normal menstrual cycles and ovulation require a stable, adequate body weight with \nenergy sufficiency (in relationship to appetite, micro- and macronutrients, food availability) \nbalanced with energy expenditure from physical activity and resting metabolic rate 16. Relative \nenergy deficiency (called RED-S when related to sporting activity) 17, plus social, emotional, and \nother individual-specific imbalances are related to development of adaptive, reversible \nhypothalamic reproductive suppressions, the most severe of which is amenorrhea, and the most \nprevalent a normal-length cycle with a short luteal phase18. Since progesterone (released in large \namounts only after ovulation/egg release in the midcycle) significantly raises the core/basal \ntemperature 19 in a step-function, and estradiol appears to have opposite effects 15, reproduction-\nenergy-temperature balance relationships in women/PORA are quite complex12.  \nEnergy balance is carefully maintained through multiple systems including changes in resting \nmetabolic rate, muscular responses (shivering) in cool environments, and sweating followed by \nevaporation in warm environments. These temperature control systems involve complex \ninteractions with the neuroendocrine and endocrine systems as well as with a host of hormonal, \nneurological and adipokine (fat-derived hormone) variables related to appetite and food intakes \nand physical activity and sleep patterns. The role of brown adipose tissue (BAT) activity remains \nunclear related to energy balance, in general, and specifically related to reproduction12,20. Mouse \nlactation (maternal provision of substantial energy to a newborn) has been reported to be \nassociated with decreased BAT activity 21.  Although other animal studies yield similar results, \nthis phenomenon has not yet been reported in humans 22.  If confirmed in humans, this change in \nBAT suppression could be understood as a mechanism that facilitates energy balance when the \nmother is providing additional calories in milk to the baby. BAT is also not confirmed to be \nrelated to adult obesity or higher body mass index (BMI, kg/m2) 12,20. However, in 55 children, \nBMI age-standardized normal weight percentage was negatively related to cool-stimulated BAT \n(r = -0.384; P = .004) 23.   \nThe BAT literature has provided some findings about both estradiol and about progesterone. The \nanimal literature suggests that estradiol stimulates or causes increased non-shivering BAT \nactivity, but that has rarely been shown in women 24,25. However, in a comprehensive study of \nnormal-weight young men plus premenopausal regularly cycling women in the confirmed \nfollicular and luteal phases, progesterone was strongly related to the elevated luteal phase \ntemperature, as is known 25. BAT-stimulated activity was also higher in women compared to \nmen, and within-woman, during the follicular than luteal phases 25. Estradiol was related to \n\nVersion4                              H23-03963                     September 25, 2024                              Page 7 of 22 \n \nincreased BAT activity in the luteal phase (r = .13, P <.05), while progesterone had no \nsignificant relationship in either menstrual cycle phase 25.   \nUnderstanding the control of temperature in humans is also made more complex by issues related \nto measurement of BAT anatomy and activity. Human studies exploded once the body’s \ndistribution of BAT, or BAT anatomy began to be documented using positron emission \ntomography (PET) of 18F fluorodeoxyglucose. That method has since become the gold standard \nmethod to measure BAT. However, as this test relies on glucose, anatomy doesn’t always \nparallel activity, especially, apparently, in states of insulin resistance 12. For that reason, experts \nare now recommending that BAT activity is best assessed by measuring changes following \nstandardized non-shivering cooling as assessed using infrared thermography 26. Another human \nstudy of BAT activity in women with PCOS measured supraclavicular (BAT anatomy) skin \ntemperatures versus deltoid (control) skin temperatures and found the greatest differences during \nsleep 27. A further study used Z-spectral imaging in women with PCOS and showed low BAT \nactivity 28. Animal models of PCOS have also shown lower BAT activity 29. \nAs mentioned, androgenic polycystic ovary syndrome (PCOS) 30 and endometriosis (EndoM) 31 \nhave been proposed to be diametrically opposite reproductive disturbances 32.  PCOS is a \nmultidimensional women’s reproductive and metabolic condition characterized by androgen \nexcess, long/irregular menstrual cycles and/or multiple follicular ovarian cysts30 and tends to be \nassociated with obesity and insulin resistance.  EndoM is also a common premenopausal \nreproductive condition in approximately 10% of women; it is characterized by ectopic \nendometrial-like tissue in the pelvis, ovary or other anatomical sites. EndoM is associated with \nchronic abdominal/pelvic pain and infertility 33. By contrast to women with PCOS, women with \nEndoM tend to have lower or normal body weights 34 35.  Despite the strong diversities of these \ntwo conditions 32, the literature also has documented that those with PCOS have EndoM more \ncommonly than do controls 36,37. In addition, both EndoM38 and PCOS39 are associated with \ninfertility, as well as with ovulatory disturbances and lower-than-normal progesterone levels.  \nWomen with PCOS tend to develop insulin resistance, Type 2 Diabetes Mellitus (T2DM) and \ncentral or visceral obesity with increased waist circumference (WC).  Although, in general, BMI \nis inversely related to the BAT anatomical area or activity, it is now increasingly clear that \ninsulin resistance is associated with decreased BAT activity 12,40.  A recent review asserted that \nhigher BMI and insulin resistance might not change the PET assessment of BAT anatomy but \nmay decrease BAT activity12 (as discussed earlier).  BAT, by the new radiographic technique of \nZ-spectral imaging, showed both smaller area and possibly decreased activity the higher the fat \ncontent of the brown adipose tissue 28. That investigation studied Chinese participants; 13 \nwomen had PCOS as well as being overweight/obese, 19 women were controls and 17 were \nmen; none had diabetes, but all with PCOS had insulin resistance 28. Results showed greater fat \nwithin brown adipose tissue in those with PCOS; that may be related to lower BAT activity 28. \nThe other BAT study in humans with PCOS measured resting (non-stimulated) supraclavicular \nand upper arm temperatures as a simple way of estimating BAT activity in humans, with the \nbiggest difference being recorded during sleep 27. This cross-sectional study without cycle-phase \ndocumentation in 47 women with PCOS (mean BMI 29, waist circumference 101 cm) and 11 \nwomen controls (mean BMI 25, WC 77) found that PCOS supraclavicular temperatures, \nespecially at night, were lower than in controls. The lower nighttime supraclavicular than deltoid \ntemperatures in PCOS than in controls had a significant negative relationship with free \n\nVersion4                              H23-03963                     September 25, 2024                              Page 8 of 22 \n \ntestosterone levels; however, the temperature difference in the two skin areas was minimally \npositively related to BMI 27. An important observation, however, was that control, upper arm \ntemperatures did not differ between the three groups. Another human study using PET showed \nthat BAT anatomy had a lower area in women with PCOS than in BMI- and age-matched \ncontrols 41. Thus, at least BAT area was negatively related to central adiposity 41.  \nAs mentioned previously, one of the anthropomorphic differences between PCOS and EndoM is \nthat the latter tend to be of normal weight 42.  Thus, this has increased speculation that PCOS and \nEndoM may differ in BAT activity. Women with PCOS tend to have more prevalent \noverweight/obesity, increased WC (that reflects increased visceral as well as subcutaneous fat), \nand metabolic syndrome. All of these are associated with insulin resistance 43. The key question \nin support of the divergent relationships of PCOS and EndoM then is:  What is the activity of \nBAT in women with EndoM?   \nThe purpose of this research is to document brown adipose tissue cold but non-shivering activity \nfor the first time in women with known endometriosis. In addition, we will determine whether \nBAT activity in women with EndoM differs from healthy control women who are of similar \nweight and similarly menstrual cycle characteristics.  \n1.2 Relevant Literature and Data \nEndometriosis (EndoM) is a relatively common (9% of a population-based sample of women at \nage 44 44) condition in which ectopic endometrium-like tissue found outside the uterus is \nassociated with severe menstrual cramps (secondary dysmenorrhea), pain with intercourse (deep \ndyspareunia), pain with defecation (dyschezia) and urination (dysuria) and also with fatigue and \nchronic pelvic pain 45.  EndoM has been described as an “estrogen-dependent chronic \ninflammatory process” with a deficiency of progesterone receptors 46.  EndoM is understood to \nbe progesterone-resistant, but this assertion is not well-documented. It is also commonly \nassociated with primary infertility 47. Many symptomatic women spend years and see multiple \nphysicians before being diagnosed with EndoM much less, before being effectively treated 48. \nThe pathophysiologic etiology of EndoM is currently unclear but it is associated with early \nmenarche, heavy menstruation, shorter cycles and nulliparity 49; all of these reproductive \ncharacteristics are associated with higher estradiol levels. It is currently unclear whether part of \nthe estrogen-progesterone imbalance 50 in EndoM is not only related to higher estradiol values \nbut also to more prevalent subclinical ovulatory disturbances (normal length and clinically \nnormal menstruation but short or insufficient luteal phases, or anovulation [SOD]) 51,52.  \nNo prospective studies of ovulatory characteristics in untreated women with documented EndoM \nare available. Compared with control women, if those with EndoM have higher estradiol \nexposures we would expect them to have shorter cycles and shorter follicular phases 53. Older \nstudies of women with infertility showed that a late decline in basal temperature variables at the \ntime of flow was associated with endometriosis 54. But without a reliable assessment of ovulation \nand luteal phase length in these data it is difficult to understand their importance or specificity.  \nEndometriosis is also associated with an overall increased risk for cancers despite adjusting for \nsurgeries that have removed the uterus and ovaries; these increased cancers in a population-based \nlongitudinal study, included breast, ovarian and hematological malignancies (particularly  \nHodgkin’s lymphoma) 55. Cancer-related mutations have been found in deep endometriosis 56 \n\nVersion4                              H23-03963                     September 25, 2024                              Page 9 of 22 \n \nalthough a full genetic interpretation of this observation is not yet clear. An endometriosis family \nhistory is sometimes positive. \nDietary intakes and energy expenditures in endometriosis have not been well-studied but the \nobservation of a lower body weight and lower BMI than in controls is consistent 45,57 .  We know \nof no reports of BAT anatomy or cold-stimulated, non-shivering BAT activity in women with \nEndoM.  \nWith increasing interest in measuring human BAT anatomy and activity, there is evidence that \nmany common characteristics (age\n26, sex25) and clinical conditions influence or cause \nabnormalities in these measurements. The evidence that BAT, teleologically was considered the \n“hibernating gland”58 raises questions about seasonal variations in BAT cool-stimulated activity.  \nAny increased activity of the sympathetic nervous system (SNS) such as in chronic pain59 or the \nhypothalamic-pituitary-adrenal (HPA) axis as in depression 60 will increase BAT activity. In \nparticular the phenomenon known as central pain sensitization syndrome 6 is a state of \nhypervigilance with elevated SNS and HPA stimulation. Those with attention \ndeficit/hyperactivity disorder (ADHD) who are being treated with medications such as \nMethylphenidate (Ritalin) would have elevated BAT activity.  The nicotine in smoking/vaping \nhas been shown to increase BAT activity through the HPA axis 7. Insulin resistance, and Type 2 \nDiabetes Mellitus will decrease BAT anatomy 12; it is not yet entirely clear that they would \ndecrease BAT activity.  The increasingly popular “cold water bathing,” or the more traditional \nuse of saunas followed by cold water or snow exposure, both stimulate BAT activity. Because of \nthe involvement of both SNS and HPA in athletic training and competition, these would also \nlikely increase BAT activity. Thus, in doing a cross-sectional, controlled study it is important to \navoid these potential confounders. It is also key, as much as possible, that participants in both \nexperimental (EndoM) and control groups have similar BMI, waist circumference and age \nvariables, as well as regular normal-length menstrual cycles.  \n2 STUDY OBJECTIVES \nPrimary research question:  Is the standardized cool-stimulated, non-shivering activity of BAT \nby infrared thermography similar in regularly cycling women with EndoM and in regularly \ncycling, healthy controls?   \nThe primary hypothesis is that standardized BAT activity (stimulated by non-shivering cooling) \nusing infrared thermography will be higher in regularly menstruating women with endometriosis \ncompared with healthy, similar-age and BMI-range regularly menstruating women/PORA \ncontrols.  \n2.1 Primary Objective  \nThe primary objective of this research is to determine whether the increase in standardized \nbrown adipose tissue (BAT) activity random-ordered in the follicular and luteal phases following \nnon-shivering cooling is the same in regularly cycling women with endometriosis (EndoM) and \nin healthy, regularly cycling controls.   \n \n \n\nVersion4                              H23-03963                     September 25, 2024                              Page 10 of 22 \n \n2.2 Secondary Objectives  \nThe secondary objectives, that arise from the above literature review and these data, are to \ndetermine whether the following outcomes differ between women with EndoM and healthy \ncontrols: \n1) Indicators of in utero androgen exposure (anogenital distance, or the ratio of Digit 2:4 \nlengths, or both measures).  \n2) Menstrual Cycle Diary© experiences (especially feelings of self-worth, feeling of energy, \nand outside stresses) recorded over two complete menstrual cycles.  \n3) Menstrual cycle, luteal phase lengths and ovulatory characteristics by QBT© in two complete \ncycles.    \n4) Premenstrual/luteal phase levels of, progesterone, testosterone, cortisol, and DHEAS.  \n5) Self-reported 3-day dietary intakes (i.e., energy and macronutrient intakes) \n6)  Whether in those with EndoM and controls, there is a systematic difference between \nrandom-ordered BAT activity in the mid-follicular versus the mid-luteal phase.   \nIn addition, in the entire study cohort we will determine: \n1) The accuracy of a screening reproductive questionnaire based on the baseline CaMos \nquestionnaire and SF-36 self-rated health, plus the sensitivity and specificity of each variable for \npredicting two consecutive, normally ovulatory (≥10-day luteal phase) menstrual cycles.   \n2) The relationship of cooling-stimulated BAT activity with the proportion of body fat and non-\nbone lean (muscle) by body dual energy X-ray absorptiometry testing in 20 \nparticipants/group \n3) Whether cooling-stimulated BAT will vary by season.  \n4) Whether cooling-stimulated BAT, by cycle phase will differ by whether it is the first or the \nsecond test. \n3.1 INVESTIGATION PLAN \n1) Recruitment—we will use accessible and credible websites ( www.cemcor.ubc.ca; reachbc.ca, \netc.), social media, REACH BC, VCHRI, public talks and tear-tab strategic postering (all \ninformation ethics approved) as well as contacts through agencies and organizations related to \nendometriosis health care and/or support.  \n2) Screening—we invite all people approaching researchers, to tell each about the study and \ninvite them to review the Consent Form.  \nIf potential participants verbally say they have read the consent form and agree, we will screen \nwith a screening questionnaire, and the Central Sensitization Inventory\n6 (CSI; completed online \nin REDCap) to determine all inclusion and exclusion criteria and make appropriate decisions \nabout inclusion.  \n\nVersion4                              H23-03963                     September 25, 2024                              Page 11 of 22 \n \n3) Those volunteering in the EndoM Group will be asked if they have had surgery to diagnose \nendometriosis and what month and year that occurred. If yes, and more than 3 months but \nless than five years previously, they will be eligible. If no, they also will be asked if they \nhave had an ultrasound or an MRI showing that they have endometriosis in an ovary \n(endometrioma). If yes, they are eligible, but if no, they will be ineligible.                       \nEligible participants in each group will complete  the interviewer-administered CaMos \nquestionnaire (adapted from the CaMos instruments, www.camos.org)3 by video \nconferencing with answers directly entered by the interviewer into REDCap. They will \nindividually complete the generic Health-Related Quality of Life instrument, SF-3661 \n(directly onto the secure database, REDCap).  \n4) For those volunteering as Controls, the questionnaires as describe above will also be \ncompleted. Variables considered likely associated with ovulation are:  gyn-age >12, a normal \nreproductive history without surgeries or past amenorrhea, lack of androgen excess or \ninfertility, age at menarche between 12 and 13 years, parity >0, normal and stable BMI \nwithout weight cycling, no report of anxiety, depression or sleep problems, if they live with \nother adults rather than alone, and rate their health as good, very good or excellent. Those \nwith very abnormal reproductive histories will thanked. All will be told that if their first cycle \nis not ovulatory, they will not be continued in the study.  \n5) The overall plan of the study will be discussed with each volunteer. It involves monitoring \nDiary and first morning temperature over two complete cycles plus part of a third, and two \nin-person visits that will each last about 2 hours. Given that the order of the follicular and \nluteal phase testing for BAT activity will be randomized \na. The first in-person visit will be in the luteal/premenstrual phase or the mid-\nfollicular phase of the second cycle when the baseline body measures, and first \nBAT activity will be assessed followed by fingerpick blood spot collection.  \nb. The second in-person visit will be either in the luteal/premenstrual phase of the \nsecond cycle or in the mid-follicular phase of the third cycle during which we will \ndo the second BAT activity assessment and finger prick blood spot collection.                                                                                                               \nOn that occasion, the first 20/group of women with complete data will also have a \nwhole body DXA for assessment of bone density and % body fat and muscle \n(performed at the Hip Health Centre less than a block from the research offices). \nAll will be invited to complete the final questionnaire on REDCap, provided with \ntheir honorarium of $100.00, a DivaCup® (menstrual cup, if they wish), and an \nauthor-signed copy of the informative, award-winning novel about the menopause \ntransition, Estrogen’s Storm Season— stories of perimenopause.   \n6) Each participant will receive telephone, and email support to complete the Menstrual Cycle \nDiary©8 (Diary) forms (4 blank paper forms will be mailed to them), including online video \nresources http://www.cemcor. ca/resources/daily-menstrual-cycle-diary. They will also be \nprovided with (by mail) a single-batch digital oral thermometer and instructed in collecting \ntheir temperature in the morning on first awakening \nhttps://www.cemcor.ubc.ca/resources/qualitative-basal-temperature-qbt-method-ovulation-\ndetection. Participants will be asked to record their morning temperature at the bottom of the \nDiary every day (in the evening before bed as they are completing the Diary).  \n7) In order for the coordinator to keep track of each participant’s cycles, each will be requested \nto send each complete Diary with its temperature data by fax or email to the coordinator at \nthe end of each cycle.  It will be evaluated for completion and its ovulatory characteristics \n\nVersion4                              H23-03963                     September 25, 2024                              Page 12 of 22 \n \nwill be assessed by the twice-validated Quantitative Basal Temperature© (QBT©) method4,5. \nResults will determine if that participant, if in the Control Group, needs to repeat a cycle or \ncan be scheduled for testing in the second cycle. Participants will be provided with their own \ncycle length and ovulatory information about each cycle.  \n8) At the first in-person visit, the participants will be instructed in completing the 3-day diet \ndiary and provided with the paper form and instructions. They will each be reminded by a \ntelephone/email about 4 days before their scheduled second-in-person visit to begin the Diet \nDiary. At the second visit, they will return the completed Diary and the coordinator/research \nwill be able to confirm and clarify any missing or confusing entries with the participant.  \n9) Cool activation of Brown Adipose Tissue will use the method of Levy 62. (See Methods \nbelow) \n3.1.1 Duration of Study Participation \nThe study duration per participant will usually be up to 70 days, or over 2.2 menstrual cycles. \nThe total estimated time to participate is about 10-11 hours over about 2.2 months. This research \ntime includes five minutes/day for temperature and Diary (5-6 hours), screening questionnaire (.5 \nhours), initial questionnaire (~1.2 hours), first in-person study visit, including physical measures, \n2:4-digit measures, bloodspot test and BAT (1.5 hours) and final visit (1.5 hours).   \n3.2 Study Population \n3.2.1 Inclusion Criteria for Women with Endometriosis  \n1)  Community-dwelling women or PORA ages 19-40 years \n2)  Body Mass Index (BMI) 18.5-28  \n3)  Normal-length (21-35 days), predictable menstrual cycles \n4)  History of endometriosis defined as surgical diagnosis in the last 5 years (but not in the last \nthree months) or current imaging diagnosis of ovarian endometriosis (on ultrasound or MRI). \n(Person-reported diagnoses of endometriosis are reliable)1.  \n5) Women can enroll even if using a Kyleena IUD for more than 60 days, or a Mirena IUD for 5 \nyears or more and have regular month apart menstrual cycles \n3.2.2 Inclusion Criteria for Control Women \n1) Community-dwelling women or PORA ages 19-40 years.   \n2) Body mass index 18.5-28  \n3) Normal-length (21-35 days), predictable menstrual cycles \n4) Women as controls will be healthy with a normal reproductive history by CaMos baseline \nquestionnaire, with normal-length, predictable and proven ovulatory menstrual cycle in the \nfirst cycle by twice-validated Quantitative Basal Temperature methods4,5 although a short \nluteal phase is accepted.  \n5) Women can enroll even if using a Kyleena IUD for more than 60 days, or a Mirena IUD for \n5 years or more and have regular month apart menstrual cycles  \n\nVersion4                              H23-03963                     September 25, 2024                              Page 13 of 22 \n \n3.2.3 Exclusion Criteria for both groups \n1) Reporting daily chronic pain > 3/10 on a 11-point numeric rating scale at any of several sites \nand/or a Central Sensitization Inventory score ≥406. \n2) Participants with PCOS, clinical androgen excess, hysterectomy, both ovaries removal or a \ngynecological diagnosis other than EndoM. \n3) Any use of combined hormonal contraceptives (CHC) except Kyleena (>60 days), and \nMirena (≥5 years) IUD, ovarian hormonal agonists or antagonists within the last three months \n(3-mo).  \n4) Current or ongoing mental health issues, addictions or major situational stress that are likely \nto interfere with completion of this trial \n5) Planning pregnancy in the next two months. \n6) Diabetes Mellitus (T1DM or T2DM) or known insulin resistance.  \n7) Cold-water bathing, use of medications with adrenergic stimulating or blocking activity, or \nintense physical activity or exercise training (< 300 minutes of moderate or vigorous activity \nper week over the last three months). \n8) Current use of nicotine delivered by any method (smoking, vaping, chewing)7.  \n9) Unwilling or unable to consistently take and record first morning oral temperatures (for \nQBT© assessment) and keep the Menstrual Cycle Diary© 8, an 18-item interval scale tool \ncollecting reproductive and daily life experiences and for which normative data related to \novulation are available 9,10. \nStudy Procedures \n4 STUDY EVALUATION AND MEASUREMENTS \n4.1 Screening and Monitoring Evaluations and Measurements \n1) Height and weight will be measured in light clothing, in stocking feet on a balance beam with \na stadiometer (with each result being a mean of three repeated measures).  \n2) Waist circumference will be measured with a non-stretch, cloth tape-measure half way \nbetween the lowest rib and iliac crest, while standing with quiet breathing; the final will be the \nmean of three assessments in cm. Hip circumference will be measured at the widest lateral \ndimension of the hip, with the same tape, while standing with relaxed breathing. The mean of \nthree measures in cm will be final.   \n3) Quantitative Basal Temperature© (QBT©) will use the Mean Temperature analysis method \n4 to assess whether or not ovulation occurred and the length of the luteal phase (with ≥10 days \nbeing normal). It will be recorded using a single-batch set of digital thermometers (tested \ncoefficient of variation vs a mercury standard thermometer = .1°C) that is recorded first thing in \nthe morning. The temperature will be recorded at the bottom of the Diary in the evening. \n4) Menstrual Cycle Diary©8 (Diary) a self-report tool about diverse women’s experiences \nthroughout the menstrual cycle will be analyzed as previously reported using Principal \nComponents Analysis for each group and comparison of factor loading by group9.  We will \nretrieve cycle lengths from this tool. This is an 18-item ordinal scaled instrument including two \n\nVersion4                              H23-03963                     September 25, 2024                              Page 14 of 22 \n \nvalidated flow assessments 63; other experiences such as interest in sex and cramps have \npreviously been reported 9,10.  Each participant will complete the Diary in the evening just before \nsleep. \n5) Ad libitum 3-day Diet Diary:  Participants will be asked to record their dietary intake for 3 \ndays (2 weekdays and 1 weekend) using a study-specific form (paper and pen). Participants will \nbe instructed on correct methods of recording dietary data (i.e., including information on cooking \nmethod, serving size, added oils/sugar, time of meal, etc.) and will be asked to track dietary \nintake for two weekdays and one weekend day. These data will be entered into and analyzed \nusing the FoodProcessor® Nutrition Analysis software (ESHA Research, Chicago, IL, USA) to \nobtain information on energy, macronutrient, and micronutrient intake.   \n6)  Appetite: Participants will complete two validated and standard questionnaires to assess \nappetite ‘traits’ – or aspects of appetite that are not dependent on momentary physical or \nemotional states. These include the 18-item short form Three-Factor Eating Questionnaire 64 \n(measures dietary restraint, disinhibition, general hunger) and the 35-item Adult Eating Behavior \nQuestionnaire 65 (measures Food responsiveness, general hunger, emotional overeating, \nenjoyment of food, satiety responsiveness, food fussiness, emotional undereating, and slowness \nin eating).  \n7) Exercise: We have a number of questions about daily activity on the baseline comprehensive \n(CaMos©) questionnaire. In addition, participants will complete the short form International \nPhysical Activity Questionnaire (IPAQ)66 on RedCap.  \n8) Assessments of in utero androgen exposure will include:  \na. Three measurements of the second and fourth digits (D) of the dominant hand and their \nratio in all participants. The lengths of 2D and 4D will be measured from the midpoint of each \nproximal crease to the distal finger-tip using a Helio caliper 67. The difference of 2D to 4D will \nbe obtained by subtracting 4D from 2D. The ratio of 2D:4D will be obtained by dividing 2D by \n4D lengths.   \nb. The anogenital distance measurements will be optional (indicated by a check box on the \nconsent form) and performed only in those indicating acceptance, in a secure location, in the \nsupine position by a woman physician following standard procedures 68.  \n9) Brown Adipose Tissue Activity stimulated by cooling will be measured using standard \nthermographic techniques 26,62,69 (see Appendix 1 for the detailed Protocol) after ≥4 hours \nfasting (morning or afternoon—both mid-follicular and luteal phase assessments within-woman \nat the same time of day) in a quiet environment without windows or heaters. The participant will \nconverse with the coordinator during which she will be queried about strenuous exercise in the \npast 24 hours, any recent caffeine, and about the quality of the previous night’s sleep. She will be \nshown the cooling suit and explained about the process of the study including that she must tell \nus when she feels like she will soon start to shiver.  \nCooling equipment consists of a shirt and pants through which ice water circulates (BCS4 \nCooling Unit W/3 PC Suit M-S from MEG-ENG).  The participant will be asked to put it on \n(over a provided tank top and their own shorts or underpants).  At baseline after measures of \nroom temperature and humidity and about 10 minutes of participant relaxation, the infrared \nthermographic camera (FLIR E54, from ITM Instruments, Teledyne FLIR) will first assess the \n\nVersion4                              H23-03963                     September 25, 2024                              Page 15 of 22 \n \ntemperature of the midpoint of the top of the sternum (a control point). Then we will position the \nfocus of the camera at an angle to best visualize the area above the clavicle, one meter from first \nthe Left ( L) and then the Right ( R ) supraclavicular regions with thermal pictures to be taken at \nfive-minute intervals for 15 minutes (see Appendix 2 for the data-collection form). The mean of \nall values will be considered the baseline measurement at the three sites.  \nOnce all baseline measures are complete the cooling suit will be turned on. Every five minutes \nthe participant will have measures of BAT in each supraclavicular fossa and in the upper sternum \n(control) recorded. The participant will also frequently answer a question about shivering— if \nresponses are negative, all three measurements will be repeated and recorded. This will continue \nfor about 30 minutes unless the participant shivers. In that case the cooling unit pump will be \nturned off and when she stops shivering the imaging will begin again as above. The mean of the \nfinally selected two temperatures will be used.  \n \nThe heart rate per minute will be recorded at four different times: during the relaxation phase, the \nbaseline phase, the cooling phase of the BAT test, and at the end of the visit. \n10) Capillary hormone levels:  For participant convenience and to allow flexibility in \nscheduling, progesterone, testosterone, and other hormones will be measured in dried blood spots \n(DBS) 70,71. To decrease the potential sympathetic activity associated with this, each collection \nwill follow the BAT activity analysis. The hand to provide the sample will be warmed by \nexternal and gentle means prior to the fingerpick.  \nDBS involves a finger-prick of soap and water washed, warm fingers following hand and arm \nmovements, external warming and lowering the hand a few minutes. The puncture will be by \nstandard T2DM-like methods. The fresh blood sample will be collected using the 20 µL \ncollector, and an investigator will immediately allow the sample from the capillary tube to fill the \ncircles on the Whatman #903 filter paper (FDA approved). We will allow the filter paper to dry \nat room temperature for at least 2 hours.  \nThese hormones (progesterone, testosterone, cortisol, and DHEAS) will be analyzed with \nmicrosample methods that have been validated.  \nDBS will be processed by following way --the filter paper will have the ID # and date. We will \ndry the filter paper, place in a sealed zip-lock bag with ID # and date. We will store in the \n4°C fridge before shipping. When the study is complete, we will transport the frozen samples to \nVictoria General Hospital where the Dr. Chen's lab (UBC affiliated site within Island Health) \nwill analyze, the hormones using validated laboratory methods. \n11) Central Sensitization Inventory (CSI)6 will be a required screening tool (performed by the \ninterested person in REDCap). Each participant must have a score below 40 in order to be \neligible. In addition, on the telephone screening questionnaire each potential participant will be \nasked if they have daily pain.  If they respond “no” they are eligible.  If they respond yes, we will \nask what is their level of pain on a 0-10 scale. If they say it is > 3 they will become ineligible.  \n\nVersion4                              H23-03963                     September 25, 2024                              Page 16 of 22 \n \n5 STATISTICAL CONSIDERATIONS \n5.1 Primary Endpoint \nWe will examine the BAT activity data, acquired random-ordered in the mid-follicular and the \nmid-luteal phase/premenstrual phase from final mean of the two BAT examinations in each \nparticipant with EndoM and each control. If these data are normally distributed, and the two \ngroups are not different in BAT relevant variables we will perform a parametric comparison of \nthese data using a non-paired T test. If there are differences of relevance between the two groups \nwe will use regression analysis to assess whether BAT activity differs in participants with \nendometriosis versus controls.  \n5.2 The secondary objectives, that arise from the above literature review and these data, are to \ndetermine whether the following outcomes differ between women with EndoM and healthy \ncontrols: \ni. Indicators of in utero androgen exposure (anogenital distance, or the ratio of Digit 2:4 lengths, \nor both measures).  \nii. Menstrual Cycle Diary© experiences (especially feelings of self-worth, feeling of energy, and \noutside stresses) recorded over two complete menstrual cycles.  \niii. Menstrual cycle, luteal phase lengths and ovulatory characteristics by QBT© in two complete \ncycles.    \niv. Luteal/Premenstrual phase levels of progesterone, testosterone, cortisol, and DHEAS.  \nv. Self-reported 3-day dietary intakes (i.e., energy and macronutrient intakes) \nvi.  Whether in those with EndoM and controls, there is a systematic difference between random-\nordered BAT activity in the mid-follicular versus the mid-luteal phase of ovulatory cycles.   \n5.2a In addition, in the entire study cohort we will determine: \n1) The accuracy of a screening reproductive questionnaire based on the baseline CaMos \nquestionnaire and SF-36 self-rated health, plus the sensitivity and specificity of each variable for \npredicting two consecutive, normally ovulatory (≥10-day luteal phase) menstrual cycles.   \n2) The relationship of cooling-stimulated BAT activity with the proportion of body fat and non-\nbone lean (muscle) by body dual energy X-ray absorptiometry testing in 20 participants/group \n3) Whether cooling-stimulated BAT will vary by season.  \n4) Whether cooling-stimulated BAT, by cycle phase, will differ by whether it is the first or the \nsecond test. \nWe will evaluate the sensitivity and specificity of the reproductive questionnaire screening \nprocess to determine its accuracy. The association between BAT activity and body fat and \nmuscle will be analyzed through correlation analysis. Depending on the data distribution, the \ncooling-stimulated BAT activity within subjects will be assessed using the Paired-t test or the \nWilcoxon Signed Rank test. Furthermore, multivariate analyses will be conducted to estimate \n\nVersion4                              H23-03963                     September 25, 2024                              Page 17 of 22 \n \ncooling-stimulated BAT activity, adjusting for relevant variables. All statistical analyses will be \nperformed using R and SPSS, with a significance level set at p < 0.05. \n6 STUDY ADMINISTRATION \n6.1 Data Collection and Management \n1.       The primary database will be a specially created one in REDCap, a secure online form \nused by UBC into which participants can upload questionnaires and the results can be \nentered into a study database and cleaned before providing the final database to the \nstatistician. Confidentiality.  All participants will be given an ID number without any \nrelationship to personal information. The master list decoding the ID, personal identity and \ncontact information for each participant will be in a locked office cabinet, in a locked \noffice and a secure building (Diamond Centre, UBC space). All researchers dealing with \nthe data will have their electronic equipment encrypted for security.   \n2.      Security.  The data will be online in the K-drive that is backed up every five minutes   \nthrough the University of British Columbia Med-IT.  \n6.2 Regulatory and Ethical Considerations \nWe will achieve Clinical Research ethics approval for this protocol through the UBC CREB with \nharmonization with the Women’s and Children’s and Simon Fraser University ethics groups.  \n7 PUBLICATION Choices \n Potential journals to which we will submit are:  Journal of Clinical Endocrinology and \nMetabolism, Human Reproduction, Clinical Endocrinology, Journal of Obstetrics and \nGynecology, Fertility and Sterility, Journal of Endometriosis and Pelvic Pain Disorders, \nWomen’s Reproductive Health, Reproductive Biology and Endocrinology, Biology of \nReproduction, Women’s Health (Sage publisher), PLOS One or PLOS Medicine or BMJ Sexual \nand Reproductive Health.  \n8 REFERENCES \n \n1 Shafrir, A. L. et al. Validity of self-reported endometriosis: a comparison across four \ncohorts. Hum Reprod 36, 1268-1278 (2021). https://doi.org:10.1093/humrep/deab012 \n2 Nisenblat, V., Bossuyt, P. M., Farquhar, C., Johnson, N. & Hull, M. L. Imaging \nmodalities for the non-invasive diagnosis of endometriosis. Cochrane Database Syst Rev \n2, CD009591 (2016). https://doi.org:10.1002/14651858.CD009591.pub2 \n3 Kreiger, N. et al. The Canadian Multicentre Osteoporosis Study (CaMos): Background, \nrationale, methods. Canadian Journal Aging 18, 376-387 (1999).  \n4 Prior, J. C., Vigna, Y. M., Schulzer, M., Hall, J. E. & Bonen, A. Determination of luteal \nphase length by quantitative basal temperature methods: validation against the midcycle \nLH peak. Clinical & Investigative Medicine 13, 123-131 (1990).  \n5 Bedford, J. L., Prior, J. C., Hitchcock, C. L. & Barr, S. I. Detecting evidence of luteal \nactivity by least-squares quantitative basal temperature analysis against urinary \n\nVersion4                              H23-03963                     September 25, 2024                              Page 18 of 22 \n \nprogesterone metabolites and the effect of wake-time variability. Eur.J Obstet Gynecol \nReprod Biol. 146, 76-80 (2009).  \n6 Orr, N. L. et al. Central sensitization inventory in endometriosis. Pain 163, e234-e245 \n(2022). https://doi.org:10.1097/j.pain.0000000000002351 \n7 Mano-Otagiri, A., Iwasaki-Sekino, A., Ohata, H., Arai, K. & Shibasaki, T. Nicotine \nsuppresses energy storage through activation of sympathetic outflow to brown adipose \ntissue via corticotropin-releasing factor type 1 receptor. Neurosci Lett 455, 26-29 (2009). \nhttps://doi.org:10.1016/j.neulet.2009.03.054 \n8 Prior, J. C. in Reproductive Endocrinology, Surgery and Technology   (eds E. Y. Adashi, \nJ. A. Rock, & Z. Rosenwaks)  1077-1091 (Raven Press, 1996). \n9 Macbeth, A. B., Goshtasebi, A., Mercer, G. W. & Prior, J. C. Does Interest in Sex Peak at \nMid-Cycle in Ovulatory Menstrual Cycles of Healthy, Community-Dwelling Women? \nAn 11-month Prospective Observational Study. Women's Reproductive Health 8, 79-91 \n(2021). https://doi.org:10.1080/23293691.2021.1901519 \n10 Bann, S., Goshtasebi, A., Shirin, S. & Prior, J. C. A one-year observational cohort study \nof menstrual cramps and ovulation in healthy, normally ovulating women. Sci Rep 12, \n4738 (2022). https://doi.org:10.1038/s41598-022-08658-3 \n11 Vollman, R. F. in Major Problems in Obstetrics and Gynecology, Vol 7 Vol. 1  (ed E. A. \nFriedman)  11-193 (W.B. Saunders Company, 1977). \n12 Carpentier, A. C., Blondin, D. P., Haman, F. & Richard, D. Brown Adipose Tissue-A \nTranslational Perspective. Endocr Rev 44, 143-192 (2023). \nhttps://doi.org:10.1210/endrev/bnac015 \n13 Magallon, D. T. & Masters, W. H. 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Adaptive, reversible, hypothalamic reproductive suppression: More than \nfunctional hypothalamic amenorrhea. Frontiers in Endocrinology 13 (2022). \nhttps://doi.org:10.3389/fendo.2022.893889 \n19 Landau, R. L., Bergenstal, D. M., Lugibihl, K. & Kascht, M. E. The metabolic effects of \nprogesterone in man. J Clin Endocrinol.Metab 15, 1194-1215 (1955).  \n20 Trayhurn, P. Origins and early development of the concept that brown adipose tissue \nthermogenesis is linked to energy balance and obesity. Biochimie 134, 62-70 (2017). \nhttps://doi.org:10.1016/j.biochi.2016.09.007 \n\nVersion4                              H23-03963                     September 25, 2024                              Page 19 of 22 \n \n21 Trayhurn, P., Douglas, J. B. & Mcguckin, M. M. Brown Adipose-Tissue Thermogenesis \nIs Suppressed during Lactation in Mice. Nature 298, 59-60 (1982). https://doi.org:DOI \n10.1038/298059a0 \n22 Krol, E. & Speakman, J. R. 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L. & Crespi, B. J. Endometriosis and polycystic ovary syndrome are \ndiametric disorders. Evol Appl 14, 1693-1715 (2021). https://doi.org:10.1111/eva.13244 \n33 Taylor, H. S., Kotlyar, A. M. & Flores, V. A. Endometriosis is a chronic systemic \ndisease: clinical challenges and novel innovations. The Lancet 397, 839-852 (2021). \nhttps://doi.org:10.1016/s0140-6736(21)00389-5 \n34 Crespi, B. Variation among human populations in endometriosis and PCOS A test of the \ninverse comorbidity model. Evol Med Public Health 9, 295-310 (2021). \nhttps://doi.org:10.1093/emph/eoab029 \n35 Ferrero, S., Anserini, P., Remorgida, V. & Ragni, N. Body mass index in endometriosis. \nEur J Obstet Gynecol Reprod Biol 121, 94-98 (2005). \nhttps://doi.org:10.1016/j.ejogrb.2004.11.019 \n36 Kujanpaa, L. et al. 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Drug Discovery Today: Disease Models (Part B) 32, 71-82 (2020). \nhttps://doi.org:https://doi.org/10.1016/j.ddmod.2020.11.008 \n40 Kaikaew, K., Grefhorst, A. & Visser, J. A. Sex Differences in Brown Adipose Tissue \nFunction: Sex Hormones, Glucocorticoids, and Their Crosstalk. Front Endocrinol \n(Lausanne) 12, 652444 (2021). https://doi.org:10.3389/fendo.2021.652444 \n41 Oliveira, F. R. et al. Brown adipose tissue activity is reduced in women with polycystic \novary syndrome. Eur J Endocrinol 181, 473-480 (2019). https://doi.org:10.1530/EJE-19-\n0505 \n42 Reis, F. M. et al. Superficial Peritoneal Endometriosis: Clinical Characteristics of 203 \nConfirmed Cases and 1292 Endometriosis-Free Controls. Reprod Sci 27, 309-315 (2020). \nhttps://doi.org:10.1007/s43032-019-00028-1 \n43 Sun, Y. et al. Association of Normal-Weight Central Obesity With All-Cause and Cause-\nSpecific Mortality Among Postmenopausal Women. 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Is There a Correlation between the Second-to-Four Digit Ratio (2D:4D) \nand Endometriosis? Results of a Case-Control Study. J Clin Med 12 (2023). \nhttps://doi.org:10.3390/jcm12052040 \n\nVersion4                              H23-03963                     September 25, 2024                              Page 22 of 22 \n \n68 Mira-Escolano, M. P. et al. Longer anogenital distance is associated with higher \ntestosterone levels in women: a cross-sectional study. BJOG 121, 1359-1364 (2014). \nhttps://doi.org:10.1111/1471-0528.12627 \n69 Brasil, S. et al. A systematic review on the role of infrared thermography in the Brown \nadipose tissue assessment. Rev Endocr Metab Disord 21, 37-44 (2020). \nhttps://doi.org:10.1007/s11154-020-09539-8 \n70 Shirtcliff, E. A., Reavis, R., Overman, W. H. & Granger, D. A. Measurement of gonadal \nhormones in dried blood spots versus serum: verification of menstrual cycle phase. \nHorm.Behav. 39, 258-266 (2001).  \n71 Petsos, P., Ratcliffe, W. A., Heath, D. F. & Anderson, D. C. Comparison of blood spot, \nsalivary and serum progesterone assays in the normal menstrual cycle. Clinical \nEndocrinology 24, 31-38 (1986).  \n \nAPPENDICES \nAppendix I—Brown Adipose Tissue Cold- Activated Protocol—Endometriosis BAT Activity \nStudy \nAppendix 2— Thermal Imaging Data Collection—Endometriosis and Brown Adipose Activity  \nStudy","source_license":"CC0","license_restricted":false}