Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Introduction: The United States has a growing population of transgender adolescents aged 12-21y presenting for medical transition via gender affirming hormone therapy (HT). Transgender people do not identify with their sex assigned at birth while cisgender people do. HT involves estrogen for trans females, those who identify as female, and testosterone for trans males, those who identify as male. Initiation of HT is beginning in adolescence and is a lifelong commitment for many transgender individuals. Little is known about how HT affects ASCVD risk factors in transgender adolescents. Studies of cisgender adults show cardioprotective effects of estrogen, and an estimated 37% of normal-weight cis gender adolescents have at least 1 ASCVD risk factor; but little investigation exists for transgender people, especially adolescents. Although some studies involving the transgender population have reproducible results, few include adolescents and comparison to acceptable ASCVD risk factor profiles. Guidelines for transgender health recommend laboratory assessment before starting HT and annually. This study expands on current knowledge and informs clinical practice by comparing ASCVD risk biomarkers of transgender adolescents taking HT to acceptable clinical values for adolescents. Hypothesis: Estrogen therapy for trans females will ameliorate ASCVD risk factors while testosterone therapy for trans males will worsen ASCVD risk factors. Methods: Retrospective chart review identified 300 transgender patients receiving HT at an academic adolescent medicine gender clinic. Of those 300, 50 trans female and 50 trans male were randomly selected for evaluation of ASCVD risk parameters at baseline and 1 year after initiating HT. ASCVD risk factors were compared to the International Diabetes Federation guidelines for metabolic syndrome. Parameters included lipids, glucose, and blood pressure. BMI ≥30 was used as proxy for obesity. Results: At baseline, at least one abnormal value was found for 50% (n=25, of these 5 had obesity) of trans females and 66% (n=33, of these 13 had obesity) of trans males. At 1 year, at least one abnormal value was found for 30% (n=15) of trans females and 70% (n=35) of trans males. At baseline, 50% (n=25) of trans females and 82% (n=41) of trans males, and at 1 year 20% (n=10) of trans females and 48% (24) of trans males had all parameters measured. Paired t-tests (α=.05) showed no significant change in ASCVD risk factors for trans females, and significant decrease in HDL and significant increase in BMI for trans males after 1 year of HT. Conclusions: The prevalence of having at least 1 ASCVD risk factor is greater among transgender adolescents than normal weight cisgender adolescents. HT may worsen HDL and BMI for trans males. We recommend baseline parameters for all and follow up and lab surveillance for patients with abnormal baseline parameters.

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Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate nontreatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease. Video LinkThe video component of this article can be found at https://www.jove.com/video/57182/ 9,10,11. One example is animal integration, where animals must be stably integrated into the mouse colony, therefore normalizing genetic background and diet composition, to avoid non-treatment related effects. Further, many EE studies have been completed