Sex as a Biological Variable in Preclinical Animal Studies with an Infectious Disease Exemplar.

Sex as a biological variable (SABV) has historically been overlooked in biomedical research, creating a critical gap in our understanding of how sex influences physiological and therapeutic outcomes. This is particularly important in infectious diseases such as Clostridioides difficile infection, where sex has been identified as an independent risk factor for disease development. Emerging evidence has suggested that sex- and hormones play a protective role against disease susceptibility. Despite evidence suggesting the importance of sex in this disease these variables remain poorly integrated into preclinical safety assessment and therapeutic development. This dissertation addresses this gap by implementing a rigorous, sex-inclusive safety framework, which was developed in a first-line animal model using C57BL/6 mice to evaluate the safety of a novel therapy against C. difficile, High temperature requirement-A (HtrA). This body of work investigates the contribution of sex-associated biological factors to preclinical safety assessments, evaluating whether therapeutic responses differ between males and females across biological systems. Across multiple systems including gastrointestinal integrity, epithelial barrier function, visceral nociception, microbiome recovery and reproductive endpoints, HtrA was well tolerated in both male and female mice. Importantly, reproductive assessments, including ovarian follicle development and testicular histology, demonstrated no evidence of endocrine disruption. Incorporating both sexes did not introduce confounding variability; rather, it strengthened the robustness and interpretability of the dataset, supporting SABV as a critical component of preclinical safety evaluation. To extend this framework into a second, more translationally relevant model, the influence of sex hormones on the vaginal ecosystem was characterized in rhesus macaques across the menstrual cycle. Immune profiling revealed stable peripheral populations (CD45, CD4, CD8, CD56, CD123), with a hormone-dependent increase in CD45 expression associated with elevated estradiol levels. Vaginal microbiota composition remained stable across individuals, with increases in Mobiluncus spp. observed only during peak progesterone levels rather than throughout the secretory phase. This work establishes a framework for integrating SABV into preclinical research and demonstrates its applicability across both first- and second-line animal models. By addressing sex bias and incorporating hormone-driven biology, these studies enhance the rigor, reproducibility and translational relevance of preclinical research, particularly in areas historically underrepresenting female physiology.

Department dc:contributor.department

Translational Medicine

Year dc:date.issued

2026

Author dc:creator

• Tozer, Kyla

Advisor dc:contributor.supervisor

• Sheth, Prameet

• Translational Medicine
• Sex as a Biological Variable

Statement dc:rights

• Attribution-NonCommercial-NoDerivatives 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc-nd/4.0/

Language dc:language.iso

eng

Handle dc:identifier.uri

https://hdl.handle.net/1974/36551

OAI identifier oai:identifier

oai:queensu.scholaris.ca:1974/36551