Investigation of Candida albicans diversity in an infertile cohort using next generation genetic analysis

Abstract

Vaginal candidiasis affects 75% of all women at least once during their life and Candida albicans has been implicated in infertility. In vitro studies also show that Candida albicans influences spermatozoa quality suggesting an impact on male factor infertility. However, few studies have investigated Candida diversity and their impact on infertility. This PhD study has evaluated, (i) Candida prevalence and diversity from oral and female/male reproductive tract specimens collected from 20 couples (40 individuals) of differing fertility status; (ii) sensitivity profiles obtained for different species using the antifungal drugs fluconazole and amphotericin-B; (iii) different phenotypes related to fitness and virulence for different C. albicans strains, and evaluation extended to Candida species from the CTG clade; (iii) Diploid strain type (DST) for C. albicans strains identified using Multilocus sequence typing MLST approach, considering karyotypic level changes; and (iv) immune responses of the cohort. Candida prevalence and diversity were assessed in the ovulation phase of the menstrual cycle. Identification to species level was obtained based on an ITS2 molecular approach and 35 bp signal were used for preliminary diversity assessment. Multiple sampling was considered to extend our knowledge about Candida genetic diversity from participants with different health status, thus 1–6 samples /individual of Candida species were collected. A clinical break point (CBP) and epidemiological cut off value (ECV) were determined using the Clinical Laboratory Standards Institute (CLSI) M27-A3 method. Further, sterol profile was obtained for subset species demonstrating resistance to fluconazole. Biofilm formation, growth rate, and phospholipase activity was measured for different C. albicans genotypes based on ITS2 and Candida species from the CTG clade. An in vivo model was used to assess the degree of Candida species virulence. Whole genome sequencing (WGS) was used in this current study to obtain MLST profile and determine the DST, copy number variant across chromosomes, and mating locus type. The clade designations of current strains were assessed using 1,380 C. albicans strains. The putative founders of strains were defined. We also extended our understanding of possible predisposing factors for VVC and RVVC by investigating the immune response for both genders, using MNCs derived from each participant, and levels of IL-6, IL-10 and IL-17 were assessed upon SC5314 interaction with mononuclear cells (MNCs) obtained from each participant. Key findings include that Candida species, mainly C. albicans, were prevalent in infertile women. The majority of women who reported VVC/RVVC were simultaneously colonized in both the oral vaginal sites with the same DST. DSTs were participant specific, and occasional intra-strain diversity for individuals were observed. Pronounced genetic diversity was detected in couples undergoing anti-fungal treatment. Differences in immune response based on participant sex rather than fertility status was observed