Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC) Finds Patient Age and Sperm Morphology Are Associated With Centriole Quality

Abstract

A large proportion of infertility and miscarriage causes are unknown. One potential cause is a defective sperm centriole, a subcellular structure essential for sperm motility and embryonic development. Yet, the extent to which centriolar maladies contribute to male infertility is unknown due to the lack of a convenient way to assess centriole quality. We developed a robust, location-based, ratiometric assay to overcome this roadblock, the Fluorescence-based Ratiometric Assessment of Centrioles (FRAC). We performed a case series study with semen samples from 33 patients, separated using differential gradient centrifugation into higher-grade (pellet) and lower-grade (interface) sperm fractions. Using a reference population of higher-grade sperm from infertile men with morphologically standard sperm, we found that 79% of higher-grade sperm of infertile men with substandard sperm morphology have suboptimal centrioles (P = 0.0005). Moreover, tubulin labeling of the sperm distal centriole correlates negatively with age (P = 0.004, R = −0.66). These findings suggest that FRAC is a sensitive method and that patient age and sperm morphology are associated with centriole quality.

Abnormal Centriolar Biomarker Ratios Correlate with Unexplained Bull Artificial Insemination Subfertility – a Pilot Study

“…The latter result suggests the fertile bulls' low-quality sperm population has lower quality centrioles relative to the fertile bulls' high-quality sperm. Similar lower centriole quality results were reported with sperm of infertile men (Turner et al, 2021) but not in fertile men or men with unexplained infertility (Jaiswal et al, 2022).…”

“…1). This technique found that patients diagnosed with teratozoospermia (poor morphology) had lower centriole quality (Turner et al, 2021), as was suggested earlier by electron microscopy studies (Alosilla Nakamura et al, 2002;Porcu, 2003;Rawe et al, 2002;Sha et al, 2017;Terada et al, 2009). Also, FRAC identi es male factor infertility in couples with unexplained infertility (Jaiswal et al, 2022).…”

“…A key point in the validation process is de ning a threshold (cutoff) that would allow clinical use of the test with con dence. We developed FRAC testing and demonstrated that FRAC can detect differences between men with different infertility types (Jaiswal et al, 2022;Turner et al, 2021). It should be noted that some of the distributions of the mean ratios in the fertile bulls have a slightly skewed distribution, implying that they are not gaussian.…”

“…Tubulin is a building block of PC, DC, and Axoneme microtubules (reviewed in Guichard et al, 2023), and its labeling is more intense in the centrioles than in the axoneme of human sperm (Fishman et al, 2018;Turner et al, 2021). Acetylated tubulin is a posttranslational modi cation of tubulin that makes the microtubules more exible and stable (Nekooki-Machida and Hagiwara, 2020; Portran et al, 2017).…”

The Proximal Centriole-Like Structure Anchors the Centriole to the Sperm Nucleus

“…This is consistent with our finding that poc1 mutant flies have little to no sperm in their seminal vesicles, as sperm must be able to swim in order to reach the seminal vesicle. In humans, sperm with abnormal morphology have reduced expression of POC1B mRNA (Platts et al, 2007), and abnormal levels of POC1B at the centrioles were found in patients with unexplained infertility (Turner et al, 2021). Furthermore, the atypical centriole in human and bovine sperm has an enrichment of POC1B {Fishman, 2018 #53} (Khanal et al, 2021).…”