Assessment of DNA structure and integrity in the human spermatozoon

Román Montañana, Claudia ORCID: 0000-0001-6672-3381 (2020). Assessment of DNA structure and integrity in the human spermatozoon. University of Birmingham. Ph.D.

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Abstract

Male fertility is routinely assessed by basic semen analysis, but this is poorly predictive of fertility outcome. One promising advanced diagnostic is assessing sperm DNA damage, but there is a lack of method standardisation, clinical thresholds and comparison data for different assays. The objective of this study was to validate standardised methods for the assessment of sperm DNA quality using TUNEL, Acridine orange (AO) and Chromomycin A3 (CMA3) in slide-based assays; and assess prognostic value for fertility and miscarriage.

The data obtained reveals that using differing optimal mounting solution for different assays is key to reliable results. The use of DNA fragmentation inductors such as DNase and hydrogen peroxide in donor samples confirmed and validated the use of AO and TUNEL for detecting DNA damaged cells.

A novel semi-automated computer-based scoring system for fluorescence microscopy images was devised and compared with visual operator results for intra-assay variability of AO and TUNEL assays. This system allowed objective and consistent results free of operator subjectivity.

The assessment of TUNEL, AO and CMA3 values in a subset of patients from the HABSelect trial showed no correlation between the assays corroborating that different assays measure different aspects of DNA quality. The number of patient samples assessed were insufficiently powered to draw firm conclusions related to clinical outcome, but we believe they are useful in making a case for further investigations in the field.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):