DETERMINATION OF 5-METHYLCYTOSINE, 5- HYDROXYMETHYLCYTOSINE, AND 5-FORMYLCYTOSINE EXPRESSION IN MOBILE AND IMMOBILE SPERM FROM ONE EJACULATION: ANALYSIS OF RELATIONSHIPS WITH SPERM PROPERTIES USING PARTIAL LEAST SQUARE REGRESSION

Dr. M. Katayama, Dr. S. Kaneko, Dr. T. Tsukimura, Dr. K. Takamatsu, Dr. T. Togawa

Abstract


AIM: To investigate prospective selection markers of donor sperm used in the assisted reproductive technology (ART) of intracytoplasmic sperm injection (ICSI), we measured the concentration of three epigenetic alteration markers; 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and 5-formylcytosine (5fC), in mobile (i.e., normal shape, motility, morphology) and immobile (i.e., no motility, abnormal shape) sperm from one ejaculation. METHODS: Mobile and immobile sperm were separated from the seminal plasma of one ejaculation using the original monopolistic centrifugal sedimentation separation method for sperm isolation. Differences in the three prospective epigenetic alteration markers between mobile and immobile sperm were assessed using the Student's t-test. Principal component analysis (PCA) and partial least squares regression (PLS) were performed to determine the relationships between sperm properties and markers, enabling the extraction of most prospective selective markers for ICSI.

RESULTS: Examined markers in mobile and immobile sperm (n = 13) were 5mC_Mobile (0.32‒6.47%), 5hmC_Mobile (0.3‒2.9%), 5fC_Mobile (0.011‒0.159%), and 5mC_Immob (0.37‒11.7%), 5hmC_Immob (0.6‒20.1%), and 5fC_Immob (0.001‒0.315%), respectively. The expression difference between mobile and immobile sperm was significant for 5mC (P = 0.0443, 95% confidence interval) and 5fC (P = 0.0204), while that of 5hmC (P = 0.0773, 95% confidence interval) was significant by 90% confidence interval. Regression analysis showed a correlation of 5mC_Mobile with sperm morphology (r = 0.3514). PCA identified 5mC_Mobile and 5hmC_Mobile as the most suitable factors to identify sperm properties. PLS analysis determined that 5mC_Mobile, 5fC_Mobile, and 5fC_Immob were related to sperm motility, 5mC_Immob to sperm morphology, and 5mC_Mobile and 5fC_Mobile to sperm concentration. 5hmC_Mobile (PLS 2, 3, 4‒5, 6) was related to sperm morphology with a high variable importance in projection (VIP) value of 1.4. Overall, concentration of 5hmC_Mobile in donor sperm appears to be the best sperm selection marker for ICSI.

Keywords


epigenetic markers; 5-methylcytosine; 5- hydroxymethylcytosine; 5- formylcytosine; sperm, t-test; PCA; PLS

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