Detailed cell-level analysis of sperm nuclear quality among the different hypo-osmotic swelling test (HOST) classes

Abstract

Purpose: We studied the quality differences between the different hypo-osmotic swelling test (HOST) classes, as measured by criteria of DNA fragmentation, DNA decondensation, and nuclear architecture. The aim was to find particular HOST classes associated with good-quality metrics, which may be potentially used in ICSI (intra-cytoplasmic sperm injection).

Methods: Ten patients from the Department of Reproductive Medicine at Tenon Hospital (Paris, France) were included. Their semen samples were collected and divided into two fractions: one was incubated in a hypo-osmotic solution as per HOST protocol and sorted by sperm morphology, and a second was incubated without undergoing the HOST protocol to serve as an unsorted baseline. Three parameters were assessed: DNA fragmentation (TUNEL assay), DNA decondensation (chromomycin A3 assay), and nuclear architecture (FISH, with telomeric and whole chromosome painting probes). The different HOST classes were evaluated for these three parameters, and statistical analysis was performed for each class versus the unsorted non-HOST-treated sperm. Results with p<0.05 were considered statistically significant.

Results: For each of the parameters evaluated, we found significant differences between HOST-selected spermatozoa and non-selected spermatozoa. Overall, spermatozoa of HOST classes B and B+ exhibited the highest quality based on four metrics (low DNA fragmentation, low DNA decondensation, short inter-telomeric distance, and small chromosome 1 territory area), while spermatozoa of HOST classes A and G exhibited the poorest quality by these metrics.

Conclusion: In addition to their pathophysiological interest, our results open possibilities of sperm selection prior to ICSI, which may allow for optimization of reproductive outcomes in heretofore unstudied patient populations.

Keywords: DNA decondensation; DNA fragmentation; Host; Hypo-osmotic swelling test; ICSI; Nuclear architecture; Sperm selection.

Fig. 1

The HOST classification. The HOST classification, as proposed in the WHO Laboratory Manual for Examination and Processing of Human Sperm (starting from the 3rd edition in 1992), with the additional B+ class, described by our team [3]

Fig. 2

Nuclear quality analysis was performed with four different assays. The spermatozoa from the different HOST classes, as well as unsorted spermatozoa, were evaluated based on 4 different parameters: A DNA fragmentation with a TUNEL assay, causing the sperm nuclei with significant DNA breaks to exhibit a green fluorescence (1), absent in spermatozoa with intact DNA (2). B DNA decondensation, with a chromomycin A3 assay, with which spermatozoa with decondensed DNA exhibit bright yellow fluorescence (1), as opposed to their condensed nuclei associated with dull orange fluorescence. C Inter-telomeric distance, using telomeric probes for the two arms of chromosomes 1. D Chromosomal territory area for chromosome 1, using a whole chromosome painting probe specific of that chromosome

Fig. 3

Evaluation of the different HOST classes, compared to that of unsorted, pre-HOST, spermatozoa, based on four nuclear quality parameters. Data shown corresponds to all the subjects together. Results with p<0.05 were considered statistically significant. A DNA fragmentation: Compared to unsorted spermatozoa (US), classes B, C, D/E, and F exhibit lower DNA fragmentation rates, and class A exhibits higher DNA fragmentation rates. B DNA decondensation: Compared to US, classes B and B+ exhibit lower decondensation rates, and classes A and G exhibit higher decondensation rates. C Inter-telomeric distance: The y axis corresponds to the mean inter-telomeric distance for chromosome 1 in relationship to the head’s length. Compared to US, classes B and B+ exhibit shorter inter-telomeric distances, and classes A, D/E, and G exhibit longer inter-telomeric distances. D Proportion of spermatozoa with joint telomeres: Compared to US, classes B and B+ exhibit a higher proportion of joint telomeres, and classes A, F, and G exhibit a lower proportion. E Chromosomal territory area: The y-axis corresponds to the mean chromosome 1 area, in relation to the total nuclear area. Compared to US, classes A, F, and G exhibit a larger chromosomal territory area for chromosome 1

Fig. 4

Review of the past and current studies assessing sperm quality based on HOST class. This figure summarizes the results of the present study as well as of others on the respective quality of the HOST classes, based on the following parameters: DNA fragmentation, DNA decondensation, phosphatidyl-serine externalization, aneuploidy, chromosomal segregation in rearrangement carriers, inter-telomeric distance, and chromosomal territory area. For each parameter and study, the results were normalized: 100% means that the HOST class showed the highest score for a given parameter, and 0% means it showed the lowest score. We highlight the relatively higher quality of spermatozoa belonging to the B and B+ HOST classes, and the relatively poorer quality of those belonging to the A, G, and possibly C, D/E, and F classes