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. 2023 Nov 28;33(1):36.
doi: 10.1186/s12610-023-00211-0.

External quality assessment scheme for sperm DNA fragmentation: a pilot study in China

Yan Zheng  1   2 , Ying-Bi Wu  1   2 , Ye-Lin Jia  1   2 , Li-Juan Ying  1   2 , Ting-Ting Yang  1   2 , Qing-Yuan Cheng  1   2 , Jiao Qin  1   2 , Chen Luo  1   2 , Lin Yu  3   4 , Fu-Ping Li  5   6
Affiliations

External quality assessment scheme for sperm DNA fragmentation: a pilot study in China

Yan Zheng et al. Basic Clin Androl. .

Abstract
in English, French

Background: The aim of this article is to establish an external quality assessment (EQA) scheme for sperm Deoxyribonucleic acid (DNA) fragmentation (SDF) detection, and to assess the feasibility of the scheme. In addition, this article provides some case analysis of abnormal results in order to really help improve the performance of the laboratory.

Results: In 2021 and 2022, 10 and 28 laboratories in China volunteered to participate in the EQA program respectively. Two samples were selected for EQA each year, a large spread of results was obtained for the four samples, and the highest values were 13.7, 4.2, 8.0 and 4.0 times the lowest respectively. The coefficients of variation (CVs) were very high for the four samples, at 46.6%, 30.1%, 26.7% and 30.3%, respectively. The CVs of the samples with high SDF values were lower than those of the samples with low SDF values. There was no significant difference between the results of sperm chromatin structure assay (SCSA) and sperm chromatin dispersion (SCD). For the 10 laboratories that participated in EQA in 2021 and 2022, the CVs of low SDF value samples and high SDF value samples decreased from 46.6% and 30.1% in 2021 to 32.5% and 22.7% in 2022, respectively.

Conclusion: This is the first study to evaluate the EQA program on SDF, which involved a number of laboratories and was demonstrated to be feasible. It is recommended that all laboratories participate in the EQA of SDF to ensure the accuracy of the results.

RéSUMé: CONTEXTES: L’objectif de cet article est d’établir un système externe d’évaluation de la qualité (EEQ) pour la détection de la fragmentation de l’ADN des spermatozoïdes (SDF) et d’évaluer la faisabilité de ce système. En outre, cet article fournit une analyse de cas de résultats anormaux afin d’aider réellement à améliorer les performances du laboratoire. RéSULTATS: En 2021 et 2022, respectivement 10 et 28 laboratoires en Chine se sont portés volontaires pour participer au programme EEQ. Deux échantillons ont été sélectionnés chaque année pour l’EEQ ; un large éventail de résultats a été obtenu pour les quatre échantillons, et les valeurs les plus élevées étaient respectivement de 13,7, 4,2, 8,0 et 4,0 fois les plus faibles. Les coefficients de variation (CV) étaient très élevés pour les quatre échantillons, soit respectivement 46,6 %, 30,1 %, 26,7 % et 30,3 %. Les CV des échantillons avec des valeurs de SDF élevées étaient inférieurs à ceux des échantillons avec de faibles valeurs de SDF. Il n’y avait pas de différence significative entre les résultats du test de structure de la chromatine des spermatozoïdes (SCSA) et ceux de la dispersion de la chromatine des spermatozoïdes (SCD). Pour les 10 laboratoires qui ont participé à l’EEQ en 2021 et 2022, les CV des échantillons à faible valeur de SDF et ceux des échantillons à valeur élevée de SDF ont diminué, passant respectivement de 46,6 % et 30,1 % en 2021 à 32,5 % et 22,7 % en 2022. CONCLUSIONS: Il s’agit de la première étude à évaluer le programme externe d’évaluation de la qualité (EEQ) de l’analyse de la SDF, qui a impliqué un certain nombre de laboratoires, et qui s’est avéré réalisable. Il est recommandé que tous les laboratoires participent à l’EEQ de la SDF afin d’en assurer l’exactitude des résultats.

Keywords: External quality control; Sperm DNA fragmentation; Sperm chromatin dispersion; Sperm chromatin structure assay.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Distribution of spermatozoa under a microscope after thawing. Phase-contrast microscopy was performed with a green filter at 20 × magnification. Spermatozoa were unstained
Fig. 2
Fig. 2
Results of SCSA and SCD for four samples (2021A, 2021B, 2022A and 2022B). The 25th and 75th percentiles are represented by boxes, with the median value, while the 10th and 90th percentiles are represented by whiskers. DFI, DNA fragmentation index; SCSA, sperm chromatin structure assay; SCD, sperm chromatin dispersion
Fig. 3
Fig. 3
The CVs of SCSA and SCD in four samples (2021A, 2021B, 2022A and 2022B). CV, coefficient of variation; SCSA, sperm chromatin structure assay; SCD, sperm chromatin dispersion
Fig. 4
Fig. 4
Comparison of the mean bias of 10 laboratories in 2021 and 2022. The 10 points on the left are the mean bias of two samples of 10 participants in 2021. The 10 points on the right are the mean bias of the two samples in 2022 for those 10 participants
Fig. 5
Fig. 5
A Dot plot with lower voltage settings for flow cytometry. The voltage was low and the boundary of the sub-populations was unclear. B Dot plot with normal voltage settings for flow cytometry. The voltage was normal and the boundary of the sub-populations was clear. FSC: forward scatter; SSC: side scatter
Fig. 6
Fig. 6
A Dot plot with a sample size of 25 µl for flow cytometry. When 25 µl of the semen sample was added, the denaturation effect of the low-pH (1.2) detergent solution on sperm chromatin was weakened, and the result of sperm DNA fragmentation was low. B Dot plot with a sample size of 5 μl for flow cytometry. When 5 μl of the semen sample was added, the acid denaturation effect of the acid treatment solution was good and the result of sperm DNA fragmentation was accurate. PerCP-Cy5.5: red AO fluorescence of sperm with broken DNA; FIFC: green AO fluorescence of DNA stainability
Fig. 7
Fig. 7
A Dot plot with fixed gating analysis for flow cytometry. The fixed gating of the flow cytometry analysis software was biased to the right, and the result of sperm DNA fragmentation was reduced. B Dot plot with manual gating analysis for flow cytometry. Sperm with abnormal DNA fragmentation can be well included by manual gating. PerCP-Cy5.5: red AO fluorescence of sperm with broken DNA; FIFC: green AO fluorescence of DNA stainability
Fig. 8
Fig. 8
A Dot plot with low speed for flow cytometry. At low speed, a single cell passes through the sample tube, and cells appear on a diagonal. B Dot plot with high speed for flow cytometry. At high speed, multiple cells pass through the sample tube at the same time, and the cells are clustered together in an off-diagonal distribution. FSC: forward scatter; SSC: side scatter
See this image and copyright information in PMC

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