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. 2023 Aug;40(8):1855-1864.
doi: 10.1007/s10815-023-02838-4. Epub 2023 Jun 10.

Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting

Cheng-Teng Hsu #  1 , Chun-I Lee  2   3   4   5 , Fong-Sian Lin  1 , Fang-Zong Wang  1 , Hui-Chen Chang  1 , Tse-En Wang  1 , Chun-Chia Huang  2   5 , Hui-Mei Tsao  2 , Maw-Sheng Lee  2   3   4   5 , Ashok Agarwal  6   7   8
Affiliations

Live motile sperm sorting device for enhanced sperm-fertilization competency: comparative analysis with density-gradient centrifugation and microfluidic sperm sorting

Cheng-Teng Hsu et al. J Assist Reprod Genet. 2023 Aug.

Abstract

Purpose: A live motile sperm sorting device (LensHooke® CA0) developed to prevent the deleterious effects of centrifugation was evaluated comparatively with conventional density-gradient centrifugation (DGC) and microfluidic-based device (Zymot) in sperm selection.

Methods: Semen samples from 239 men were collected. CA0 under different incubation intervals (5, 10, 30, and 60 min) and temperatures (20, 25, and 37℃) was conducted. The sperm quality in CA0-, DGC-, and Zymot-processed samples was then comparatively evaluated. Semen parameters included concentration, motility, morphology, motion kinematics, DNA fragmentation index (DFI), and the rate of acrosome-reacted sperm (AR).

Results: Total motility and motile sperm concentration increased in a time- and temperature-dependent manner and the total motility peaked for 30 min at 37℃. In paired analysis, CA0 showed significantly higher total motility (94.0%), progressive motility (90.8%), rapid progressive motility (83.6%), normal morphology (10.3%), and lower DFI (2.4%) and AR (4.7%) than the other two methods in normozoospermic samples (all p < 0.05). For non-normozoospermic samples, CA0 had significantly better results than the other two methods (total motility 89.2%, progressive motility 80.4%, rapid progressive motility 74.2%, normal morphology 8.5%, DFI 4.0%, and AR 4.0%; all p < 0.05).

Conclusion: CA0 yielded spermatozoa with enhanced sperm fertilization potentials; DFI was minimized in samples processed by CA0. CA0 was effective for both normal and abnormal semen samples due to its consistent selection efficiency.

Keywords: Sperm DNA fragmentation; Sperm motility; Sperm preparation; Sperm quality; Sperm retrieval.

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Conflict of interest statement

This study was supported by Bonraybio Co., Ltd. C.T.H. is the owner of Bonraybio Co., Ltd. and F.S.L., F.Z.W., H.C.C., and T.E.W. are employees of Bonraybio Co., Ltd. The employer (C.I.L., C.C.H., and H.M.T.) of Lee Women’s Hospital has collaborated with Bonraybio Co., Ltd. with regard to the IRB approval for providing human samples to complete this collaborative project. A.A. reports no financial support from Bonraybio Co., Ltd. Bonraybio Co., Ltd. reports patent applications “Sperm sorting device and method” licensed in Taiwan (application number: 111110406; 111209204), China (application number: 202210427414.7; 202220949418.7; 202222325590.8), the USA (application no: 17/815,484), and Europe (application no: 22188408.3). The remaining authors have no conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1
CA0 sorts neat semen sample based on sperm motility. a The LensHooke® CA0 sperm-separation device consists of three components: lower chamber (gray), upper chamber (dark gray) with a built-in membrane filter and fabricated retrieval port, and a cover (light gray). b The separation steps are as follows: (1) place the lower chamber on a flat surface, (2) add 1 mL neat semen sample (shown in gray) until the lower chamber is filled, (3) place the upper chamber on top of the lower chamber, (4) add 0.9 mL sperm wash media (shown in pink) into the upper chamber, (5) close the CA0 device with the cover and incubate the assembly at 37℃, and (6) aspirate 0.5 mL sperm suspension from the retrieval port of the upper chamber. c Motile sperm (sperm head shown in yellow) vertically swim along the wall of the collection area within the lower chamber. Self-propelling sperm pass through a built-in microporous membrane filter in the upper chamber, whereas immotile sperm (head in gray) and debris (grayish round structures) sediment go to the bottom of the lower chamber
Fig. 2
Fig. 2
CA0 performance in total motility and motile sperm concentration with different incubation times and incubation temperatures. Nineteen normozoospermic samples with normal sperm parameters and 40 non-normozoospermic samples with abnormal sperm concentration, total motility, normal morphology, or combined, based on the WHO 5th lower reference limit, were processed. Each semen sample was split into five aliquots. One aliquot was allocated as the control without sperm separation (Neat), and other four aliquots were placed into the CA0 device and incubated for 5, 10, 30, and 60 min, respectively. Values of (a) total motility (%) and (b) motile sperm concentration (M/mL) were measured. Next, 23 normozoospermic samples and 38 non-normozoospermic samples were prepared. A liquefied semen aliquot was placed in the collection tube as the control (Neat); three liquefied semen aliquots were placed into the CA0 device and incubated at 20, 25, and 37℃ for 30 min respectively. Values of (c) total motility (%) and (d) motile sperm concentration (M/mL) were measured. Box plots illustrate the data distribution, with five horizontal lines representing the 10th, 25th, 50th, 75th, and 90th percentiles of a variable. The values outside the 10th and 90th percentile are plotted as individual diamonds (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)
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