Selection of functional human sperm with higher DNA integrity and fewer reactive oxygen species

Abstract

Fertilization and reproduction are central to the survival and propagation of a species. Couples who cannot reproduce naturally have to undergo in vitro clinical procedures. An integral part of these clinical procedures includes isolation of healthy sperm from raw semen. Existing sperm sorting methods are not efficient and isolate sperm having high DNA fragmentation and reactive oxygen species (ROS), and suffer from multiple manual steps and variations between operators. Inspired by in vivo natural sperm sorting mechanisms where vaginal mucus becomes less viscous to form microchannels to guide sperm towards egg, a chip is presented that efficiently sorts healthy, motile and morphologically normal sperm without centrifugation. Higher percentage of sorted sperm show significantly lesser ROS and DNA fragmentation than the conventional swim-up method. The presented chip is an easy-to-use high-throughput sperm sorter that provides standardized sperm sorting assay with less reliance on operators's skills, facilitating reliable operational steps.

Keywords: DNA integrity; advance reproductive technologies; reactive oxygen species; reproduction; sperm sorting.

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1

Macro- Microfluidic Sperm Sorter (MSS) for selection of functional human sperm with higher DNA integrity and fewer reactive oxygen species. (a) The photo of the MSS showing inlet, filter and two PMMA chambers. The MSS was filled with color dye to enhance contrast. (b) The illustration demonstrates the MSS design and working principle. The MSS consists of one inlet for the injection of raw unprocessed semen sample and two PMMA chambers separated by nuclepore track-etched membrane filter. The most healthy and motile sperm swim through the filter leaving unhealthy dead sperm in the bottom chamber. (c) SEM images of polycarbonate nuclepore track-etched membrane filters of different micropore diameters, i) 3 μm ii) 5 μm and iii) 8 μm. These images shows the comparative size of various filter pores and sperm. The scale bar is 10 μm.

Figure 2

Sperm sorting, motility and viability analysis. (a) %Motility of human sperm isolated using different pore diameter filters 3 μm, 5 μm and 8 μm. Sperm were retrieved at three time points, i.e 15, 30, and 45 min. Motility of the sperm sorting by using filters was significantly higher than stock sample motility (*p-value < 0.05 between stock semen and sorted sperm, N=3). (b) Retrieval efficiency of sorted sperm using 3 different chips. Retrieval efficiency was saturated after 30 min time point (*p-value < 0.05 between time points, N=3). Simulation results for (c) 5 μm chip and (d) 8 μm chip. Error bars represent standard error of the mean in (a-d). (e) Representative fluorescent live/dead (green/red) images of unsorted and sorted sperm using 3 different MSS chips.

Figure 3

Diluted sperm sorting and motility analysis using MSS. (a) Semen sample was diluted with HTF+1% BSA buffer at 1:4 ratio. %Motility of diluted human sperm isolated using different pore diameter filters 3 μm, 5 μm and 8 μm. Sperm were retrieved at three time points, i.e 15, 30, and 45 min. Motility of the sperm sorting by using filters was significantly higher than diluted stock motility (*p-value < 0.05 between stock semen and sorted sperm, N=3). (b) Retrieval efficiency of sorted sperm using 3 different MSS chips (*p-value < 0.05 between time points, N=3). Simulation results for (c) 5μm chip and (d) 8μm chip. Error bars represent standard error of the mean.

Figure 4

Sperm velocity analysis. (a) Curvilinear velocity (VCL), (b) Straight line Velocity (VSL), and (c) Average path velocity (VAP) of stock and sorted sperm using 3, 5, and 8μm MSS chips. The sperm analyzed for velocity parameters were collected from retrieval chamber of the chips after 30 min time point. The sperm sorted using 5 and 8 μm chips showed significantly improved velocity parameters compared to unprocessed stock semen (*p-value < 0.05 between stock semen and sorted sperm, N=10-20) .Error bars represent standard error of the mean.

Figure 5

Morphological and nuclear maturity analysis of sperm. (a) Representative brightfield images of sperm using optical microscope and an oil immersed 100x objective. Total magnification was 1000x. The table summarizes the criteria and typical comments used to assess sperm morphology. Five sperm are shown in these images whereas only 1 is morphologically normal. (b) Plots shows normal morphology (%) for stock and sorted sperm. Sperm sorted using 8 μm MSS showed more percentage of normal sperm compared to stock and 5 μm MSS (N=3). (c) Representative grayscale images of sperm stained with aniline blue and a table to show condition of chromatin. (d) Mature sperm percentage calculated for sperm from stock, 5μm and 8μm MSS (N=3). Error bars represent standard error of the mean.

Figure 6

Analysis of reactive oxygen species (ROS) generation and DNA fragmentation in sorted sperm using flowcytometer. (a) Sperm sorted using 3, 5, and 8 μm chips showed significantly lesser ROS generation compared to swim-up method (*p-value < 0.05 between swim-up and other sperm samples, N=4). Sperm sorted using 3 and 5 μm chips showed significantly lesser ROS generation compared to washing method (#p -value < 0.05 between raw semen sample and others N=4). (b) Sperm sorted using 3, 5, 8 μm chips showed significantly lesser DNA fragmentation compared to unsorted semen sample (*p-value < 0.05 between swim-up and other sperm samples, N=6). Sperm sorted using 8 μm chips showed lesser DNA fragmentation compared to swim-up (p-value < 0.06). Error bars represent standard error of the mean.