Successful selection of mouse sperm with high viability and fertility using microfluidics chip cell sorter

Cell sorting via flow cytometry is a powerful tool to select subpopulations of cells in many biological fields. Selection of fertilisation-prone sperm is a critical step to ensure a stable and high fertilisation rate in in vitro fertilisation (IVF). However, a combination of conventional cell sorting and IVF system has not been established because of severe mechanical damages to the sperm during the sorting process. A cell sorter with microfluidics chip technology that lessens cell damage during cell sorting may address this problem. We evaluated the effects of microfluidics chip cell sorting on the sperm using the parameters, such as motility and fertility, and found this cell sorting method had minimal harmful effect on the sperm. Then, sperm were selected by a marker for acrosome reaction and showed higher fertilisation rate than that of the population of acrosome-intact sperm. Embryo derived from these sperm developed normally. These results indicated that microfluidics chip cell sorting can select fertile sperm to improve IVF technique.

. Effect of sorting on sperm motility. Epididymal sperm were sorted using a signal distribution of forward scattered light (FSC) and side scattered light (SSC) (A), and sperm motility was analysed using a HTM-IVOS. Motility is the ratio of sperm moving 5 µm/s to total sperm (B). Progressive motility is the ratio of sperm moving at 50 µm/s to total sperm and with straightness greater than 50% (C). Path velocity (VAP) is average velocity of the smoothed sperm path (D). Progressive velocity (VSL) is the average velocity measured in a straight line from the beginning to the end of the sperm track (E). Track speed (VCL) is the average velocity measured over the actual point to point sperm track (F). Lateral amplitude (ALH) is the mean width of the head oscillation as the sperm swims (G). Beat frequency (BCF) is the frequency of sperm heads crossing the average sperm path in either direction (H). Straightness (STR) is a measure of the departure of the average sperm path from a straight line (ratio of VSL/VAP) (I). Linearity (LIN) is a measure of the departure of the actual sperm track from a straight line (ratio of VSL/VCL) (J). Elongation is the ratio of head width to head length (K). Values are given as the mean ± SD (n = 7). *p < 0.05 compared with sort (−). We acknowledged that the copyright of  Effect of sorting on sperm fertility and developmental ability. Sperm were pre-incubated for 60 min, and sperm suspensions were diluted using mHTF to adjust the flow rate to approximately 400 events/s. Then, cell sorting was performed using forward scattered light (FSC) and side scattered light (SSC). After sorting, the sorted sperm suspensions were collected from the sorting reservoir and used as in vitro fertilisation (IVF) medium. Oocytes were introduced into the IVF medium. After IVF, the two-cell embryos were either cultured in KSOM/AA medium or transferred into the oviducts of pseudopregnant ICR females (10 embryos/oviducts). (B) The fertilisation rate was calculated as the number of two-cell embryos (A) divided by the number of inseminated oocytes x 100. (G) The developmental rate was calculated as the number of embryos at several stages-4-cell (D), morulae (E) and blastcysts (F)-divided by the number of two-cell embryos (C) x 100. (H) Two-cell embryos developed normally into live pups. I) the birth rate was calculated as the (number of live pups divided by the number of transferred two-cell embryos) x 100. Values are given as the mean ± SD (n = 3-6). *p < 0.05 compared with sort (−).
www.nature.com/scientificreports www.nature.com/scientificreports/ To examine the viability of the sorted sperm, the motility of sorted and unsorted sperm was compared. Although motile sperm were obtained after the cell sorting, the percentages of motile sperm and progressive motile sperm in the sorted sperm were lower than in the unsorted sperm (Figs. 1B,C). Conversely, other parameters of the sorted sperm were equivalent to those of the unsorted sperm ( Fig. 1D-K).
Fertility and developmental abilities of the sorted sperm. To examine the fertility of the sorted sperm, they were used in IVF, and it was found that in comparison to unsorted sperm, sorted sperm fertilised with oocytes and fertilised eggs developed normally up to the blastocyst stage in in vitro culture ( Fig. 2A-G). When the embryos were transferred to the pseudopregnant females, we obtained normal pups with birth rate similar to those of the unsorted sperm ( Fig. 2G-I). These observations suggest that sperm sorted by the microfluidics chip cell sorter have normal fertilisation and developmental ability. www.nature.com/scientificreports www.nature.com/scientificreports/ Selection of acrosome-reacted sperm by cell sorting. To examine the effect of sperm selection based on acrosome reaction (AR) on fertilisation ability, sperm were stained with FITC-labelled PNA and sorted based on the signal distribution of forward scattered light (FSC) and signal intensity of FITC (acrosome-intact, FITC negative: acrosome-reacted, FITC positive). The sperm were classified into three groups based on the signal intensity of FITC [acrosome-reacted low (AR-low), acrosome-reacted middle (AR-middle) and acrosome-reacted high (AR-high)] and collected separately ( Fig. 3C-E). Observation under fluorescence microscopy revealed that more than 80% of the acrosome-reacted sperm were in the AR-middle and AR-high groups (Fig. 3J). Conversely, in the AR-low group, the population of acrosome-reacted sperm was reduced by almost half. Therefore, acrosome-reacted sperm were enriched by the microfluidics chip cell sorter.
Then, to compare motility of the sorted three groups of sperm, they were analysed by computer-assisted sperm analyser in multiple parameters. All the parameters of sperm motility were not different among the three groups (Fig. 4).

Fertility and developmental abilities of acrosome-reacted sperm.
To study the effect of sperm sorting using a marker of AR on fertilisation ability, we conducted IVF using the AR-low, AR-middle and AR-high sperm. The fertilisation rate of the AR-high sperm was higher than was that of the AR-low sperm (Fig. 5A). The . Effect of selection on sperm motility. After selection, sperm motility was analysed using an HTM-IVOS. Motility is the ratio of sperm moving 5 µm/s to total sperm (A). Progressive motile is the ratio of sperm moving at 50 µm/s to total sperm and swith traightness higher than 50% (B). Path velocity (VAP) is the average velocity of the smoothed sperm path (C). Progressive velocity (VSL) is the average velocity measured in a straight line from the beginning to the end of the sperm track (D). Track speed (VCL) is the average velocity measured over the actual point to point sperm track (E). Lateral amplitude (ALH) is the mean wdth of the head oscillation as the sperm swims (F). Beat frequency (BCF) is the frequency of sperm heads crossing the average sperm path in either direction (G). Straightness (STR) is a measure of the departure of the average sperm path from a straight line (ratio of VSL/VAP) (H). Linearity (LIN) is a measure of the departure of the actual sperm track from a straight line (ratio of VSL/VCL) (I). Elongation is the ratio of head width to head length (J). Values are given as the mean ± SD (n = 8-10). *p < 0.05 compared with AR-low. (2020) 10:8862 | https://doi.org/10.1038/s41598-020-65931-z www.nature.com/scientificreports www.nature.com/scientificreports/ two-cell embryos developed normally into blastocysts and pups (Fig. 5B). When the embryos were transferred to the pseudopregnant females, we obtained normal pups with similar rate among three groups (Fig. 5C). These observations suggest that AR-based sperm sorting is effective to select fertilisation-prone sperm.

Discussion
Sperm selection by flow cytometry is usually used for measuring DNA content [11][12][13] . Difference in the DNA content between X-and Y-chromosome sperm has been applied in the sex selection method used for rabbits, pigs, swine, cattle, sheep and horses [14][15][16] , combined with assisted reproductive technologies, such as IVF, artificial insemination or intracytoplasmic sperm injection to produce offsprings [17][18][19] . However, to the best of our knowledge, there have been no reports on sperm selection using flow cytometry and the use of the sorted sperm to produce embryos in mice using IVF.
In this study, we demonstrated that the microfluidics chip cell sorter is useful for sperm selection. The selected sperm showed in vitro fertility and full developmental ability. To the best of our knowledge, this may be the first report that sorted mouse sperm could produce healthy embryos and pups via IVF.
In addition, we demonstrated that sperm selection of AR-high sperm indicated higher fertilisation rate than that of AR-low sperm (Fig. 5). AR is a morphological change in sperm that occurs during sperm capacitation [20][21][22][23] and is essential for fertilisation 24 . AR is induced by physiological and chemical stimulations such as interaction of ZP, progesterone or calcium ionophore [25][26][27] . However, the timing of AR is flexible and largely unknown in vivo. In mice sperm, it was clarified that most fertilising sperm initiate AR before contacting ZP and acrosome-reacted sperm can pass through ZP and fertilise eggs 28 . On the other hand, acrosome-intact and acrosome-reacted sperm are heterogeneously present with oocytes in IVF medium. We hypothesised that the presence of acrosome-intact sperm negatively affects to success of fertilisation. In this study, we first demonstrated that the sorted sperm from the AR-high group indicated higher fertilisation ability than those from the AR-low group in IVF. This finding implies that the selection of AR sperm is an important step to achieve fertilisation. www.nature.com/scientificreports www.nature.com/scientificreports/ In this study, we demonstrated that FITC-labelled PNA sperm sorted using the microfluidic cell sorter had good motility, fertilisation and developmental abilities. FITC is the most widely used fluorescence probe conjugated with a small compound, protein or an antibody 29 . A study indicated that FITC does not impair the motility and fertilising functions of human sperm 30 . These results suggest that FITC and FITC-labelled compounds are useful to label sperm with normal functions. However, further investigations are needed to safely use the technique in industrial and medical applications.
Recently, Umehara reported a novel technique of sex selection based on Toll-like receptors 7/8 (TLR7/8) 31 . The TLR7/8 ligand suppressed the motility of X-chromosome-bearing sperm. The difference in motility between Xand Y-chromosome-bearing sperm after reagent treatment was applicable to produce pups of selected sex. Using the technique, 83% (XY, male) or 81% (XX, female) of pups were of the expected sex. Selection of TLR7/8-positive or TLR7/8-negative sperm by the microfluidic cell sorter may be a new application of sex sorting for IVF and embryo transfer and may improve the success rate of sex selection.
In conclusion, we showed that the microfluidics chip cell sorter is helpful in sorting and selecting mouse sperm. Application of the technology can improve the IVF technique by selecting good quality sperm. We consider that this sperm sorting technique represents an innovative approach to solving technical problems with reproductive technology used in various mammalian species. Reagents and media. BSA-free Toyoda Yokoyama Hosi medium, a modified Krebs-Ringer bicarbonate solution (fTYH) with 0.75 mM methyl-beta-cyclodextrin and 1.0 mg/mL polyvinyl alcohol (Sigma) (cTYH), was used as a medium for pre-incubation of sperm [32][33][34] . Calcium-enhanced human tubal fluid (mHTF) was used as the fertilisation medium [35][36][37] , and potassium simplex optimised medium (KSOM) was used for in vitro culture and embryo transfer 38 .

Animals
Sperm sorting. Enumeration and sorting of sperm were performed using the microfluidics chip cell sorter (On-Chip Sort, On-chip Biotechnologies, Japan). The fluid channel was pre-washed with mHTF, and sperm samples were dissolved in mHTF so that the flow rate was approximately 400 events/s. The sperm were gated into scatter and collected in the collection reservoir. Collected sperm were fixed into 200 µL added mHTF and put it on the dish and covered with mineral oil. The dish was using as a fertilisation dish and the sperm was assessed the motility.
Sperm selection based on AR. Sorting of sperm was conducted using the microfluidics chip cell sorter The fluid channel was pre-washed with mHTF, and sperm samples were dissolved in mHTF so that the flow rate was approximately 400 events/s. The sperm were gated into scatter or fluorescence intensity of FITC-labelled PNA (Figs. 3A,B) and collected in the collection reservoir. We classified them into three groups-AR-low, AR-middle and AR-high-by intensity of FITC ( Fig. 3C-E). Collected sperm were fixed into 200 µL added mHTF and put it on the dish and covered with mineral oil. The dish was using as a fertilisation dish and the sperm was assessed the motility and the AR.
Assessment of sperm motility. Sperm motility was assessed using a computer-assisted sperm analyser (IVOS Sperm Analyzer, Hamilton-Thorne Research Co. Ltd., USA) 39 . Sperm were incubated in cTYH for 60 min at 37 °C under 5% CO 2 in air. Sperm suspensions were diluted 50 times in mHTF, incubated for 5 min at 37 °C and sorted using the microfluidics chip cell sorting system described previously. After sorting, sperm suspensions were placed in a disposable sperm analysis chamber (Hamilton-Thorne Research) and analysed using the IVOS system. We analysed the following sperm motility parameters: percentage of motile sperm (motile sperm moved more than 5-µm/s), percentage of motile sperm with progressive motility (motile sperm with progressive motility were denoted by a path velocity> 50 µm/s and a straightness ratio> 50%) and a marker of hyperactivation [lateral amplitude of head (ALH): this is the average value of the maximum swing width of the sperm head]. In addition, path velocity (VAP), progressive velocity (VSL), track speed (VCL), beat frequency (BCF), straightness (STR), Linearity (LIN) and Elongation were measured. During motility analysis, the sperm analysis chamber was warmed at 37 °C. To analyse the motile parameters, 200-1000 sperm were examined in each experiment. The experiments were independently from 7 to 10 times. Assessment of acrosome-reacted sperm. Acrosome-reacted sperm were evaluated using the peanut agglutinin FITC method 40 . Epididymal sperm were collected in 90 µl of cTYH, and the sperm suspensions were incubated for 60 min at 37 °C under 5% CO 2 in air. Then, 10 µl of fTYH containing FITC-labelled PNA (10 µg/mL) was added to the drop of sperm suspension and incubated for 10 min. Sperm suspensions were diluted 50 times in mHTF and incubated for 5 min at 37 °C. Following incubation, sperm selection was conducted using the microfluidics chip cell sorting system and acrosome-reacted sperm (FITC-labelled PNA positive) were observed under fluorescence microscopy (Keyence Co. Ltd., Japan) to count the number of acrosome-intact sperm [AR(−)] (Fig. 3F,G) and acrosome-reacted sperm [AR (+),] (Fig. 3H,I). Percentage of acrosome-reacted sperm was calculated by the number of AR (+) sperm divided by the number of AR (+) sperm and number of AR (−) sperm.