Hypo-Osmotic Swelling (HOS Test)
Vitality test using hypo-osmotic swelling
As an alternative to dye exclusion, the hypo-osmotic swelling test may be used to assess vitality. This is useful when staining of spermatozoa must be avoided, e.g. when choosing spermatozoa for intracytoplasmic sperm injection (ICSI). The hypo-osmotic swelling test presumes that only cells with intact membranes (live cells) can swell in hypotonic solutions. Spermatozoa with intact membranes swell within 5 minutes in hypo-osmotic medium, and all flagellar shapes are stabilized by 30 minutes.
• Use 30 minutes incubation for routine diagnostics.
• Use 5 minutes incubation when spermatozoa are to be processed for therapeutic use.
Preparing the reagents 1. Swelling solution for diagnostic purposes: dissolve 0.735 g of sodium citrate dihydrate and 1.351 g of D-fructose in 100 ml of purified water.
• 1-ml aliquots of this solution can be frozen at –20 °C.
2. For spermatozoa intended for Medically Assisted Reproduction (MAR), dilute the culture medium to be used 1+1 (1 : 2) with appropriate sterile, purified water.
Postive = >60% swelling
Procedure
1. Thaw the frozen swelling solution and mix well before use.
2. Warm 1 ml of swelling solution or 1 ml of 1+1 (1 : 2) diluted medium in a closed microcentrifuge tube at 37 °C for 5 minutes.
3. Mix the ejaculate well.
4. Remove a 100-µl aliquot of ejaculate and add to the swelling solution. Mix gently by drawing it in and out of the pipette.
5. Incubate at 37 °C for exactly 5 minutes or 30 minutes (see above), then transfer a 10-µl aliquot to a clean slide and cover with a 22 mm × 22 mm coverslip.
6. Remix the semen sample, remove a replicate aliquot, mix with swelling solution, and prepare a replicate slide, as above.
7. Examine each slide with phase contrast optics at ×200 or ×400 magnification.
8. Tally the number of unswollen (dead) and swollen (vital) cells with the aid of a laboratory counter.
9. Evaluate 200 spermatozoa in each replicate, to achieve an acceptably low sampling error.
Scoring
1. Swollen spermatozoa are identified by changes in the shape of the cell, as indicated by coiling of the tail.
2. Live cells are distinguished by evidence of swelling of the sperm tail; score all forms of swollen tails as live spermatozoa.
Postive = >60% swelling
Negative = < 50% swelling