Chapter 3

The Hypo-osmotic Swelling Test for Evaluation

of Sperm Membrane Integrity
Sivakumar Ramu and Rajasingam S. Jeyendran

Abstract
A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in
sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-
osmotic swelling (HOS) test evaluates the functional integrity of the sperm’s plasma membrane and also
serves as a useful indicator of fertility potential of sperm. The HOS test predicts membrane integrity by
determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its
environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or
“swell.” A higher percentage of swollen sperm indicates the presence of sperm having a functional and
intact plasma membrane. Here, we present the detailed protocol for performing the HOS test and explain
the results for interpretation.

Key words: Sperm membrane integrity, Fertility, Hypotonic solution, HOS test, Sperm function

1. Introduction

In the diagnosis of male infertility, a number of sperm characteristics

are evaluated. A test for assessing the functional integrity of the
sperm membrane is one important measure. Not only sperm mem-
brane integrity is important for its metabolism, but also a correct
change in the dynamics of the membrane is required for successful
union of the male and female gametes, i.e., for sperm capacitation,
acrosome reaction, and binding of the spermatozoon to the egg
surface. Thus, the integrity and functional activity of the sperm
membrane are of fundamental importance in the fertilization
process, and assessment of membrane function may be a useful
indicator of the fertilizing potential of spermatozoa.
When exposed to hypo-osmotic stress, water and small-
molecular-weight compounds and elements will attempt to enter

Douglas T. Carrell and Kenneth I. Aston (eds.), Spermatogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 927,
DOI 10.1007/978-1-62703-038-0_3, © Springer Science+Business Media, LLC 2013

21
22 S. Ramu and R.S. Jeyendran

Fig. 1. Schematic representation of various morphological changes of human spermatozoa exposed to hypo-osmotic
stress. (a) Sperm with unaltered morphology. (b–g) Sperm with different types of tail swelling indicated by hatched area.
Figure originally published in ref. 1 reproduced with permission.

into the sperm to reach osmotic equilibrium. This inflow of fluids

will increase sperm volume, and the plasma membrane will bulge
(balloon) to achieve a minimum surface-to-volume ratio (1). The
sperm tail appears to be particularly susceptible to such hypo-
osmotic stress, and based on the vigor of the sperm, different
patterns of tail swelling are observed (Fig. 1). These induced
changes to sperm tail morphology are visible with phase-contrast
microscopy. This ballooning effect is often referred to as “swell-
ing.” It can be assumed that the ability of the sperm tail to swell in
the presence of a hypo-osmotic stress is a sign that transport of
fluid across the membrane occurs normally, i.e., a sign of both
structural and functional integrity of the membrane as opposed to
only structural integrity of the cell membrane, which is the basis
for eosin Y exclusion in the vital staining test (2). The hypo-osmotic
swelling (HOS) test is useful when performing a semen analysis in
the diagnosis of the infertile male (1, 3–5). In addition to provid-
ing valuable information on sperm viability, this test is widely used
to select sperm for intracytoplasmic sperm injection (ICSI) in cases
of asthenozoospermia (6). Here we provide in detail the protocol
for performing the HOS test and the interpretation of the results.

2. Materials

Prepare all solutions using distilled water and analytical grade

reagents. Prepare and store all reagents at the recommended
temperature.

2.1. Hypo-osmotic 1. Solution A: 1.5 mM fructose.

Solution Preparation Weigh 2.7 g fructose (MW 180.16) in a sterile beaker.
Add 100 ml distilled H2O.
 3 The Hypo-osmotic Swelling Test for Evaluation of Sperm Membrane Integrity 23

2. Solution B: 1.5 mM sodium citrate.

Weigh 1.47 g sodium citrate (MW 294.1) in a sterile beaker.
Add 100 ml distilled H2O.
3. Mix equal volume of Solutions A and B, and store in 1 ml ali-
quots using disposable, polypropylene tubes at −20°C until use
or up to 1 year (see Note 1).

2.2. Semen Sample Collect the semen samples by masturbation or other recommended
procedure and allow the sample to liquefy completely (~30 min)
(see Note 2).

2.3. Additional 1. Distilled H2O.

Supplies Needed 2. Glass slide with coverslip.
3. 37°C incubator (optional).
4. Laboratory cell counter.
5. Measuring cylinder.
6. Phase contrast microscope.
7. Pipette.
8. Polypropylene tubes.
9. Sterile beaker.
10. Weighing balance.

3. Methods

3.1. HOS Test 1. Incubate tube containing 1.0 ml of hypo-osmotic (HOS) solu-
tion at 37°C for 10 min.
2. Add 0.1 ml of thoroughly mixed, liquefied semen to tube con-
taining HOS solution, and mix gently.
3. Incubate semen/HOS solution mixture for at least 30 min,
but not longer than 3–4 h, at 37°C (see Note 3).
4. After incubation at 37°C mix the tube gently, place a drop
onto a microscopic slide, and cover the drop with coverslip
(see Note 4).
5. Place prepared slide on microscope and observe under phase
contrast (400×) for the spermatozoa with swollen tails as
shown in Fig. 1 (see Note 5).

3.2. Sperm Swelling Hypo-osmotic stress will induce several distinct categories of swell-
Pattern ing in the sperm tail region as described below (Figs. 1 and 2):
1. Tip: Very tip of the tail is swollen; rest of the tail is normal.
2. Hairpin swelling: Tail swells at mid piece and main piece
junction with tip swelling or without tip swelling.
24 S. Ramu and R.S. Jeyendran

Fig. 2. Spermatozoa were either unexposed (a) or exposed (b–d) to hypo-osmotic stress for 30 min at 37°C. A representative
slide prepared and observed under phase contrast microscope displaying various forms of tail swelling. The pictures were
taken using a Nikon microscope with digital imaging system (magnification, ×650).

3. Shortened and thickened tail: Tail swells, constricting surface,

causing shortening.
4. Partly or completely enveloped sperm tail: Tail “balloons” from
swelling.
5. Count a total of 200 sperm (at least 100) per sample, including
both swollen and non-swollen sperm.
6. Calculate swollen sperm as a percentage of total sperm number
counted.
7. Report as percentage of swollen sperm.

3.3. Results Measured as a percentage of sperm tails observed as swollen:

and Interpretation Record the results in percentage swollen on an observation sheet
like one shown below:

Interpretation
Result
Sample ID (% swollen) Normal Equivocal Abnormal
³60 % 50–59 % <50 %

Since only a single variable (membrane function) is measured, caution

is advised. For example, the HOS test is “normal” but some other
sperm parameter may be defective rendering the sperm infertile.
The abnormal result is more important than the normal result.
 3 The Hypo-osmotic Swelling Test for Evaluation of Sperm Membrane Integrity 25

4. Notes

1. The HOS test solution should be clear. If you notice any turbidity,
discard and prepare a fresh stock.
2. For highly viscous specimens, ejaculate may be diluted with an
equal volume of medium or forced in and out of a 3 ml syringe
attached to an 18-gauge needle until the sample becomes more
pliable.
3. The HOS test can be performed at ambient temperature for
the same period without much change in the expected results.
4. Using a Kim wipe, gently pat down the coverslip to remove the
excess fluid and form a thin film of one layer of sperm.
5. Count cells under 400× magnification, and appropriate phase
for accurate assessment of the tail swelling. If bright field is used,
then count coiled tails prior to HOS test and subtract the per-
cent of coiled tails obtained prior to the HOS test from the
percent of swollen sperm following the HOS test.

References
1. Jeyendran RS et al (1984) Development of an
assay to assess the functional integrity of the
human sperm membrane and its relationship to
other semen characteristics. J Reprod Fertil
70:219–228
2. Schrader SM et al (1986) Sperm viability: a
comparison of analytical methods. Andrologia
18:530–538
3. Van der Ven HH et al (1986) Correlation
between human sperm swelling in hypoos-
motic medium (hypoosmotic swelling test)
and in vitro fertilization. J Androl 7:
190–196
4. Jeyendran RS et al (1992) The hypoosmotic
swelling test: an update. Arch Androl 29:
105–116
5. Hossain A et al (2010) Spontaneously devel-
oped tail swellings (SDTS) influence the
accuracy of the hypo-osmotic swelling test
(HOS-test) in determining membrane integ-
rity and viability of human spermatozoa. J Assist
Reprod Genet 27:83–86
6. Verheyen G et al (1997) Comparison of differ-
ent hypo-osmotic swelling solutions to select
viable immotile spermatozoa for potential use
in intracytoplasmic sperm injection. Hum
Reprod Update 3:195–203