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Plasma membrane integrity of
cryopreserved human sperm:
an investigation of the results of the
hypoosmotic swelling test, the water test,
and eosin-Y staining
Ming-Huei Lin, M.D., Mahmood Morshedi, Ph.D., Chartchai Srisombut, M.D.,
Ahmed Nassar, M.D., and Sergio Oehninger, M.D.
Department of Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia
Medical School, Norfolk, Virginia and Department of Obstetrics and Gynecology, Reproductive Endocrinology
and Infertility Unit, Mackay Memorial Hospital, Taipei, Taiwan, Republic of China
Objective: [1] To examine the relationship between sperm membrane integrity and motion parameters before
and after cryopreservation; [2] to determine the capacity of the membrane integrity tests to predict the outcome
of cryopreservation in fertile and infertile men; and [3] to examine the degree of agreement between tail and
head membrane integrity of testicular and ejaculated immotile sperm cryopreserved for intracytoplasmic
sperm injection.
Design: Prospective study.
Setting: Academic tertiary care institution.
Patient(s): Fertile donors and normozoospermic oligozoospermic, and asthenozoospermic subfertile men.
Intervention(s): Semen samples were cryopreserved and thawed for analysis.
Main Outcome Measure(s): Sperm membrane integrity and computer-assisted motion parameters.
Result(s): The hypoosmotic swelling test and water test had a significant and positive correlation in the fresh
and cryopreserved ejaculates of all groups. The results of the hypoosmotic swelling test correlated positively
with the percent motility in the fresh ejaculates of fertile and subfertile men. None of the membrane integrity
tests correlated with the cryosurvival rate in any group. In the ejaculated and testicular samples with no
postcryopreservation motility, the simultaneous assessment of hypoosmotic swelling test and eosin showed
that of 33% sperm exhibiting coiling with the hypoosmotic swelling test, only 9% were eosin negative,
whereas 24% were eosin positive.
Conclusion(s): [1] The water test may be a simpler replacement for the hypoosmotic swelling test; [2] none
of the membrane integrity tests predicted sperm motility after cryopreservation; and [3] there was a high
degree of disagreement between the hypoosmotic swelling test and eosin in the samples with no postcryo-
preservation motility. (Fertil Sterilt1998;70:1148–55. ©1998 by American Society for Reproductive Med-
icine.)
Key Words: Sperm, cryopreservation, membrane integrity, motility
Semen cryopreservation offers several ad-
vantages over the use of fresh semen in donor
insemination programs, including ease of use,
assurance of the availability over an ex-
tended period of time and possibility to rule
out infection with human immunodeficiency
virus. Furthermore, cryopreservation of semen
can be performed before radiation therapy, che-
motherapy, vasectomy, or other events that may
cause irreversible damage to the male gonads.
It is important that cryopreservation is cur-
rently a standard procedure used to preserve
ejaculated, epididymal, and testicular sperm re-
covered at the time of urologic interventions
coupled with intracytoplasmic sperm injection
(ICSI). However, cryopreservation results in
reduced motility and fertility of human semen
(1, 2). Freezing-thawing results in damage to
the plasma membrane and acrosome of human
spermatozoa as evidenced by significant ultra-
Received May 8, 1998;
revised and accepted July
21, 1998.
Presented in part at the
53rd Annual Meeting of the
American Society for
Reproductive Medicine,
Cincinnati, Ohio, October
18–22, 1997.
Reprint requests:
Mahmood Morshedi, Ph.D.,
The Jones Institute for
Reproductive Medicine,
601 Colley Avenue,
Norfolk, Virginia 23507.
(FAX: 757-446-8998;
E-mail: mahmood@jones1
.evms.edu).
FERTILITY AND STERILITYt
VOL. 70, NO. 6, DECEMBER 1998
Copyright ©1998 American Society for Reproductive Medicine
Published by Elsevier Science Inc.
Printed on acid-free paper in U.S.A.
0015-0282/98/$19.00
PII S0015-0282(98)00351-3
1148
structural changes demonstrated by electron microscopy (3).
Therefore, a simple and reliable test to predict the outcome
of cryopreservation is desirable.
Various tests have been developed to assess the functional
and structural integrity of the sperm plasma membrane. The
World Health Organization (WHO) (4) stated that “sperm
vitality is reflected in the proportion of spermatozoa that are
‘alive’ as determined by either dye exclusion or osmoregu-
latory capacity under hypo-osmotic conditions.” One of
these methods is based on the principle that dead cells with
damaged plasma membranes take up supravital stains such
as eosin-Y, whereas living cells cannot be stained because
their membranes actively exclude the dye (5).
The eosin-Y staining makes it possible to differentiate
spermatozoa that are immotile but alive from those that are
dead. There is agreement that this assay reflects sperm mem-
brane integrity (particularly of the head region because it is
this area that takes up the stain) and is often referred to as a
“vitality” test (6). Jeyendran et al. (6) studied the functional
integrity of sperm membranes with use of a hypoosmotic
solution composed of equal parts of fructose and sodium
citrate. The ability of the sperm tail to swell and/or coil in the
presence of a hypoosmotic solution demonstrates that influx
of water across the membrane occurs normally and gives
information on the integrity and compliance of the tail mem-
brane (7).
According to the WHO (4), the hypoosmotic swelling test
should not be used as a sperm function test but may be used
as a “vitality” test. More recently, the hypoosmotic swelling
test was used to select sperm with a functionally intact
membrane in a population of completely immotile sperm for
ICSI (8). Lomeo and Giambersio (9) developed a simpler
technique, the water test, using distilled water as a hypoos-
motic medium to evaluate the integrity of the sperm mem-
brane. They demonstrated that the water test is as reliable as
hypoosmotic swelling test is for evaluating sperm membrane
integrity. Both methodologies can be adapted for the pro-
cessing of samples with very few spermatozoa.
The success with ICSI also has resulted in a significant
increase in the number of testicular and epididymal aspira-
tion attempts resulting in retrieval of sluggish and immotile
spermatozoa. In many of these instances, spermatozoa also
have been cryopreserved, leading to increased chances of
conception through repeated ICSI attempts. Recent findings
have indicated that some of these spermatozoa may gain
motility on incubation under defined conditions (10). How-
ever, in many instances immotile sperm are the only avail-
able source for ICSI. In situations like this, ICSI has either
been canceled or a vitality test such as hypoosmotic swelling
test has been used to select spermatozoa for injection. Recent
experience has demonstrated that the only sperm factor that
can significantly influence the results of ICSI is the immo-
tility or vitality of the sperm.
The objectives of the present study were as follows: [1] to
examine the relationship between the hypoosmotic swelling
test, water test, and eosin-Y staining (tests that examine
sperm plasma membrane integrity through different ap-
proaches) and sperm motion characteristics before and after
cryopreservation; [2] to assess how the hypoosmotic swell-
ing test, water test, and eosin-Y staining correlate with each
other and to determine their capacity to predict cryopreser-
vation outcome. Ejaculates from fertile and subfertile men
were examined for this purpose; and [3] to examine the
degree of agreement (or lack of) between the results of
hypoosmotic swelling test, representing sperm tail mem-
brane integrity and eosin staining, indicative of the integrity
of the sperm head membrane, in a selected group of ejacu-
lated and testicular samples with no postcryopreservation
sperm motility using a simultaneous hypoosmotic swelling
test-eosin evaluation. This is an important practical issue at
the time of ICSI because often hypoosmotic swelling test is
used to select sperm with tail membrane integrity disregard-
ing the assessment of the head membrane.
MATERIALS AND METHODS
Semen Samples
A total of 71 semen samples were prospectively collected
for the first part of this study after approval of the Institu-
tional Review Board of Eastern Virginia Medical School.
Twenty-seven of these samples were obtained from fertile
donors (group A). Twenty-two samples were collected by
men consulting for infertility with normozoospermia (group
B). Another 22 samples were collected by men consulting
for infertility with asthenozoospermia and/or oligozoosper-
mia (group C) defined following WHO criteria (4).
Semen samples were collected by masturbation in sterile
specimen cups after 3–4 days of sexual abstinence and
allowed to liquefy for 30 minutes at room temperature.
Sperm concentration and motility parameters were analyzed
by computer-assisted semen analysis (CASA) with the Cell-
Soft Semen Analyzer (Cryo Resources Ltd., New York, NY)
following parameter settings described previously (11).
Sperm morphology was assessed by strict criteria (12) fol-
lowing staining with Diff-Quik (Dade Diagnostics, Aguada,
PR). All men had morphology scores with .4% normal
forms (“good prognosis pattern”) (12). All samples evalu-
ated had ,1310
6
leukocytes (peroxidase staining), were
negative for antisperm antibodies (mixed antiglobulin reac-
tion [MAR] test, Fertility Technologies, Natick, MA), and
had negative cultures for Ureaplasma and Mycoplasma spe-
cies.
In the second part of the study, 10 cryopreserved semen
and/or testicular sperm samples with no postcryopreserva-
tion motility also were evaluated (group D). Semen samples
were collected by patients with malignancies before chemo-
therapy in six cases. Testicular sperm were received for
cryopreservation and for future use in ICSI (n 54, cases of
FERTILITY & STERILITYt1149
nonobstructive azoospermia). In all cases studied, motility of
the samples before cryopreservation was ,5%. All evalua-
tions were performed on the portion of the sample set aside
for postthaw evaluation after obtaining patients consent.
Sperm Tests
The hypoosmotic swelling test was performed after esti-
mation of standard semen parameters, and the percentage of
sperm with coiled tails was recorded. An aliquot of 0.1 mL
of liquefied semen was added to 1 mL of hypoosmotic
solution. This solution was prepared by dissolving 7.35 g of
sodium citrate and 13.5 g of fructose in 1 kg of deionized
distilled water (osmolality of 150 mOsm/kg), filter sterilized,
and frozen in 1-mL aliquots for use as required. After incu-
bation for 30 minutes at 37°C in 5% CO
2
, the coiling of the
sperm tail representing an intact flagellar membrane was
evaluated according to the guidelines reported by Jeyendran
et al. (8).
The water test was performed by mixing 10
m
L of semen
and 40
m
L of distilled water on a microscope slide and
covered with a thin 24 330-mm coverslip. The mixture was
incubated for 5 minutes in a moist chamber at 37°C before it
was examined with a microscope at 3400 magnification. At
least 100 sperm were examined, and the percentage of sperm
that showed coiled tails was calculated for both the hypoos-
motic swelling test and water test (6, 9).
The eosin-Y staining (0.5% wt/vol) was performed by
mixing 15
m
L of semen with 5
m
L of the stain on a micro-
scope slide; immotile spermatozoa that were stained (posi-
tive) and unstained (negative) within 1–2 minutes after ad-
dition of the stain (wet prep) were assessed (4). Only the
immotile fraction was evaluated (because all motile sperma-
tozoa are vital and, consequently, exclude the dye), and the
results were expressed as the percentage of unstained or
negative sperm (therefore having an intact head membrane).
Eosin-Y staining, hypoosmotic swelling test, and water test
were performed on each fresh semen sample after liquefac-
tion in men from groups A, B, and C.
Thawed samples from patients from group D were eval-
uated for the degree of spontaneous coiling and the eosin
staining as described above. Samples also were evaluated
with a combined hypoosmotic swelling test-eosin method.
Postthaw samples were incubated with the hypoosmotic
swelling test medium and mixed with the eosin stain after
incubation for the combined evaluation of hypoosmotic
swelling test and eosin results. The standard hypoosmotic
swelling test (as described above) or a micromethod used by
Liu et al. (13) were used to assess tail coiling in these thawed
samples. At least 100 spermatozoa or as many as could be
found were evaluated.
Because the water test showed an excellent correlation
with hypoosmotic swelling test (see Results), no combined
water test-eosin staining was attempted in these latter cases.
For the three sperm membrane integrity tests, results were
analyzed blindly in coded slides. For each slide, all sperma-
tozoa identified from five randomly selected fields were
assessed in a systematic fashion.
Sperm Cryopreservation-Thawing
To minimize the effect of variable sperm concentrations
on sperm viability postcryopreservation (14), all samples
were washed before cryopreservation. Each liquefied semen
sample was mixed with a volume of Ham’s F-10 medium
(Fertility Technologies, Natick, MA) supplemented with
0.5% human serum albumin (HSA; Irvine Scientific, Santa
Ana, CA) equal to 3 times the volume of semen. The mixture
then was aliquoted into a minimum of two 15-mL conical
centrifuge tubes (VWR Scientific, Bridgeport, NJ) and cen-
trifuged at 380 3gfor 10 minutes. The supernates were
discarded, and the pellets were resuspended in 0.5 mL of the
same medium.
For the samples of men from groups A, B, and C, an
analysis of sperm concentration was performed, and the
sperm concentration was adjusted to approximately 50 3
10
6
/mL. The cryopreservation procedure involved gradual
dilution of the washed semen with a volume of freezing
medium (Irvine Scientific) equal to one half of the washed
semen volume. The freezing medium contained TES and
tris(hydroxymethyl)aminomethane (Tris) buffers, sodium ci-
trate, fructose, 12% (vol/vol) glycerol, and 20% (vol/vol)
heat-inactivated egg yolk. The cryovials (0.4 mL/vial) were
then suspended 5 cm above the liquid nitrogen for vapor
freezing for 30 minutes before loading on precooled canes
and plunging them directly into liquid nitrogen.
The thawing procedure was performed after $1 day cry-
ostorage. Each sample was thawed rapidly in a 42°C water
bath for 3 minutes and allowed to equilibrate to room tem-
perature 15 minutes (quick freeze-quick thaw). In previous
studies (15, 16) we showed that this rate of thawing matches
more closely the freezing rate used and results in optimal
recovery of motile sperm for artificial insemination and
excellent fertilization rates under IVF conditions. A dupli-
cate standard semen analysis then was performed on the
postthaw sample as described above. Eosin-Y staining, hy-
poosmotic swelling test, and water test also were performed
in the thawed samples.
Coiling of the sperm tail that is usually observed during
the freezing-thawing process in a small proportion of sper-
matozoa was assessed in each specimen by scoring the
percentage of coiled sperm in the absence of hypoosmotic
solution. This value was then subtracted from the total hy-
poosmotic swelling test scores. The sperm cryosurvival rate
was calculated by dividing percent postthaw motility by
percent fresh motility 3100.
To cryopreserve poor-quality samples (i.e., testicular
sperm or ejaculated sperm with very low or sluggish motil-
ity), each sample was mixed with Ham’s F-10 medium
containing 0.5% HSA, divided into small volumes and cen-
1150 Lin et al. Membrane integrity of cryopreserved sperm Vol. 70, No. 6, December 1998
trifuged at 290 3gfor 5 minutes. Supernates were sepa-
rated and examined for the presence of sperm. Pellets were
resuspended into a small volume (typically 100
m
L) of
Ham’s F-10 medium with 0.5% HSA. The cryopreservation
involved gradual dilution of the washed sample with a vol-
ume of freezing medium (Irvine Scientific) equal to one half
of its volume and aliquoting of the mixture into several
1.2-mL cryovials in small volumes. Samples were then cryo-
preserved in liquid nitrogen vapor as described above.
Samples containing red blood cells and/or tissue remnants
were centrifuged at 190 3gfor 1 minute. The supernatant
was removed and subjected to further washing and cryo-
preservation as described above. The pellet was resuspended
in a small volume of Ham’s F-10 medium with 0.5% HSA,
mixed with the freezing medium, and cryopreserved in sep-
arate vials labeled accordingly. Our experience with this type
of samples indicates an excellent sperm survival in samples
with a motility of .5%.
Statistical Analysis
Statistical significance between the mean values of the
different tests obtained before and after freezing was as-
sessed with use of paired Student’s t-test. Correlations be-
tween the sperm membrane integrity tests and basal semen
parameters, between fresh and postthaw semen samples, and
between fertile donors and subfertile patients were assessed
with use of Pearson’s correlation coefficient. A Pvalue of
,.05 was considered statistically significant. Results are
presented as means 6SEM.
RESULTS
The semen analysis data and the results of three sperm
membrane integrity (or vitality) tests obtained from the fresh
and post-thaw samples of groups A, B, and C are shown in
Table 1. The percentage of swollen-coiled sperm (intact tail
membrane) obtained in hypoosmotic swelling test and water
test were not statistically significant between the fresh sam-
ples of groups A, B, and C. The percentage of immotile
sperm not stained with eosin-Y (intact head membrane) also
was not statistically significant between fresh samples of
group A and B. However, a statistically significant differ-
ence was found between groups A and C (P,.02) and
between groups B and C (P,.05).
The fresh samples of groups A and B showed no statis-
tically significant differences in percent motility, motile con-
centration, velocity, linearity, and motility index (percent
motility 3mean curvilinear velocity), whereas group C
showed significantly poorer motility characteristics than
those of groups A and B. There was a significant and positive
correlation between hypoosmotic swelling test and water test
and between hypoosmotic swelling test and percent motility
in the fresh samples of all three groups (Table 2).
As expected, cryopreservation resulted in a significant
reduction in sperm motion parameters and in the results of
the sperm membrane integrity tests. Patients from groups B
and C showed a significantly lower percentage of postthaw
motile sperm than those of group A, with group C showing
TABLE 1
Sperm motility characteristics and test results of the fresh and postthaw samples in the fertile donors (group A), patients
with normal semen (group B), and patients with abnormal semen (group C).
Sperm characteristic
Group A (n 527) Group B (n 522) Group C (n 522)
Fresh
sample Postthaw
sample Fresh
sample Postthaw
sample Fresh
sample Postthaw
sample
WT (percentage coiled) 71.3 62.6 40.7 62.5* 73.6 63.0 39.9 63.5* 67.6 61.8 21.3 61.9*†‡
HOS (percentage coiled) 64.3 63.0 35.6 62.3* 65.5 64.2 29.1 63.2* 58.0 62.9 19.0 62.5*†‡
Eosin-Y stain
(percentage unstained) 63.5 62.7 34.9 62.0* 61.3 62.6 30.4 62.0* 53.1 62.9§\19.5 61.7*†‡
Percent motility 76.5 62.8 47.6 63.2* 70.3 63.2 26.8 63.6*† 40.4 64.0§\4.4 60.8*†‡
Motile concentration
(310
6
/mL) 71.6 69.3 35.0 63.3* 89.3 69.5 30.3 66.7* 8.8 61.5§\1.6 60.4*†‡
Motility index 32.0 61.8 15.9 61.2* 28.4 62.2 10.3 62.0* 14.5 61.6§\1.5 60.3*
Linear velocity (
m
m/s) 40.4 61.4 33.3 61.2* 38.6 61.8 37.3 63.1 35.4 61.3§ 31.0 62.4
Linearity 5.7 60.2 4.9 60.3* 5.4 60.3 5.6 60.3 5.7 60.3 4.9 60.4
Cryosurvival rate (%) 61.7 65.1 39.7 65.5¶ 10.9 61.6¶
Note: Only nonmotile sperm were evaluated for eosin staining. Values are means 6SEM. HOS 5hypoosmotic swelling test; WT 5water test.
*P,.05 (vs. the fresh semen samples);
†P,.05 (vs. the postthaw semen samples of group A).
‡P,.05 (vs. the postthaw semen samples of group B).
§P,.05 (vs. the fresh semen samples of group A).
\P,.05 (vs. the fresh semen samples of group B);
¶P,.01 (vs. frozen semen samples of group A).
** P,.01 (vs. the frozen semen samples of group B).
FERTILITY & STERILITYt1151
the poorest results. The postthaw results of the three sperm
membrane integrity tests were statistically significant be-
tween groups A and C, and between groups B and C, but not
between groups A and B.
There was no significant correlation between hypoos-
motic swelling test and percent motility, between water test
and percent motility, and between eosin-Y staining and
percent motility in the postthaw samples of all three groups.
However, hypoosmotic swelling test was positively corre-
lated with water test in postthaw samples of all groups (Table
2). The cryosurvival rate of group B was significantly lower
than that of group A (P,.01) but significantly higher than
that of group C (P,.001, Table 1). None of the three sperm
membrane integrity tests in fresh samples of donors and
patients (groups A, B, and C) correlated with the cryosur-
vival rate.
The relationships between sperm head membrane integ-
rity as assessed by the eosin-Y staining and the response in
the hypoosmotic swelling test and water test (indicative of
sperm tail membrane integrity) are shown in Table 3. The
hypoosmotic swelling test was positively correlated with
eosin-Y staining in the fresh samples of groups A (P,.02)
andB(P,.03). However, no correlation was found be-
tween hypoosmotic swelling test and eosin-Y stain in the
fresh samples of group C and in the postthaw samples of all
three groups. A poor correlation (P5.058) was observed
between water test and eosin-Y stain in fresh and postthaw
samples of all three groups.
Table 4 presents data obtained from evaluation of ejacu-
lated and testicular samples with no motility postcryopreser-
vation (group D). With hypoosmotic swelling test, 33.2% of
all spermatozoa exhibited positive coiling, whereas 66.8%
were coiled negative. Of the 33.2% coiled spermatozoa
(intact tail membranes), 9.2% were eosin negative (intact
head membranes) and 24% were eosin positive (abnormal
head membranes); of the 66.6% uncoiled spermatozoa, 6.6%
were eosin negative and 60% were eosin positive.
On the basis of the combined hypoosmotic swelling test-
eosin test, only 9.2% of sperm were identified as having both
tail and head intact membranes. However, there was a high
degree of variation among samples with this respect. The
results of hypoosmotic swelling test and eosin, therefore,
correlated one way or another in only 69.2% of spermatozoa.
Comparison of the eosin results (nonmotile spermatozoa)
before (72%) and after cryopreservation (82%) indicated no
drastic increase in eosin staining of spermatozoa (membrane
damage) after cryopreservation.
DISCUSSION
The WHO recommended using the hypoosmotic swelling
test as a vitality test (i.e., to gain information on the integrity
and compliance of the tail membrane) and not as a sperm
function test (4). A simpler technique, the water test, was
TABLE 2
Correlations between the hypoosmotic swelling test (HOS) and water test (WT), HOS and percent motility of fresh semen
samples, and between HOS and WT of cryopreserved-thawed semen samples in the fertile donors (group A), patients with
normal semen (group B), and patients with abnormal semen (group C).
Comparison
Group A (n 527) Group B (n 522) Group C (n 522)
rP
value rP
value rP
value
HOS vs. WT (fresh) .6038 .0009 .7155 .0004 .7196 .0002
HOS vs. percent motility (fresh) .5525 .0028 .5263 .0171 .4492 .0360
HOS vs. WT (cryopreserved/thawed) .4464 .0196 .7138 .0002 .6687 .0009
TABLE 3
Correlations between viability detected by eosin-Y stain
and results of hypoosmotic swelling test and water test in
the fresh and postthaw samples of donors (group A),
patients with normal semen (group B), and patients with
abnormal semen (group C).
Patient group,
sample
Correlation between viability detected by eosin-Y
stain and results of indicated test
Hypoosmotic
swelling test Water test
rP
value rP
value
Group A
Fresh .3413 .0152 2.0233 NS
Postthaw .0490 NS .1507 NS
Group B
Fresh .4849 .0266 .4177 NS
Postthaw .3339 NS .2392 NS
Group C
Fresh 2.0685 NS 2.0933 NS
Postthaw 2.1511 NS 2.1055 NS
Note: NS 5not significant.
1152 Lin et al. Membrane integrity of cryopreserved sperm Vol. 70, No. 6, December 1998
developed to use distilled water as a hypoosmotic medium to
evaluate the integrity of the tail sperm membrane (9). Be-
cause the tail membrane is more loosely attached to the
underlying structures than in the sperm head, the sperm tails
seem to be particularly sensitive to such swelling. Sperma-
tozoa with nonfunctioning membranes will not swell (6).
Some investigators demonstrated that the sperm coiling
scores obtained by the water test correlated well with those
obtained by the hypoosmotic swelling test (17). Our results
confirm these findings and show a good correlation between
the water test and hypoosmotic swelling test not only in fresh
semen samples but also in postthawed semen samples. These
findings suggest that the water test may be an adequate
replacement to the hypoosmotic swelling test.
Cell damage during freezing usually is ascribed to mem-
brane rupture caused by the formation of intracellular ice
crystals during rapid cooling, or by osmotic effects, or me-
chanical force from extracellular ice formation during slow
cooling. However, it is unclear to what extent membrane
damage is responsible for the deleterious defects of cryo-
preservation on motility.
Our study showed that sperm from subfertile men exhib-
ited significantly lower postthaw percent motility, motile
sperm concentration, and cryosurvival rate than those of
fertile donors. These results suggest that the sperm of sub-
fertile patients (even with comparable quality in the cases
with normozoospermia or with moderate oligoasthenozo-
ospermia) are more intolerant to the cryopreservation-thaw-
ing process than those of fertile donors. Whether a compo-
nent(s) of seminal fluid or other factor(s) related to the sperm
contributed to these differences remains to be elucidated.
The present study demonstrated that the hypoosmotic
swelling test was significantly correlated with percent mo-
tility in the fresh samples of all three groups but not in
postthaw samples. It is possible that loss of sperm tail
membrane integrity may be a major factor contributing to the
decline of sperm motility. After cryopreservation, however,
several factors, and not only tail membrane damage, may be
responsible for the loss of progressively motile sperm. There
was no correlation between eosin-Y staining and percent
motility in semen samples of the three groups (A, B, and C)
before and after cryopreservation; this suggests that disrup-
tion of head membrane integrity may not be associated
necessarily with the loss of sperm motility. However, when
an immotile spermatozoon is being used for procedures such
as ICSI, integrity of the sperm head membranes may be an
important factor to consider.
The fresh samples of donors and patients with normozo-
ospermia showed no statistically significant differences in
motility parameters and in the results of the three membrane
tests (hypoosmotic swelling test, water test, and eosin Y
staining), whereas patients with abnormal semen parameters
showed significantly poorer motility parameters and results
of eosin-Y staining. This fact suggests that the fresh samples
in donor and patients with normozoospermia may have sim-
ilar conditions of sperm head and tail membranes, whereas
patient samples with abnormal semen parameters have not
only a higher incidence of head membrane defects in the
fresh semen samples but also a higher incidence of intoler-
ance to the freezing-thawing process.
None of the three sperm membrane integrity tests (or
vitality tests) in fresh semen of donors or patients could be
used to predict the motility changes after cryopreservation-
thawing. The present data are in agreement with the results
TABLE 4
Evaluation of samples with no postcryopreservation motility and the relationship between hypoosmotic swelling test and
eosin-Y stain in these samples (group D).
Sample type
Motility (%)
(before
cryopreservation) Eosin 1(before
cryopreservation) Eosin 1(after
cryopreservation)
Coiled and
eosin 1
(combined)
Coiled and
eosin 2
(combined)
Not coiled
and eosin 1
(combined)
Not coiled
and eosin 2
(combined)
Semen* 2 79% 88% 25% 6% 69% 0%
Testicular† 1 74% 79% 8% 10% 76% 6%
Testicular 3 70% 81% 11% 4% 70% 15%
Semen 3 68% 78% 39% 24.5% 35.5% 1%
Semen 4 80% 88% 61.5% 13% 25.5% 0%
Semen 1 72% 85% 29% 7% 64% 0%
Testicular 1 83% 89% 13% 7.4% 76% 3.6%
Testicular 4 71% 79% 14% 8.2% 70% 7.8%
Semen 3 69% 76% 16.5% 8% 62% 13.5%
Semen 5 58% 78% 23% 4% 54% 19%
Mean 6SEM 2.7 60.4 72 62.3 82 61.6 24 65.1 9.2 61.9 60 65.4 6.6 62.2
Note: Combined method 5HOS-eosin.
* Patients with malignancies receiving chemotherapy.
† Patients with nonobstructive azoospermia.
FERTILITY & STERILITYt1153
of the hypoosmotic swelling test in previous studies (18, 19).
The lack of predictive power for the cryosurvival rate of
sperm by the hypoosmotic swelling test may be due to the
fact that there are many interacting factors other than the
membrane integrity of human sperm that can influence the
cryosurvival rate. These include among others, the variation
in individual semen quality, the method of cryopreservation,
the thawing procedure, and the cryoprotectants used
(1, 2, 20, 21).
In certain situations, sperm may have an intact membrane
but be immotile, and in these cases supravital stains can
differentiate immotile but structurally intact sperm (un-
stained) from those that are immotile and dead (stained) (21).
As sperm die, they lose their ability to resist the influx of
the membrane-impermeant dye. However, it is unknown
whether using the sperm corrupted by supravital stain will
lead to alterations of DNA integrity, gamete interaction,
embryogenesis, and negatively affect the outcome of assisted
reproductive technologies (21).
The hypoosmotic swelling test has been used clinically as
a tool in identifying immotile sperm with an intact mem-
brane in fresh and cryopreserved sperm for ICSI (22). In our
study, as well as in others (21), the hypoosmotic swelling
test could correctly assess the integrity of the sperm tail
membrane in fresh samples of fertile and subfertile men as
demonstrated by the positive and significant correlation with
the percent progressive motility (Table 2). However, this
was not the case for the postthaw samples of groups A, B,
and C. It is possible that the determining factor(s) in sperm
survival after cryopreservation is(are) related to the sperm
head membrane and/or other cytoplasmic-metabolic factors,
whereas the hypoosmotic swelling test mainly reflects tail
membrane intactness and perhaps maintenance of motility.
In this study we also evaluated a limited number of
ejaculated and testicular samples with no postcryopreserva-
tion sperm motility to determine the degree of agreement (or
lack of) among the various tests currently used to determine
sperm membrane integrity (or vitality). The findings of this
study could have repercussions for ICSI (Table 4). Our
results showed that if the hypoosmotic swelling test was to
be used for the selection of cryopreserved-thawed immotile
sperm, there would be only a 9% chance of selecting a coiled
sperm (intact tail membrane) with an intact sperm head
membrane (unstained) as determined by eosin. Conversely,
the chance of selecting a coiled sperm (with an intact tail
membrane) that was stained by eosin (indicating a damaged
head membrane) would be 24%. Approximately 7% of
sperm with an intact head membrane would be missed
through hypoosmotic swelling test selection because they
did not coil.
The results presented in Table 4 also revealed two more
phenomena. First, the percentage of spermatozoa identified
as having an intact membrane are vastly different when
hypoosmotic swelling test or eosin are used. Approximately
33% of immotile sperm were identified as having an intact
membrane with hypoosmotic swelling test, but the percent-
age identified with eosin was only 16.6%. Under the condi-
tions used and the type of samples evaluated, 66.6% of
spermatozoa were identified as having membrane defects
under hypoosmotic swelling test conditions. The defect rate
identified by eosin was 84%.
Another finding was the vast differences observed among
samples for the results obtained for hypoosmotic swelling
test and eosin. Consequently, it is very important to inves-
tigate if the fraction of spermatozoa exhibiting the coiling
phenomenon but that are stained with eosin is less suitable
for ICSI than the fraction with no coiling but with intact head
membranes as determined by eosin. In preliminary studies
we also used a combination fluorescent stains, which are
nucleic acid specific to assess the sperm head membrane
integrity (23). This staining method also gives us the capa-
bility of assessing the proportion of moribund sperm in each
sample which may exhibit the coiling phenomenon under
hypoosmotic swelling test conditions. Fluorophores, SYBR-14
and propidium iodide, marketed under the sperm viability kit
designation, has been made suitable for fluorescent micros-
copy.
Our preliminary studies have shown that between 15%
and 20% of fresh spermatozoa coiled under hypoosmotic
swelling test conditions show sperm head leakage and DNA
staining indicative of structural damage. Our comparison
studies also are indicating that there is a significant degree of
disagreement between eosin and SYBR-14 and propidium
and iodide, two tests to assess the integrity of sperm head
membrane.
Recently, much attention has been given to the status of
sperm plasma membrane and the nuclear DNA changes as
they relate to the apoptosis phenomenon. The impact of
various factors on the integrity of plasma membrane and
sperm DNA also has generated interest (24). There have
been reports that spermatozoa with normal morphology may
have severe nuclear damage, rendering them incapable of
supporting normal embryo development (24). Parallel and
simultaneous studies to assess the apoptosis-associated
plasma membrane lipid changes (asymmetry) in the form of
translocation of phosphatidyl serine from the inner side to
the outer layer of membrane and evaluation of the integrity
of sperm nuclear and mitochondrial DNA by in situ cell
death detection may provide further information for select-
ing viable immotile sperm for ICSI.
Recently, a simple test of measuring sperm tail:head ratio
was used to assess its relationship to the viability of nonmo-
tile fresh and cryopreserved testicular sperm as measured by
SYBR-14 and PI (25). It was found that 82.3% of viable
sperm had the tail:head ratio of .10 compared to 24.6% of
nonviable sperm exhibiting such ratio. Although this method
may not be suitable for the evaluation of spontaneously
1154 Lin et al. Membrane integrity of cryopreserved sperm Vol. 70, No. 6, December 1998
coiled spermatozoa, it may be useful to study induced coiling
(hypoosmotic swelling test) and its relationship to eosin,
apoptotic-related changes of sperm and ICSI outcome.
In conclusion, sperm from subfertile men with normal
semen parameters exhibited a poorer cryopreservation out-
come than those of fertile donors. The subfertile men with
abnormal semen parameters had the worst sperm outcome
after the freezing-thawing process. In the absence of more
accurate tests to assess sperm membrane integrity, the water
test may be an adequate replacement to the hypoosmotic
swelling test in the evaluation of sperm tail membrane in-
tactness. Because deionized water has no extra components,
it may be more suitable to select sperm at the time of ICSI.
In addition, the water test may be more amenable to
processing of single sperm or microprocessing of samples
with a few sperm. The hypoosmotic swelling test, water test,
and eosin-Y staining, vitality tests that reflect sperm plasma
membrane integrity, cannot be used to predict the motility
changes or outcome after cryopreservation of sperm in fertile
donors or subfertile patients with normal or abnormal sperm
parameters. The value of the hypoosmotic swelling test for
selecting intact cryopreserved sperm from a sample with
completely immotile sperm for micromanipulation proce-
dures, such as ICSI, may be revisited.
References
1. Keel BA, Webster BW, Roberts DK. Effect of cryopreservation on the
motility characteristics of human spermatozoa. J Reprod Fertil 1987;
81:213–20.
2. Critser JK, Huse-Benda AR, Aaker DV, Arneson BW, Ball GD. Cryo-
preservation of human spermatozoa: the effect of cryoprotectants on
motility. Fertil Steril 1988;50:314–20.
3. Woolley DM, Richardson DW. Ultrastructural injury to human sper-
matozoa after freezing and thawing. J Reprod Fertil 1978;53:389–94.
4. World Health Organization. Laboratory manual for the examination of
human semen and sperm-cervical mucus Interaction. 3rd ed. New York:
Cambridge University Press, 1993:11–13.
5. Eliasson R, Treichl L. Supravital staining of human spermatozoa. Fertil
Steril 1971;22:134–7.
6. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Crabo BG, Zaneveld
JD. Development of an assay to assess the functional integrity of the
human sperm membrane and its relationship to other semen character-
istics. J Reprod Fertil 1984;70:219–28.
7. Drevius L, Eriksson H. Osmotic swelling of mammalian spermatozoa.
Exp Cell Res 1966;42:136–56.
8. Casper RF, Cowan L, Meriano JS, Lucato ML, Jarvi KA. The hypo-
osmotic swelling test for the selection of viable sperm for intracyto-
plasmic sperm injection in men with complete asthenozoospermia.
Fertil Steril 1996;65:972–6.
9. Lomeo AM, Giambersio AM. Water-test: a simple method to assess
sperm-membrane integrity. Int J Androl 1991;14:278–82.
10. Edirisinghe WR, Junk SM, Matson PL, Yovich JL. Changes in motility
patterns during in vitro culture of fresh and frozen/thawed testicular and
epididymal spermatozoa: implications for planning treatment by intra-
cytoplasmic sperm injection. Hum Reprod 1996;11:2474–6.
11. Oehninger S, Acosta R, Morshedi M, Philput C, Swanson RJ, Acosta
AA. Relationship between morphology and motion characteristics of
human spermatozoa in semen and in the swim-up fractions. J Androl
1990;11:446–52.
12. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Veeck
LL, et al. New method of evaluating sperm morphology with predictive
value for human in vitro fertilization. Urology 1987;XXX:248–51.
13. Liu J, Tsai YL, Katz E, Compton G, Garcia JE, Baramki TA. High
fertilization rate obtained after intracytoplasmic sperm injection with
100% nonmotile spermatozoa selected by using simple modified hypo-
osmotic swelling test. Fertil Steril 1997;68:273–5.
14. Graham JK. Effect of seminal plasma on the motility of epididymal and
ejaculated spermatozoa of the ram and bull during the cryopreservation
process. Theriogenology 1994;41:1151–62.
15. Morshedi M, Oehninger S, Veeck LL, Ertunc H, Bocca S, Acosta AA.
Cryopreserved/thawed semen for in vitro fertilization: results from
fertile donors and infertile patients. Fertil Steril 1990;54:1093–9.
16. Morshedi M, Oehninger S, Blackmore P, Bocca S, Coddington G,
Hodgen G. Investigation of some biochemical and functional effects of
cryopreservation of human spermatozoa using an automated freezing-
quick thawing method. Int J Androl 1995;18:279–86.
17. Fuse H, Ohta S, Sakamoto M, Kazama T, Katayama T. Hypo-osmotic
swelling test with a medium of distilled water. Arch Androl 1993;30:
111–6.
18. Chan SYW, Craft IL, Chan YM, Leong MKH, Leung CKM. The
hypo-osmotic swelling test and cryosurvival of human spermatozoa.
Hum Reprod 1990;5:715–8.
19. Chan SYW, Pearlstone A, Uhler M, Tucker M, Greenspoon R, Leung
A, et al. Human spermatozoal tail hypo-osmotic swelling test, motility
characteristics in hypotonic saline, and survival of spermatozoa after
cryopreservation. Hum Reprod 1993;8:717–21.
20. Hammitt DG, Hade DK, Williamson RA. Survival of human sperm
following controlled- and non-controlled-rate cryopreservation. Andro-
logia 1988;21:311–7.
21. Centola GM, Raubertas RF, Mattox JH. Cryopreservation of human
semen: comparison of cryopreservatives, sources of variability, and
prediction of post-thaw survival. J Androl 1992;12:283–8.
22. Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A. Suitability of the
hypo-osmotic swelling test for assessing the viability of cryopreserved
sperm. Fertil Steril 1996;66:798–804.
23. Garner DL, Johnson LA. Viability assessment of mammalian sperm
using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276–84.
24. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyri-
bonucleic acid fragmentation is increased in poor quality semen sam-
ples and correlates with failed fertilization in intracytoplasmic sperm
injection. Fertil Steril 1998;69:528–32.
25. Marmar JL, Corson SL, Gibbs M, Huszar G. Tail to head ratio (TH) of
fresh and testicular sperm as a measure of viability. J Androl (Suppl):
Jan/Feb 1998;58.
FERTILITY & STERILITYt1155
