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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
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.
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
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). 0015-0282/98/$19.00 PII S0015-0282(98)00351-3
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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 postcryopreservation motility. (Fertil Sterilt 1998;70:1148 –55. ©1998 by American Society for Reproductive Medicine.) Key Words: Sperm, cryopreservation, membrane integrity, motility
Semen cryopreservation offers several advantages over the use of fresh semen in donor insemination programs, including ease of use, assurance of the availability over an extended period of time and possibility to rule out infection with human immunodeficiency virus. Furthermore, cryopreservation of semen can be performed before radiation therapy, chemotherapy, vasectomy, or other events that may cause irreversible damage to the male gonads.
It is important that cryopreservation is currently a standard procedure used to preserve ejaculated, epididymal, and testicular sperm recovered 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-
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 osmoregulatory 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 membrane 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 membrane (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 hypoosmotic medium to evaluate the integrity of the sperm membrane. 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 processing of samples with very few spermatozoa. The success with ICSI also has resulted in a significant increase in the number of testicular and epididymal aspiration 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). However, in many instances immotile sperm are the only available 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 immotility or vitality of the sperm. The objectives of the present study were as follows: [1] to FERTILITY & STERILITYt
examine the relationship between the hypoosmotic swelling test, water test, and eosin-Y staining (tests that examine sperm plasma membrane integrity through different approaches) and sperm motion characteristics before and after cryopreservation; [2] to assess how the hypoosmotic swelling test, water test, and eosin-Y staining correlate with each other and to determine their capacity to predict cryopreservation 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 membrane integrity and eosin staining, indicative of the integrity of the sperm head membrane, in a selected group of ejaculated 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 disregarding 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 Institutional 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 oligozoospermia (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 CellSoft Semen Analyzer (Cryo Resources Ltd., New York, NY) following parameter settings described previously (11). Sperm morphology was assessed by strict criteria (12) following staining with Diff-Quik (Dade Diagnostics, Aguada, PR). All men had morphology scores with .4% normal forms (“good prognosis pattern”) (12). All samples evaluated had ,1 3 106 leukocytes (peroxidase staining), were negative for antisperm antibodies (mixed antiglobulin reaction [MAR] test, Fertility Technologies, Natick, MA), and had negative cultures for Ureaplasma and Mycoplasma species. In the second part of the study, 10 cryopreserved semen and/or testicular sperm samples with no postcryopreservation motility also were evaluated (group D). Semen samples were collected by patients with malignancies before chemotherapy in six cases. Testicular sperm were received for cryopreservation and for future use in ICSI (n 5 4, cases of 1149
nonobstructive azoospermia). In all cases studied, motility of the samples before cryopreservation was ,5%. All evaluations were performed on the portion of the sample set aside for postthaw evaluation after obtaining patients consent.
analyzed blindly in coded slides. For each slide, all spermatozoa identified from five randomly selected fields were assessed in a systematic fashion.
Sperm Tests
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 centrifuged at 380 3 g for 10 minutes. The supernates were discarded, and the pellets were resuspended in 0.5 mL of the same medium.
The hypoosmotic swelling test was performed after estimation 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 incubation for 30 minutes at 37°C in 5% CO2, 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 mL of semen and 40 mL of distilled water on a microscope slide and covered with a thin 24 3 30-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 hypoosmotic swelling test and water test (6, 9). The eosin-Y staining (0.5% wt/vol) was performed by mixing 15 mL of semen with 5 mL of the stain on a microscope slide; immotile spermatozoa that were stained (positive) and unstained (negative) within 1–2 minutes after addition of the stain (wet prep) were assessed (4). Only the immotile fraction was evaluated (because all motile spermatozoa 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 liquefaction in men from groups A, B, and C. Thawed samples from patients from group D were evaluated 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 1150 Lin et al.
Membrane integrity of cryopreserved sperm
Sperm Cryopreservation-Thawing
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 3 106/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 citrate, 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 cryostorage. Each sample was thawed rapidly in a 42°C water bath for 3 minutes and allowed to equilibrate to room temperature 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 duplicate standard semen analysis then was performed on the postthaw sample as described above. Eosin-Y staining, hypoosmotic 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 spermatozoa 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 hypoosmotic swelling test scores. The sperm cryosurvival rate was calculated by dividing percent postthaw motility by percent fresh motility 3 100. To cryopreserve poor-quality samples (i.e., testicular sperm or ejaculated sperm with very low or sluggish motility), each sample was mixed with Ham’s F-10 medium containing 0.5% HSA, divided into small volumes and cenVol. 70, No. 6, December 1998
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). Group A (n 5 27)
Sperm characteristic WT (percentage coiled) HOS (percentage coiled) Eosin-Y stain (percentage unstained) Percent motility Motile concentration (3 106/mL) Motility index Linear velocity (mm/s) Linearity Cryosurvival rate (%)
Group B (n 5 22)
Fresh sample
Postthaw sample
Fresh sample
71.3 6 2.6 64.3 6 3.0
40.7 6 2.5* 35.6 6 2.3*
63.5 6 2.7 76.5 6 2.8 71.6 6 9.3 32.0 6 1.8 40.4 6 1.4 5.7 6 0.2
Group C (n 5 22)
Postthaw sample
Fresh sample
Postthaw sample
73.6 6 3.0 65.5 6 4.2
39.9 6 3.5* 29.1 6 3.2*
67.6 6 1.8 58.0 6 2.9
21.3 6 1.9*†‡ 19.0 6 2.5*†‡
34.9 6 2.0* 47.6 6 3.2*
61.3 6 2.6 70.3 6 3.2
30.4 6 2.0* 26.8 6 3.6*†
53.1 6 2.9§\ 40.4 6 4.0§\
19.5 6 1.7*†‡ 4.4 6 0.8*†‡
35.0 6 3.3* 15.9 6 1.2* 33.3 6 1.2* 4.9 6 0.3* 61.7 6 5.1
89.3 6 9.5 28.4 6 2.2 38.6 6 1.8 5.4 6 0.3
30.3 6 6.7* 10.3 6 2.0* 37.3 6 3.1 5.6 6 0.3 39.7 6 5.5¶
8.8 6 1.5§\ 14.5 6 1.6§\ 35.4 6 1.3§ 5.7 6 0.3
1.6 6 0.4*†‡ 1.5 6 0.3* 31.0 6 2.4 4.9 6 0.4 10.9 6 1.6¶
Note: Only nonmotile sperm were evaluated for eosin staining. Values are means 6 SEM. HOS 5 hypoosmotic swelling test; WT 5 water 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).
trifuged at 290 3 g for 5 minutes. Supernates were separated and examined for the presence of sperm. Pellets were resuspended into a small volume (typically 100 mL) of Ham’s F-10 medium with 0.5% HSA. The cryopreservation involved gradual dilution of the washed sample with a volume 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 cryopreserved in liquid nitrogen vapor as described above. Samples containing red blood cells and/or tissue remnants were centrifuged at 190 3 g for 1 minute. The supernatant was removed and subjected to further washing and cryopreservation 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 separate 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 assessed with use of paired Student’s t-test. Correlations between 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 P value of ,.05 was considered statistically significant. Results are presented as means 6 SEM. FERTILITY & STERILITYt
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 samples 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 difference 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 statistically significant differences in percent motility, motile concentration, velocity, linearity, and motility index (percent motility 3 mean 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 1151
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). Group A (n 5 27)
Comparison HOS vs. WT (fresh) HOS vs. percent motility (fresh) HOS vs. WT (cryopreserved/thawed)
Group C (n 5 22)
r
P value
r
P value
r
P value
.6038 .5525 .4464
.0009 .0028 .0196
.7155 .5263 .7138
.0004 .0171 .0002
.7196 .4492 .6687
.0002 .0360 .0009
the poorest results. The postthaw results of the three sperm membrane integrity tests were statistically significant between groups A and C, and between groups B and C, but not between groups A and B. There was no significant correlation between hypoosmotic 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 correlated 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 cryosurvival rate. The relationships between sperm head membrane integrity 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) and B (P,.03). However, no correlation was found between 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 (P 5 .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 ejaculated and testicular samples with no motility postcryopreservation (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 testeosin test, only 9.2% of sperm were identified as having both 1152 Lin et al.
Group B (n 5 22)
Membrane integrity of cryopreserved sperm
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 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). Correlation between viability detected by eosin-Y stain and results of indicated test Hypoosmotic swelling test Patient group, sample Group A Fresh Postthaw Group B Fresh Postthaw Group C Fresh Postthaw
Water test
r
P value
.3413 .0490
.0152 NS
2.0233 .1507
NS NS
.4849 .3339
.0266 NS
.4177 .2392
NS NS
2.0933 2.1055
NS NS
2.0685 2.1511
NS NS
r
P value
Note: NS 5 not significant.
Vol. 70, No. 6, December 1998
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 Semen* Testicular† Testicular Semen Semen Semen Testicular Testicular Semen Semen Mean 6 SEM
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)
2 1 3 3 4 1 1 4 3 5 2.7 6 0.4
79% 74% 70% 68% 80% 72% 83% 71% 69% 58% 72 6 2.3
88% 79% 81% 78% 88% 85% 89% 79% 76% 78% 82 6 1.6
25% 8% 11% 39% 61.5% 29% 13% 14% 16.5% 23% 24 6 5.1
6% 10% 4% 24.5% 13% 7% 7.4% 8.2% 8% 4% 9.2 6 1.9
69% 76% 70% 35.5% 25.5% 64% 76% 70% 62% 54% 60 6 5.4
0% 6% 15% 1% 0% 0% 3.6% 7.8% 13.5% 19% 6.6 6 2.2
Note: Combined method 5 HOS-eosin. * Patients with malignancies receiving chemotherapy. † Patients with nonobstructive azoospermia.
developed to use distilled water as a hypoosmotic medium to evaluate the integrity of the tail sperm membrane (9). Because 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. Spermatozoa 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 membrane rupture caused by the formation of intracellular ice crystals during rapid cooling, or by osmotic effects, or mechanical force from extracellular ice formation during slow cooling. However, it is unclear to what extent membrane damage is responsible for the deleterious defects of cryopreservation on motility. Our study showed that sperm from subfertile men exhibited significantly lower postthaw percent motility, motile sperm concentration, and cryosurvival rate than those of fertile donors. These results suggest that the sperm of subfertile patients (even with comparable quality in the cases with normozoospermia or with moderate oligoasthenozoospermia) are more intolerant to the cryopreservation-thawing process than those of fertile donors. Whether a component(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 FERTILITY & STERILITYt
swelling test was significantly correlated with percent motility 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 disruption 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 normozoospermia 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 similar 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 intolerance 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 cryopreservationthawing. The present data are in agreement with the results 1153
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 (unstained) 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 membrane 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 postcryopreservation 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 1154 Lin et al.
Membrane integrity of cryopreserved sperm
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 percentage identified with eosin was only 16.6%. Under the conditions 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 investigate 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 capability 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 microscopy. 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 selecting 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 nonmotile 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 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.
9.
In conclusion, sperm from subfertile men with normal semen parameters exhibited a poorer cryopreservation outcome 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 intactness. Because deionized water has no extra components, it may be more suitable to select sperm at the time of ICSI.
10.
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 procedures, such as ICSI, may be revisited.
14.
11.
12. 13.
15. 16.
17. 18. 19.
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. Cryopreservation of human spermatozoa: the effect of cryoprotectants on motility. Fertil Steril 1988;50:314 –20. 3. Woolley DM, Richardson DW. Ultrastructural injury to human spermatozoa 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 characteristics. 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-
FERTILITY & STERILITYt
20. 21. 22. 23. 24.
25.
osmotic swelling test for the selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia. Fertil Steril 1996;65:972– 6. Lomeo AM, Giambersio AM. Water-test: a simple method to assess sperm-membrane integrity. Int J Androl 1991;14:278 – 82. 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 intracytoplasmic sperm injection. Hum Reprod 1996;11:2474 – 6. 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. 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. 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 hypoosmotic swelling test. Fertil Steril 1997;68:273–5. 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. 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. 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 freezingquick thawing method. Int J Androl 1995;18:279 – 86. 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. 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. 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. Hammitt DG, Hade DK, Williamson RA. Survival of human sperm following controlled- and non-controlled-rate cryopreservation. Andrologia 1988;21:311–7. 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. 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. Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276 – 84. Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril 1998;69:528 –32. 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.
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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
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). 0015-0282/98/$19.00 PII S0015-0282(98)00351-3
1148
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 postcryopreservation motility. (Fertil Sterilt 1998;70:1148 –55. ©1998 by American Society for Reproductive Medicine.) Key Words: Sperm, cryopreservation, membrane integrity, motility
Semen cryopreservation offers several advantages over the use of fresh semen in donor insemination programs, including ease of use, assurance of the availability over an extended period of time and possibility to rule out infection with human immunodeficiency virus. Furthermore, cryopreservation of semen can be performed before radiation therapy, chemotherapy, vasectomy, or other events that may cause irreversible damage to the male gonads.
It is important that cryopreservation is currently a standard procedure used to preserve ejaculated, epididymal, and testicular sperm recovered 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-
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 osmoregulatory 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 membrane 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 membrane (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 hypoosmotic medium to evaluate the integrity of the sperm membrane. 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 processing of samples with very few spermatozoa. The success with ICSI also has resulted in a significant increase in the number of testicular and epididymal aspiration 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). However, in many instances immotile sperm are the only available 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 immotility or vitality of the sperm. The objectives of the present study were as follows: [1] to FERTILITY & STERILITYt
examine the relationship between the hypoosmotic swelling test, water test, and eosin-Y staining (tests that examine sperm plasma membrane integrity through different approaches) and sperm motion characteristics before and after cryopreservation; [2] to assess how the hypoosmotic swelling test, water test, and eosin-Y staining correlate with each other and to determine their capacity to predict cryopreservation 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 membrane integrity and eosin staining, indicative of the integrity of the sperm head membrane, in a selected group of ejaculated 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 disregarding 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 Institutional 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 oligozoospermia (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 CellSoft Semen Analyzer (Cryo Resources Ltd., New York, NY) following parameter settings described previously (11). Sperm morphology was assessed by strict criteria (12) following staining with Diff-Quik (Dade Diagnostics, Aguada, PR). All men had morphology scores with .4% normal forms (“good prognosis pattern”) (12). All samples evaluated had ,1 3 106 leukocytes (peroxidase staining), were negative for antisperm antibodies (mixed antiglobulin reaction [MAR] test, Fertility Technologies, Natick, MA), and had negative cultures for Ureaplasma and Mycoplasma species. In the second part of the study, 10 cryopreserved semen and/or testicular sperm samples with no postcryopreservation motility also were evaluated (group D). Semen samples were collected by patients with malignancies before chemotherapy in six cases. Testicular sperm were received for cryopreservation and for future use in ICSI (n 5 4, cases of 1149
nonobstructive azoospermia). In all cases studied, motility of the samples before cryopreservation was ,5%. All evaluations were performed on the portion of the sample set aside for postthaw evaluation after obtaining patients consent.
analyzed blindly in coded slides. For each slide, all spermatozoa identified from five randomly selected fields were assessed in a systematic fashion.
Sperm Tests
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 centrifuged at 380 3 g for 10 minutes. The supernates were discarded, and the pellets were resuspended in 0.5 mL of the same medium.
The hypoosmotic swelling test was performed after estimation 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 incubation for 30 minutes at 37°C in 5% CO2, 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 mL of semen and 40 mL of distilled water on a microscope slide and covered with a thin 24 3 30-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 hypoosmotic swelling test and water test (6, 9). The eosin-Y staining (0.5% wt/vol) was performed by mixing 15 mL of semen with 5 mL of the stain on a microscope slide; immotile spermatozoa that were stained (positive) and unstained (negative) within 1–2 minutes after addition of the stain (wet prep) were assessed (4). Only the immotile fraction was evaluated (because all motile spermatozoa 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 liquefaction in men from groups A, B, and C. Thawed samples from patients from group D were evaluated 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 1150 Lin et al.
Membrane integrity of cryopreserved sperm
Sperm Cryopreservation-Thawing
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 3 106/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 citrate, 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 cryostorage. Each sample was thawed rapidly in a 42°C water bath for 3 minutes and allowed to equilibrate to room temperature 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 duplicate standard semen analysis then was performed on the postthaw sample as described above. Eosin-Y staining, hypoosmotic 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 spermatozoa 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 hypoosmotic swelling test scores. The sperm cryosurvival rate was calculated by dividing percent postthaw motility by percent fresh motility 3 100. To cryopreserve poor-quality samples (i.e., testicular sperm or ejaculated sperm with very low or sluggish motility), each sample was mixed with Ham’s F-10 medium containing 0.5% HSA, divided into small volumes and cenVol. 70, No. 6, December 1998
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). Group A (n 5 27)
Sperm characteristic WT (percentage coiled) HOS (percentage coiled) Eosin-Y stain (percentage unstained) Percent motility Motile concentration (3 106/mL) Motility index Linear velocity (mm/s) Linearity Cryosurvival rate (%)
Group B (n 5 22)
Fresh sample
Postthaw sample
Fresh sample
71.3 6 2.6 64.3 6 3.0
40.7 6 2.5* 35.6 6 2.3*
63.5 6 2.7 76.5 6 2.8 71.6 6 9.3 32.0 6 1.8 40.4 6 1.4 5.7 6 0.2
Group C (n 5 22)
Postthaw sample
Fresh sample
Postthaw sample
73.6 6 3.0 65.5 6 4.2
39.9 6 3.5* 29.1 6 3.2*
67.6 6 1.8 58.0 6 2.9
21.3 6 1.9*†‡ 19.0 6 2.5*†‡
34.9 6 2.0* 47.6 6 3.2*
61.3 6 2.6 70.3 6 3.2
30.4 6 2.0* 26.8 6 3.6*†
53.1 6 2.9§\ 40.4 6 4.0§\
19.5 6 1.7*†‡ 4.4 6 0.8*†‡
35.0 6 3.3* 15.9 6 1.2* 33.3 6 1.2* 4.9 6 0.3* 61.7 6 5.1
89.3 6 9.5 28.4 6 2.2 38.6 6 1.8 5.4 6 0.3
30.3 6 6.7* 10.3 6 2.0* 37.3 6 3.1 5.6 6 0.3 39.7 6 5.5¶
8.8 6 1.5§\ 14.5 6 1.6§\ 35.4 6 1.3§ 5.7 6 0.3
1.6 6 0.4*†‡ 1.5 6 0.3* 31.0 6 2.4 4.9 6 0.4 10.9 6 1.6¶
Note: Only nonmotile sperm were evaluated for eosin staining. Values are means 6 SEM. HOS 5 hypoosmotic swelling test; WT 5 water 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).
trifuged at 290 3 g for 5 minutes. Supernates were separated and examined for the presence of sperm. Pellets were resuspended into a small volume (typically 100 mL) of Ham’s F-10 medium with 0.5% HSA. The cryopreservation involved gradual dilution of the washed sample with a volume 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 cryopreserved in liquid nitrogen vapor as described above. Samples containing red blood cells and/or tissue remnants were centrifuged at 190 3 g for 1 minute. The supernatant was removed and subjected to further washing and cryopreservation 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 separate 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 assessed with use of paired Student’s t-test. Correlations between 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 P value of ,.05 was considered statistically significant. Results are presented as means 6 SEM. FERTILITY & STERILITYt
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 samples 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 difference 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 statistically significant differences in percent motility, motile concentration, velocity, linearity, and motility index (percent motility 3 mean 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 1151
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). Group A (n 5 27)
Comparison HOS vs. WT (fresh) HOS vs. percent motility (fresh) HOS vs. WT (cryopreserved/thawed)
Group C (n 5 22)
r
P value
r
P value
r
P value
.6038 .5525 .4464
.0009 .0028 .0196
.7155 .5263 .7138
.0004 .0171 .0002
.7196 .4492 .6687
.0002 .0360 .0009
the poorest results. The postthaw results of the three sperm membrane integrity tests were statistically significant between groups A and C, and between groups B and C, but not between groups A and B. There was no significant correlation between hypoosmotic 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 correlated 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 cryosurvival rate. The relationships between sperm head membrane integrity 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) and B (P,.03). However, no correlation was found between 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 (P 5 .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 ejaculated and testicular samples with no motility postcryopreservation (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 testeosin test, only 9.2% of sperm were identified as having both 1152 Lin et al.
Group B (n 5 22)
Membrane integrity of cryopreserved sperm
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 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). Correlation between viability detected by eosin-Y stain and results of indicated test Hypoosmotic swelling test Patient group, sample Group A Fresh Postthaw Group B Fresh Postthaw Group C Fresh Postthaw
Water test
r
P value
.3413 .0490
.0152 NS
2.0233 .1507
NS NS
.4849 .3339
.0266 NS
.4177 .2392
NS NS
2.0933 2.1055
NS NS
2.0685 2.1511
NS NS
r
P value
Note: NS 5 not significant.
Vol. 70, No. 6, December 1998
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 Semen* Testicular† Testicular Semen Semen Semen Testicular Testicular Semen Semen Mean 6 SEM
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)
2 1 3 3 4 1 1 4 3 5 2.7 6 0.4
79% 74% 70% 68% 80% 72% 83% 71% 69% 58% 72 6 2.3
88% 79% 81% 78% 88% 85% 89% 79% 76% 78% 82 6 1.6
25% 8% 11% 39% 61.5% 29% 13% 14% 16.5% 23% 24 6 5.1
6% 10% 4% 24.5% 13% 7% 7.4% 8.2% 8% 4% 9.2 6 1.9
69% 76% 70% 35.5% 25.5% 64% 76% 70% 62% 54% 60 6 5.4
0% 6% 15% 1% 0% 0% 3.6% 7.8% 13.5% 19% 6.6 6 2.2
Note: Combined method 5 HOS-eosin. * Patients with malignancies receiving chemotherapy. † Patients with nonobstructive azoospermia.
developed to use distilled water as a hypoosmotic medium to evaluate the integrity of the tail sperm membrane (9). Because 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. Spermatozoa 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 membrane rupture caused by the formation of intracellular ice crystals during rapid cooling, or by osmotic effects, or mechanical force from extracellular ice formation during slow cooling. However, it is unclear to what extent membrane damage is responsible for the deleterious defects of cryopreservation on motility. Our study showed that sperm from subfertile men exhibited significantly lower postthaw percent motility, motile sperm concentration, and cryosurvival rate than those of fertile donors. These results suggest that the sperm of subfertile patients (even with comparable quality in the cases with normozoospermia or with moderate oligoasthenozoospermia) are more intolerant to the cryopreservation-thawing process than those of fertile donors. Whether a component(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 FERTILITY & STERILITYt
swelling test was significantly correlated with percent motility 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 disruption 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 normozoospermia 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 similar 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 intolerance 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 cryopreservationthawing. The present data are in agreement with the results 1153
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 (unstained) 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 membrane 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 postcryopreservation 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 1154 Lin et al.
Membrane integrity of cryopreserved sperm
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 percentage identified with eosin was only 16.6%. Under the conditions 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 investigate 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 capability 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 microscopy. 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 selecting 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 nonmotile 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 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.
9.
In conclusion, sperm from subfertile men with normal semen parameters exhibited a poorer cryopreservation outcome 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 intactness. Because deionized water has no extra components, it may be more suitable to select sperm at the time of ICSI.
10.
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 procedures, such as ICSI, may be revisited.
14.
11.
12. 13.
15. 16.
17. 18. 19.
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