Key Words: Sperm, cryopreservation, membrane integrity, motility

Transcription

1 FERTILITY AND STERILITY VOL. 70, NO. 6, DECEMBER 1998 Copyight 1998 Ameican Society fo Repoductive Medicine ublished by Elsevie Science Inc. inted on acid-fee pape in U.S.A. lasma membane integity of cyopeseved human spem: an investigation of the esults of the hypoosmotic swelling test, the wate test, and eosin-y staining Ming-Huei Lin, M.D., Mahmood Moshedi, h.d., Chatchai Sisombut, M.D., Ahmed Nassa, M.D., and Segio Oehninge, M.D. Depatment of Obstetics and Gynecology, The Jones Institute fo Repoductive Medicine, Easten Viginia Medical School, Nofolk, Viginia and Depatment of Obstetics and Gynecology, Repoductive Endocinology and Infetility Unit, Mackay Memoial Hospital, Taipei, Taiwan, Republic of China Received May 8, 1998; evised and accepted July 21, esented in pat at the 53d Annual Meeting of the Ameican Society fo Repoductive Medicine, Cincinnati, Ohio, Octobe 18 22, Repint equests: Mahmood Moshedi, h.d., The Jones Institute fo Repoductive Medicine, 601 Colley Avenue, Nofolk, Viginia (FAX: ; /98/$19.00 II S (98) Objective: [1] To examine the elationship between spem membane integity and motion paametes befoe and afte cyopesevation; [2] to detemine the capacity of the membane integity tests to pedict the outcome of cyopesevation in fetile and infetile men; and [3] to examine the degee of ageement between tail and head membane integity of testicula and ejaculated immotile spem cyopeseved fo intacytoplasmic spem injection. Design: ospective study. Setting: Academic tetiay cae institution. atient(s): Fetile donos and nomozoospemic oligozoospemic, and asthenozoospemic subfetile men. Intevention(s): Semen s wee cyopeseved and thawed fo analysis. Main Outcome Measue(s): Spem membane integity and compute-assisted motion paametes. Result(s): The hypoosmotic swelling test and wate test had a significant and positive coelation in the fesh and cyopeseved ejaculates of all goups. The esults of the hypoosmotic swelling test coelated positively with the pecent motility in the fesh ejaculates of fetile and subfetile men. None of the membane integity tests coelated with the cyosuvival ate in any goup. In the ejaculated and testicula s with no postcyopesevation motility, the simultaneous assessment of hypoosmotic swelling test and eosin showed that of 33% spem exhibiting coiling with the hypoosmotic swelling test, only 9% wee eosin negative, wheeas 24% wee eosin positive. Conclusion(s): [1] The wate test may be a simple eplacement fo the hypoosmotic swelling test; [2] none of the membane integity tests pedicted spem motility afte cyopesevation; and [3] thee was a high degee of disageement between the hypoosmotic swelling test and eosin in the s with no postcyopesevation motility. (Fetil Steil 1998;70: by Ameican Society fo Repoductive Medicine.) Key Wods: Spem, cyopesevation, membane integity, motility Semen cyopesevation offes seveal advantages ove the use of fesh semen in dono insemination pogams, including ease of use, assuance of the availability ove an extended peiod of time and possibility to ule out infection with human immunodeficiency vius. Futhemoe, cyopesevation of semen can be pefomed befoe adiation theapy, chemotheapy, vasectomy, o othe events that may cause ievesible damage to the male gonads. It is impotant that cyopesevation is cuently a standad pocedue used to peseve ejaculated, epididymal, and testicula spem ecoveed at the time of uologic inteventions coupled with intacytoplasmic spem injection (ICSI). Howeve, cyopesevation esults in educed motility and fetility of human semen (1, 2). Feezing-thawing esults in damage to the plasma membane and acosome of human spematozoa as evidenced by significant ulta- 1148

2 stuctual changes demonstated by electon micoscopy (3). Theefoe, a simple and eliable test to pedict the outcome of cyopesevation is desiable. Vaious tests have been developed to assess the functional and stuctual integity of the spem plasma membane. The Wold Health Oganization (WHO) (4) stated that spem vitality is eflected in the popotion of spematozoa that ae alive as detemined by eithe dye exclusion o osmoegulatoy capacity unde hypo-osmotic conditions. One of these methods is based on the pinciple that dead cells with damaged plasma membanes take up supavital stains such as eosin-y, wheeas living cells cannot be stained because thei membanes actively exclude the dye (5). The eosin-y staining makes it possible to diffeentiate spematozoa that ae immotile but alive fom those that ae dead. Thee is ageement that this assay eflects spem membane integity (paticulaly of the head egion because it is this aea that takes up the stain) and is often efeed to as a vitality test (6). Jeyendan et al. (6) studied the functional integity of spem membanes with use of a hypoosmotic solution composed of equal pats of fuctose and sodium citate. The ability of the spem tail to swell and/o coil in the pesence of a hypoosmotic solution demonstates that influx of wate acoss the membane occus nomally and gives infomation on the integity and compliance of the tail membane (7). Accoding to the WHO (4), the hypoosmotic swelling test should not be used as a spem function test but may be used as a vitality test. Moe ecently, the hypoosmotic swelling test was used to select spem with a functionally intact membane in a population of completely immotile spem fo ICSI (8). Lomeo and Giambesio (9) developed a simple technique, the wate test, using distilled wate as a hypoosmotic medium to evaluate the integity of the spem membane. They demonstated that the wate test is as eliable as hypoosmotic swelling test is fo evaluating spem membane integity. Both methodologies can be adapted fo the pocessing of s with vey few spematozoa. The success with ICSI also has esulted in a significant incease in the numbe of testicula and epididymal aspiation attempts esulting in etieval of sluggish and immotile spematozoa. In many of these instances, spematozoa also have been cyopeseved, leading to inceased chances of conception though epeated ICSI attempts. Recent findings have indicated that some of these spematozoa may gain motility on incubation unde defined conditions (10). Howeve, in many instances immotile spem ae the only available souce fo ICSI. In situations like this, ICSI has eithe been canceled o a vitality test such as hypoosmotic swelling test has been used to select spematozoa fo injection. Recent expeience has demonstated that the only spem facto that can significantly influence the esults of ICSI is the immotility o vitality of the spem. The objectives of the pesent study wee as follows: [1] to examine the elationship between the hypoosmotic swelling test, wate test, and eosin-y staining (tests that examine spem plasma membane integity though diffeent appoaches) and spem motion chaacteistics befoe and afte cyopesevation; [2] to assess how the hypoosmotic swelling test, wate test, and eosin-y staining coelate with each othe and to detemine thei capacity to pedict cyopesevation outcome. Ejaculates fom fetile and subfetile men wee examined fo this pupose; and [3] to examine the degee of ageement (o lack of) between the esults of hypoosmotic swelling test, epesenting spem tail membane integity and eosin staining, indicative of the integity of the spem head membane, in a selected goup of ejaculated and testicula s with no postcyopesevation spem motility using a simultaneous hypoosmotic swelling test-eosin evaluation. This is an impotant pactical issue at the time of ICSI because often hypoosmotic swelling test is used to select spem with tail membane integity disegading the assessment of the head membane. MATERIALS AND METHODS Semen Samples A total of 71 semen s wee pospectively collected fo the fist pat of this study afte appoval of the Institutional Review Boad of Easten Viginia Medical School. Twenty-seven of these s wee obtained fom fetile donos (goup A). Twenty-two s wee collected by men consulting fo infetility with nomozoospemia (goup B). Anothe 22 s wee collected by men consulting fo infetility with asthenozoospemia and/o oligozoospemia (goup C) defined following WHO citeia (4). Semen s wee collected by mastubation in steile specimen cups afte 3 4 days of sexual abstinence and allowed to liquefy fo 30 minutes at oom tempeatue. Spem concentation and motility paametes wee analyzed by compute-assisted semen analysis (CASA) with the Cell- Soft Semen Analyze (Cyo Resouces Ltd., New Yok, NY) following paamete settings descibed peviously (11). Spem mophology was assessed by stict citeia (12) following staining with Diff-Quik (Dade Diagnostics, Aguada, R). All men had mophology scoes with 4% nomal foms ( good pognosis patten ) (12). All s evaluated had leukocytes (peoxidase staining), wee negative fo antispem antibodies (mixed antiglobulin eaction [MAR] test, Fetility Technologies, Natick, MA), and had negative cultues fo Ueaplasma and Mycoplasma species. In the second pat of the study, 10 cyopeseved semen and/o testicula spem s with no postcyopesevation motility also wee evaluated (goup D). Semen s wee collected by patients with malignancies befoe chemotheapy in six cases. Testicula spem wee eceived fo cyopesevation and fo futue use in ICSI (n 4, cases of FERTILITY & STERILITY 1149

3 nonobstuctive azoospemia). In all cases studied, motility of the s befoe cyopesevation was 5%. All evaluations wee pefomed on the potion of the set aside fo postthaw evaluation afte obtaining patients consent. Spem Tests The hypoosmotic swelling test was pefomed afte estimation of standad semen paametes, and the pecentage of spem with coiled tails was ecoded. An aliquot of 0.1 ml of liquefied semen was added to 1 ml of hypoosmotic solution. This solution was pepaed by dissolving 7.35 g of sodium citate and 13.5 g of fuctose in 1 kg of deionized distilled wate (osmolality of 150 mosm/kg), filte steilized, and fozen in 1-mL aliquots fo use as equied. Afte incubation fo 30 minutes at 37 C in 5% CO 2, the coiling of the spem tail epesenting an intact flagella membane was evaluated accoding to the guidelines epoted by Jeyendan et al. (8). The wate test was pefomed by mixing 10 L of semen and 40 L of distilled wate on a micoscope slide and coveed with a thin mm coveslip. The mixtue was incubated fo 5 minutes in a moist chambe at 37 C befoe it was examined with a micoscope at 400 magnification. At least 100 spem wee examined, and the pecentage of spem that showed coiled tails was calculated fo both the hypoosmotic swelling test and wate test (6, 9). The eosin-y staining (0.5% wt/vol) was pefomed by mixing 15 L of semen with 5 L of the stain on a micoscope slide; immotile spematozoa that wee stained (positive) and unstained (negative) within 1 2 minutes afte addition of the stain (wet pep) wee assessed (4). Only the immotile faction was evaluated (because all motile spematozoa ae vital and, consequently, exclude the dye), and the esults wee expessed as the pecentage of unstained o negative spem (theefoe having an intact head membane). Eosin-Y staining, hypoosmotic swelling test, and wate test wee pefomed on each fesh semen afte liquefaction in men fom goups A, B, and C. Thawed s fom patients fom goup D wee evaluated fo the degee of spontaneous coiling and the eosin staining as descibed above. Samples also wee evaluated with a combined hypoosmotic swelling test-eosin method. ostthaw s wee incubated with the hypoosmotic swelling test medium and mixed with the eosin stain afte incubation fo the combined evaluation of hypoosmotic swelling test and eosin esults. The standad hypoosmotic swelling test (as descibed above) o a micomethod used by Liu et al. (13) wee used to assess tail coiling in these thawed s. At least 100 spematozoa o as many as could be found wee evaluated. Because the wate test showed an excellent coelation with hypoosmotic swelling test (see Results), no combined wate test-eosin staining was attempted in these latte cases. Fo the thee spem membane integity tests, esults wee analyzed blindly in coded slides. Fo each slide, all spematozoa identified fom five andomly selected fields wee assessed in a systematic fashion. Spem Cyopesevation-Thawing To minimize the effect of vaiable spem concentations on spem viability postcyopesevation (14), all s wee washed befoe cyopesevation. Each liquefied semen was mixed with a volume of Ham s F-10 medium (Fetility Technologies, Natick, MA) supplemented with 0.5% human seum albumin (HSA; Ivine Scientific, Santa Ana, CA) equal to 3 times the volume of semen. The mixtue then was aliquoted into a minimum of two 15-mL conical centifuge tubes (VWR Scientific, Bidgepot, NJ) and centifuged at 380 g fo 10 minutes. The supenates wee discaded, and the pellets wee esuspended in 0.5 ml of the same medium. Fo the s of men fom goups A, B, and C, an analysis of spem concentation was pefomed, and the spem concentation was adjusted to appoximately /ml. The cyopesevation pocedue involved gadual dilution of the washed semen with a volume of feezing medium (Ivine Scientific) equal to one half of the washed semen volume. The feezing medium contained TES and tis(hydoxymethyl)aminomethane (Tis) buffes, sodium citate, fuctose, 12% (vol/vol) glyceol, and 20% (vol/vol) heat-inactivated egg yolk. The cyovials (0.4 ml/vial) wee then suspended 5 cm above the liquid nitogen fo vapo feezing fo 30 minutes befoe loading on pecooled canes and plunging them diectly into liquid nitogen. The thawing pocedue was pefomed afte 1 day cyostoage. Each was thawed apidly in a 42 C wate bath fo 3 minutes and allowed to equilibate to oom tempeatue 15 minutes (quick feeze-quick thaw). In pevious studies (15, 16) we showed that this ate of thawing matches moe closely the feezing ate used and esults in optimal ecovey of motile spem fo atificial insemination and excellent fetilization ates unde IVF conditions. A duplicate standad semen analysis then was pefomed on the postthaw as descibed above. Eosin-Y staining, hypoosmotic swelling test, and wate test also wee pefomed in the thawed s. Coiling of the spem tail that is usually obseved duing the feezing-thawing pocess in a small popotion of spematozoa was assessed in each specimen by scoing the pecentage of coiled spem in the absence of hypoosmotic solution. This was then subtacted fom the total hypoosmotic swelling test scoes. The spem cyosuvival ate was calculated by dividing pecent postthaw motility by pecent fesh motility 100. To cyopeseve poo-quality s (i.e., testicula spem o ejaculated spem with vey low o sluggish motility), each was mixed with Ham s F-10 medium containing 0.5% HSA, divided into small volumes and cen Lin et al. Membane integity of cyopeseved spem Vol. 70, No. 6, Decembe 1998

4 TABLE 1 Spem motility chaacteistics and test esults of the fesh and postthaw s in the fetile donos (goup A), patients with nomal semen (goup B), and patients with abnomal semen (goup C). Goup A (n 27) Goup B (n 22) Goup C (n 22) Spem chaacteistic Fesh ostthaw Fesh ostthaw Fesh ostthaw WT (pecentage coiled) * * * HOS (pecentage coiled) * * * Eosin-Y stain (pecentage unstained) * * * ecent motility * * * Motile concentation ( 10 6 /ml) * * * Motility index * * * Linea velocity ( m/s) * Lineaity * Cyosuvival ate (%) Note: Only nonmotile spem wee evaluated fo eosin staining. Values ae means SEM. HOS hypoosmotic swelling test; WT wate test. *.05 (vs. the fesh semen s);.05 (vs. the postthaw semen s of goup A)..05 (vs. the postthaw semen s of goup B)..05 (vs. the fesh semen s of goup A)..05 (vs. the fesh semen s of goup B);.01 (vs. fozen semen s of goup A). **.01 (vs. the fozen semen s of goup B). tifuged at 290 g fo 5 minutes. Supenates wee sepaated and examined fo the pesence of spem. ellets wee esuspended into a small volume (typically 100 L) of Ham s F-10 medium with 0.5% HSA. The cyopesevation involved gadual dilution of the washed with a volume of feezing medium (Ivine Scientific) equal to one half of its volume and aliquoting of the mixtue into seveal 1.2-mL cyovials in small volumes. Samples wee then cyopeseved in liquid nitogen vapo as descibed above. Samples containing ed blood cells and/o tissue emnants wee centifuged at 190 g fo 1 minute. The supenatant was emoved and subjected to futhe washing and cyopesevation as descibed above. The pellet was esuspended in a small volume of Ham s F-10 medium with 0.5% HSA, mixed with the feezing medium, and cyopeseved in sepaate vials labeled accodingly. Ou expeience with this type of s indicates an excellent spem suvival in s with a motility of 5%. Statistical Analysis Statistical significance between the mean s of the diffeent tests obtained befoe and afte feezing was assessed with use of paied Student s t-test. Coelations between the spem membane integity tests and basal semen paametes, between fesh and postthaw semen s, and between fetile donos and subfetile patients wee assessed with use of eason s coelation coefficient. A of.05 was consideed statistically significant. Results ae pesented as means SEM. RESULTS The semen analysis data and the esults of thee spem membane integity (o vitality) tests obtained fom the fesh and post-thaw s of goups A, B, and C ae shown in Table 1. The pecentage of swollen-coiled spem (intact tail membane) obtained in hypoosmotic swelling test and wate test wee not statistically significant between the fesh s of goups A, B, and C. The pecentage of immotile spem not stained with eosin-y (intact head membane) also was not statistically significant between fesh s of goup A and B. Howeve, a statistically significant diffeence was found between goups A and C (.02) and between goups B and C (.05). The fesh s of goups A and B showed no statistically significant diffeences in pecent motility, motile concentation, velocity, lineaity, and motility index (pecent motility mean cuvilinea velocity), wheeas goup C showed significantly pooe motility chaacteistics than those of goups A and B. Thee was a significant and positive coelation between hypoosmotic swelling test and wate test and between hypoosmotic swelling test and pecent motility in the fesh s of all thee goups (Table 2). As expected, cyopesevation esulted in a significant eduction in spem motion paametes and in the esults of the spem membane integity tests. atients fom goups B and C showed a significantly lowe pecentage of postthaw motile spem than those of goup A, with goup C showing FERTILITY & STERILITY 1151

5 TABLE 2 Coelations between the hypoosmotic swelling test (HOS) and wate test (WT), HOS and pecent motility of fesh semen s, and between HOS and WT of cyopeseved-thawed semen s in the fetile donos (goup A), patients with nomal semen (goup B), and patients with abnomal semen (goup C). Goup A (n 27) Goup B (n 22) Goup C (n 22) Compaison HOS vs. WT (fesh) HOS vs. pecent motility (fesh) HOS vs. WT (cyopeseved/thawed) the pooest esults. The postthaw esults of the thee spem membane integity tests wee statistically significant between goups A and C, and between goups B and C, but not between goups A and B. Thee was no significant coelation between hypoosmotic swelling test and pecent motility, between wate test and pecent motility, and between eosin-y staining and pecent motility in the postthaw s of all thee goups. Howeve, hypoosmotic swelling test was positively coelated with wate test in postthaw s of all goups (Table 2). The cyosuvival ate of goup B was significantly lowe than that of goup A (.01) but significantly highe than that of goup C (.001, Table 1). None of the thee spem membane integity tests in fesh s of donos and patients (goups A, B, and C) coelated with the cyosuvival ate. The elationships between spem head membane integity as assessed by the eosin-y staining and the esponse in the hypoosmotic swelling test and wate test (indicative of spem tail membane integity) ae shown in Table 3. The hypoosmotic swelling test was positively coelated with eosin-y staining in the fesh s of goups A (.02) andb(.03). Howeve, no coelation was found between hypoosmotic swelling test and eosin-y stain in the fesh s of goup C and in the postthaw s of all thee goups. A poo coelation (.058) was obseved between wate test and eosin-y stain in fesh and postthaw s of all thee goups. Table 4 pesents data obtained fom evaluation of ejaculated and testicula s with no motility postcyopesevation (goup D). With hypoosmotic swelling test, 33.2% of all spematozoa exhibited positive coiling, wheeas 66.8% wee coiled negative. Of the 33.2% coiled spematozoa (intact tail membanes), 9.2% wee eosin negative (intact head membanes) and 24% wee eosin positive (abnomal head membanes); of the 66.6% uncoiled spematozoa, 6.6% wee eosin negative and 60% wee eosin positive. On the basis of the combined hypoosmotic swelling testeosin test, only 9.2% of spem wee identified as having both tail and head intact membanes. Howeve, thee was a high degee of vaiation among s with this espect. The esults of hypoosmotic swelling test and eosin, theefoe, coelated one way o anothe in only 69.2% of spematozoa. Compaison of the eosin esults (nonmotile spematozoa) befoe (72%) and afte cyopesevation (82%) indicated no dastic incease in eosin staining of spematozoa (membane damage) afte cyopesevation. DISCUSSION The WHO ecommended using the hypoosmotic swelling test as a vitality test (i.e., to gain infomation on the integity and compliance of the tail membane) and not as a spem function test (4). A simple technique, the wate test, was TABLE 3 Coelations between viability detected by eosin-y stain and esults of hypoosmotic swelling test and wate test in the fesh and postthaw s of donos (goup A), patients with nomal semen (goup B), and patients with abnomal semen (goup C). atient goup, Coelation between viability detected by eosin-y stain and esults of indicated test Hypoosmotic swelling test Wate test Goup A Fesh NS ostthaw.0490 NS.1507 NS Goup B Fesh NS ostthaw.3339 NS.2392 NS Goup C Fesh.0685 NS.0933 NS ostthaw.1511 NS.1055 NS Note: NS not significant Lin et al. Membane integity of cyopeseved spem Vol. 70, No. 6, Decembe 1998

6 TABLE 4 Evaluation of s with no postcyopesevation motility and the elationship between hypoosmotic swelling test and eosin-y stain in these s (goup D). Sample type Motility (%) (befoe cyopesevation) Eosin (befoe cyopesevation) Eosin (afte cyopesevation) Coiled and eosin (combined) Coiled and eosin (combined) Not coiled and eosin (combined) Not coiled and eosin (combined) Semen* 2 79% 88% 25% 6% 69% 0% Testicula 1 74% 79% 8% 10% 76% 6% Testicula 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% Testicula 1 83% 89% 13% 7.4% 76% 3.6% Testicula 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 SEM Note: Combined method HOS-eosin. * atients with malignancies eceiving chemotheapy. atients with nonobstuctive azoospemia. developed to use distilled wate as a hypoosmotic medium to evaluate the integity of the tail spem membane (9). Because the tail membane is moe loosely attached to the undelying stuctues than in the spem head, the spem tails seem to be paticulaly sensitive to such swelling. Spematozoa with nonfunctioning membanes will not swell (6). Some investigatos demonstated that the spem coiling scoes obtained by the wate test coelated well with those obtained by the hypoosmotic swelling test (17). Ou esults confim these findings and show a good coelation between the wate test and hypoosmotic swelling test not only in fesh semen s but also in postthawed semen s. These findings suggest that the wate test may be an adequate eplacement to the hypoosmotic swelling test. Cell damage duing feezing usually is ascibed to membane uptue caused by the fomation of intacellula ice cystals duing apid cooling, o by osmotic effects, o mechanical foce fom extacellula ice fomation duing slow cooling. Howeve, it is unclea to what extent membane damage is esponsible fo the deleteious defects of cyopesevation on motility. Ou study showed that spem fom subfetile men exhibited significantly lowe postthaw pecent motility, motile spem concentation, and cyosuvival ate than those of fetile donos. These esults suggest that the spem of subfetile patients (even with compaable quality in the cases with nomozoospemia o with modeate oligoasthenozoospemia) ae moe intoleant to the cyopesevation-thawing pocess than those of fetile donos. Whethe a component(s) of seminal fluid o othe facto(s) elated to the spem contibuted to these diffeences emains to be elucidated. The pesent study demonstated that the hypoosmotic swelling test was significantly coelated with pecent motility in the fesh s of all thee goups but not in postthaw s. It is possible that loss of spem tail membane integity may be a majo facto contibuting to the decline of spem motility. Afte cyopesevation, howeve, seveal factos, and not only tail membane damage, may be esponsible fo the loss of pogessively motile spem. Thee was no coelation between eosin-y staining and pecent motility in semen s of the thee goups (A, B, and C) befoe and afte cyopesevation; this suggests that disuption of head membane integity may not be associated necessaily with the loss of spem motility. Howeve, when an immotile spematozoon is being used fo pocedues such as ICSI, integity of the spem head membanes may be an impotant facto to conside. The fesh s of donos and patients with nomozoospemia showed no statistically significant diffeences in motility paametes and in the esults of the thee membane tests (hypoosmotic swelling test, wate test, and eosin Y staining), wheeas patients with abnomal semen paametes showed significantly pooe motility paametes and esults of eosin-y staining. This fact suggests that the fesh s in dono and patients with nomozoospemia may have simila conditions of spem head and tail membanes, wheeas patient s with abnomal semen paametes have not only a highe incidence of head membane defects in the fesh semen s but also a highe incidence of intoleance to the feezing-thawing pocess. None of the thee spem membane integity tests (o vitality tests) in fesh semen of donos o patients could be used to pedict the motility changes afte cyopesevationthawing. The pesent data ae in ageement with the esults FERTILITY & STERILITY 1153

7 of the hypoosmotic swelling test in pevious studies (18, 19). The lack of pedictive powe fo the cyosuvival ate of spem by the hypoosmotic swelling test may be due to the fact that thee ae many inteacting factos othe than the membane integity of human spem that can influence the cyosuvival ate. These include among othes, the vaiation in individual semen quality, the method of cyopesevation, the thawing pocedue, and the cyopotectants used (1, 2, 20, 21). In cetain situations, spem may have an intact membane but be immotile, and in these cases supavital stains can diffeentiate immotile but stuctually intact spem (unstained) fom those that ae immotile and dead (stained) (21). As spem die, they lose thei ability to esist the influx of the membane-impemeant dye. Howeve, it is unknown whethe using the spem coupted by supavital stain will lead to alteations of DNA integity, gamete inteaction, embyogenesis, and negatively affect the outcome of assisted epoductive technologies (21). The hypoosmotic swelling test has been used clinically as a tool in identifying immotile spem with an intact membane in fesh and cyopeseved spem fo ICSI (22). In ou study, as well as in othes (21), the hypoosmotic swelling test could coectly assess the integity of the spem tail membane in fesh s of fetile and subfetile men as demonstated by the positive and significant coelation with the pecent pogessive motility (Table 2). Howeve, this was not the case fo the postthaw s of goups A, B, and C. It is possible that the detemining facto(s) in spem suvival afte cyopesevation is(ae) elated to the spem head membane and/o othe cytoplasmic-metabolic factos, wheeas the hypoosmotic swelling test mainly eflects tail membane intactness and pehaps maintenance of motility. In this study we also evaluated a limited numbe of ejaculated and testicula s with no postcyopesevation spem motility to detemine the degee of ageement (o lack of) among the vaious tests cuently used to detemine spem membane integity (o vitality). The findings of this study could have epecussions fo ICSI (Table 4). Ou esults showed that if the hypoosmotic swelling test was to be used fo the selection of cyopeseved-thawed immotile spem, thee would be only a 9% chance of selecting a coiled spem (intact tail membane) with an intact spem head membane (unstained) as detemined by eosin. Convesely, the chance of selecting a coiled spem (with an intact tail membane) that was stained by eosin (indicating a damaged head membane) would be 24%. Appoximately 7% of spem with an intact head membane would be missed though hypoosmotic swelling test selection because they did not coil. The esults pesented in Table 4 also evealed two moe phenomena. Fist, the pecentage of spematozoa identified as having an intact membane ae vastly diffeent when hypoosmotic swelling test o eosin ae used. Appoximately 33% of immotile spem wee identified as having an intact membane with hypoosmotic swelling test, but the pecentage identified with eosin was only 16.6%. Unde the conditions used and the type of s evaluated, 66.6% of spematozoa wee identified as having membane defects unde hypoosmotic swelling test conditions. The defect ate identified by eosin was 84%. Anothe finding was the vast diffeences obseved among s fo the esults obtained fo hypoosmotic swelling test and eosin. Consequently, it is vey impotant to investigate if the faction of spematozoa exhibiting the coiling phenomenon but that ae stained with eosin is less suitable fo ICSI than the faction with no coiling but with intact head membanes as detemined by eosin. In peliminay studies we also used a combination fluoescent stains, which ae nucleic acid specific to assess the spem head membane integity (23). This staining method also gives us the capability of assessing the popotion of moibund spem in each which may exhibit the coiling phenomenon unde hypoosmotic swelling test conditions. Fluoophoes, SYBR-14 and popidium iodide, maketed unde the spem viability kit designation, has been made suitable fo fluoescent micoscopy. Ou peliminay studies have shown that between 15% and 20% of fesh spematozoa coiled unde hypoosmotic swelling test conditions show spem head leakage and DNA staining indicative of stuctual damage. Ou compaison studies also ae indicating that thee is a significant degee of disageement between eosin and SYBR-14 and popidium and iodide, two tests to assess the integity of spem head membane. Recently, much attention has been given to the status of spem plasma membane and the nuclea DNA changes as they elate to the apoptosis phenomenon. The impact of vaious factos on the integity of plasma membane and spem DNA also has geneated inteest (24). Thee have been epots that spematozoa with nomal mophology may have sevee nuclea damage, endeing them incapable of suppoting nomal embyo development (24). aallel and simultaneous studies to assess the apoptosis-associated plasma membane lipid changes (asymmety) in the fom of tanslocation of phosphatidyl seine fom the inne side to the oute laye of membane and evaluation of the integity of spem nuclea and mitochondial DNA by in situ cell death detection may povide futhe infomation fo selecting viable immotile spem fo ICSI. Recently, a simple test of measuing spem tail:head atio was used to assess its elationship to the viability of nonmotile fesh and cyopeseved testicula spem as measued by SYBR-14 and I (25). It was found that 82.3% of viable spem had the tail:head atio of 10 compaed to 24.6% of nonviable spem exhibiting such atio. Although this method may not be suitable fo the evaluation of spontaneously 1154 Lin et al. Membane integity of cyopeseved spem Vol. 70, No. 6, Decembe 1998

8 coiled spematozoa, it may be useful to study induced coiling (hypoosmotic swelling test) and its elationship to eosin, apoptotic-elated changes of spem and ICSI outcome. In conclusion, spem fom subfetile men with nomal semen paametes exhibited a pooe cyopesevation outcome than those of fetile donos. The subfetile men with abnomal semen paametes had the wost spem outcome afte the feezing-thawing pocess. In the absence of moe accuate tests to assess spem membane integity, the wate test may be an adequate eplacement to the hypoosmotic swelling test in the evaluation of spem tail membane intactness. Because deionized wate has no exta components, it may be moe suitable to select spem at the time of ICSI. In addition, the wate test may be moe amenable to pocessing of single spem o micopocessing of s with a few spem. The hypoosmotic swelling test, wate test, and eosin-y staining, vitality tests that eflect spem plasma membane integity, cannot be used to pedict the motility changes o outcome afte cyopesevation of spem in fetile donos o subfetile patients with nomal o abnomal spem paametes. The of the hypoosmotic swelling test fo selecting intact cyopeseved spem fom a with completely immotile spem fo micomanipulation pocedues, such as ICSI, may be evisited. Refeences 1. Keel BA, Webste BW, Robets DK. Effect of cyopesevation on the motility chaacteistics of human spematozoa. J Repod Fetil 1987; 81: Citse JK, Huse-Benda AR, Aake DV, Aneson BW, Ball GD. Cyopesevation of human spematozoa: the effect of cyopotectants on motility. Fetil Steil 1988;50: Woolley DM, Richadson DW. Ultastuctual injuy to human spematozoa afte feezing and thawing. J Repod Fetil 1978;53: Wold Health Oganization. Laboatoy manual fo the examination of human semen and spem-cevical mucus Inteaction. 3d ed. 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Cyopesevation of human semen: compaison of cyopesevatives, souces of vaiability, and pediction of post-thaw suvival. J Andol 1992;12: Esteves SC, Shama RK, Thomas AJ J, Agawal A. Suitability of the hypo-osmotic swelling test fo assessing the viability of cyopeseved spem. Fetil Steil 1996;66: Gane DL, Johnson LA. Viability assessment of mammalian spem using SYBR-14 and popidium iodide. Biol Repod 1995;53: Lopes S, Sun JG, Juisicova A, Meiano J, Caspe RF. Spem deoxyibonucleic acid fagmentation is inceased in poo quality semen s and coelates with failed fetilization in intacytoplasmic spem injection. Fetil Steil 1998;69: Mama JL, Coson SL, Gibbs M, Husza G. Tail to head atio (TH) of fesh and testicula spem as a measue of viability. J Andol (Suppl): Jan/Feb 1998;58. FERTILITY & STERILITY 1155