Robert Casper, M.D.
James S. Meriano, M.T., C.S.L.T.
Lisa Cowan, M.L.T., A.S.C.P.
Mara L. Lucato, B.Sc.
Keith A. Jarvi, M.D.

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, The University of Toronto, and The Toronto Centre For Advanced Reproductive Technology, Toronto, Ontario, Canada

Objective:  To determine the ability of the hypo-osmotic swelling test to select viable sperm from nonmotile sperm samples for intracytoplasmic sperm injection (ICSI).

Design:  Nonrandomized, sequential comparative study.

Patients:  Thirteen couples enrolled in our ICSI program had 16 cycles in which sperm preparations with 0% motility were obtained. Five cycles used cryopreserved epididymal sperm with complete asthenozoospermia.

Interventions:  In eight cycles, the semen samples were washed through a Percoll gradient and sperm were selected randomly for ICSI. In another eight cycles, the washed sperm were placed in a hypo-osmotic solution (75 mM fructose; 25 mM sodium citrate) and the sperm with curled tails taken up with the microinjection needle, rinsed, and used for ICSI.

Main Outcome Measures:  Fertilization rate per oocyte injected as determined by the presence of two pronuclei at 18 hours after retrieval and embryo cleavage rate per oocyte injected at 48 hours after retrieval.

Results:  With random sperm injection, the fertilization and cleavage rates were 26% and 23%, respectively. In contrast, after injection of sperm selected using the hypo-osmotic swelling test, fertilization and cleavage rates were significantly greater (43% and 39%, respectively). There were three pregnancies in the eight cycles with the hypo-osmotic swelling test-selected sperm, including two from frozen epididymal sperm.
Fertil Steril 1996;65:972-6

Key Words:  Hypo-osmotic swelling test, asthenozoospermia, intracytoplasmic sperm injection, sperm viability.

Until the advent of IVF techniques using micromanipulation, complete asthenozoospermia meant almost certain infertility. Now with the techniques of micromanipulation, particularly using intracytoplasmic sperm injection (ICSI), fertilizations and pregnancies have been reported using completely immotile sperm.

The major technical problem that has arisen with the use of immotile sperm for micromanipulation has been differentiating between live and dead sperm. In our experience, <50% of the sperm are viable in samples with complete asthenozoospermia. The standard techniques of vital dye exclusion to assess the integrity of the sperm membrane and, by inference, sperm viability, all result in the death of the sperm. We looked for other, nondamaging methods to identify live sperm for use with the ICSI procedure.

The hypo-osmotic swelling test was developed to investigate the ability of the sperm membrane to transport fluid (1). Sperm are incubated in a hypo-osmolar medium and those sperm with a functional membrane undergo swelling of the cytoplasmic space and the sperm tail fibers curl. These changes are visualized easily under light microscopy. Those sperm with damaged or chemically inactive plasma membranes will not have cytoplasmic swelling and the tails will remain uncurled. Because sperm that have a positive hypo-osmolar swelling test appear to be viable, we investigated the possibility that we could use the hypo-osmotic swelling test to identify viable sperm in a population of completely immotile sperm and use the resulting sperm for ICSI.

MATERIALS AND METHODS

Oocyte Retrieval

Multiple follicular stimulation, cycle monitoring, and oocyte retrieval were performed using standard protocols. All women were stimulated using a long GnRH-agonist protocol with 1 mg SC leuprolide acetate (LA, Lupron; Abbott Pharmaceuticals, Oakville, Ontario, Canada) starting in the midluteal phase until day 3 of the subsequent cycle. If serum E2 on day 3 was <42 pg/ml (150 pmol/L), the LA dose was cut in half and HMG (Pergonal; Serono Canada, Mississauga, Ontario, Canada or Humegon; Organon Canada, Toronto, Ontario, Canada) was started at a dose of two to four ampules daily. Human chorionic gonadotropin (10,000 IU IM Profasi; Serono) was given when at least two follicles > 1.8 cm in diameter were visualized and E2 levels were approximately 272 pg/mL (1,000 pmol/L) per mature follicle. Oocyte retrieval was performed by ultrasound-guided transvaginal aspiration under local anesthetic 36 hours after hCG administration. Follicular fluid was aspirated into heparinized HEPES-buffered human tubal fluid (HTF; Irvine Scientific, Santa Ana, CA). The oocyte-cumulus complex was identified and washed in fresh HTF supplemented with 0.5% (v/v) human serum albumin (HSA; 5% solution, USP; The Canadian Red Cross Society, Toronto, Ontario, Canada) equilibrated to 37 degrees C in 5% C0₂, 5% 0₂, 90% N₂ . Oocyte complexes were incubated in organ culture dishes (Falcon Products, Befford, MA) for approximately 1 hour before denuding.

Cumulus-Corona Cell Removal

Oocyte cumulus-corona cell complexes were placed in HEPES-buffered modified HTF-0.5% HSA medium supplemented with 80 IU/mL hyaluronidase (Type VII, from bovine testes; Sigma Chemical Company, St. Louis, MO) for 45 to 60 seconds. The oocytes then were removed and placed into fresh modified HTF-0.5% HSA for mechanical denuding. The resulting stripped oocytes were washed in fresh equilibrated HTF-0.5% HSA and the maturity and quality of the oocytes was assessed before injection as described previously (2).

Sperm Preparation

For each cycle, the raw semen specimen was assessed for sperm concentration, motility, and morphology. The sperm sample was washed in equilibrated HTF-0.5% HSA for 10 minutes at 400 x g. The resulting pellet was resuspended in 1 mL of fresh medium. The suspension was placed onto a 47%:95% Percoll gradient (Perwash; Immucor Canada, Toronto, Ontario, Canada) and centrifuged at 400 x g for 30 minutes. The pellet from the 95% gradient was retrieved and resuspended in 1 mL of medium and centrifuged again for 5 minutes. The resulting pellet was resuspended in 0.5 mL HTF-0.5% HSA and mixed gently before counting and final assessment.

In the first eight samples found to have 0% motility, sperm were selected randomly from those with normal morphology for intracytoplasmic injection. Supravital staining of sperm in six of the samples was performed using 0.5% Eosin Y stain as described previously (3). After introducing the hypo-osmotic swelling test, the test was used to select sperm for injection from the next eight specimens with 0% motility. All 16 cycles were performed over the course of 4 months. All sperm injections were performed by one of the authors (J.S.M.) who had >18 months of ICSI experience. Fertilization and cleavage rates were stable over this time period. Therefore, any differences seen between the two groups are unlikely to be a result of improved ICSI technique.

Hypo-osmotic Swelling Test Incubation

When possible, approximately 200,000 sperm were placed in a culture dish containing a hypo-osmotic solution (75 mM fructose and 25 mM sodium citrate in sterile water) and incubated at 37 degrees C for approximately 1 hour. The percentage of reactive (curled tails) and nonreactive (straight tails) sperm was assessed by counting approximately 400 to 500 cells. A 4-uL aliquot was placed in the microinjection dish under equilibrated mineral oil.

Intracytoplasmic Sperm Injection Procedure

A curled-tailed sperm was identified and aspirated into the microinjection pipette. It was then moved into a drop (4 uL under oil) of modified HTF-0.5% HSA, rinsed several times, moved again into a drop (4 uL) of polyvinylpyrrolidone (10% PVP wt/vol in modified HTF- 0.5% HSA) and rinsed repeatedly until the tail almost straightened. The sperm then was injected into the cytoplasm of the oocyte as described previously (4). The nonhypo-osmotic swelling tested sperm were placed into a 500 uL of modified HTF- 0.5% HSA and a 1-uL aliquot of this sperm mixture was mixed into a 4-uL drop of PVP before sperm injection. In both groups, only metaphase II oocytes obtained at the time of retrieval were injected. Our prior experience with injection of in vitro-matured oocytes was poor (2) and we are awaiting further research in this area before continuing with these immature oocytes. Fertilization was assessed by the presence of two distinct pronuclei at approximately 18 hours after retrieval. Cleavage was assessed at 40 hours after retrieval and transfer of a maximum of three embryos if available was performed at 48 hours (or 72 hours) postretrieval. A cumulative embryo score was calculated for each patient by modifying the protocol described by Steer et al. (5). Briefly, the number of blastomeres in each embryo was multiplied by the embryo score, transposed to give the best embryos 4/4 and the worst embryos 1/4. The scores for each embryo transferred were summed to give the cumulative embryo score. Luteal support using 100 mg natural P₄ suppositories twice daily intravaginally was started in all women on the day of ET.

Statistical Analysis

Descriptive data were compared for the two groups using the group t-test. Sperm count and morphology were not found to be distributed normally and a Mann-Whitney Rank Sum test was used to analyze these data. Fertilization and cleavage rates in the two groups were compared by the X² test with Yates' correction for continuity.

RESULTS

Eight couples with a diagnosis of male factor infertility and complete sperm immotility after semen preparation were treated by ICSI before initiation of the hypo-osmotic swelling test. Three of these couples and another five couples with complete asthenospermia were treated after introduction of the hypo-osmotic swelling test for identification of sperm with intact membranes. In all 13 couples, the female partner was normal with no evidence of any pathology associated with infertility. The semen and washed sperm parameters of the male partners of these couples are shown in Table 1. Sperm concentrations were not significantly different between the two groups. However, the percent of sperm with normal morphology was lower (P = 0.002) in the group undergoing sperm selection with the hypo-osmotic swelling test. None of the men had motile sperm before or after the Percoll gradient centrifugation. Before introducing the hypo-osmotic swelling test, six of eight samples were stained with Eosin Y dye after completion of the ICSI procedure and 30.2% of the sperm excluded the stain, indicating a structurally intact sperm membrane. The mean percentage of reactive and presumably viable sperrn in the eight samples tested with hypo-osmotic swelling test was 31.1%.

The mean age, serum E₂ concentrations on the day of hCG, and the percentage of mature oocytes collected did not differ in the couples before and after the introduction of the hypo-osmotic swelling test (Table 2). The mean fertilization rate before the hypo-osmotic swelling test was 26% and after the use of the hypo-osmotic swelling test to select sperm for injection was 43% (X² = 4.6, P < 0.05). The mean cleavage rate was 23% before the hypo-osmotic swelling test and 39% after the hypo-osmotic swelling test (X² = 4.3, P < 0.05). The embryo quality in the two groups was assessed by the method of Veeck (6) and is shown in Table 3. Grade 1 and 2 embryos, which usually are selected for transfer, represented 54.6% of the embryos in the group without the hypo-osmotic swelling test and 56.2% of the embryos in the group with the hypo-osmotic swelling test (non-significant). Embryo transfer was routinely performed at 48 hours after ICSI and three embryos were transferred in six women in the hypo-osmotic swelling test group but in only three women in the without hypo-osmotic swelling test group. Delayed ET (at 72 hours) occurred in two patients in the group without hypo-osmotic swelling test because of slow embryo development and in one patient in the group with hypo-osmotic swelling test for patient convenience (Table 2). The mean cumulative embryo score (Table 2) for the without hypo-osmotic swelling test group was 22.4 + 4.4 and for the with hypo-osmotic swelling test group was 37.8 + 4.9 (P < 0.05). Three pregnancies occurred in the eight cycles treated with ICSI and hypo-osmotic swelling test for sperm selection whereas no pregnancies occurred in the eight couples treated with ICSI without sperm selection. Two of three pregnancies occurred in couples with frozen epididymal sperm, which had 0% motility after thawing. Patient 2 delivered healthy twin boys whereas patients 3 and 7 had spontaneous abortions at 8 and 14 weeks gestation, respectively. Fetal heart activity was observed on ultrasound in both cases at 6 weeks gestation.

*Values are means + SEM include standard error of the mean (SEM).
⁺Morphology is different between the groups (P = 0.002).

* Conversion factor to SI unit: E₂,3 671.
⁺ In all cases where 3 or more embryos were available, 3 embryos were transferred. The + indicates embryos transferred at 72 hours.
⁺⁺ Values are means + SEM with percentages in parentheses.
⁺⁺⁺ Significantly different, P < 0.05.

*Grade 1 is the best quality and Grade 5 is the worst (6).
⁺Values in parentheses are percentages.

DISCUSSION

The concept of using the hypo-osmotic swelling test for selecting viable immotile sperm for use in the ICSI procedure was first proposed by Desmet et al. (Desmet B, Joris H, Nagy Z, Liu J, Bocken G, Vankelecom A, et al., abstract). However, these investigators used the sperm thus selected for injection of 1-day-old, unfertilized oocytes with resulting fertilization and cleavage rates of 3% and 2%, respectively. It appears that the test was abandoned after these poor results.

In the present study, we injected fresh metaphase II oocytes within 2 hours of stripping the cumulus-corona cells. Our results demonstrate that it is possible to use the hypo-osmotic swelling test to select sperm for ICSI from samples with 0% motility. The fertilization and cleavage rates (43% and 39%, respectively) were similar to our results during the same time period in couples with motile sperm. In contrast, random selection of sperm for injection from completely asthenospermic samples resulted in significantly poorer fertilization and cleavage rates. Of the cleaved embryos produced, the percentage of high quality embryos was similar in each group. However, because of the higher fertilization rate in the group with sperm selection by hypo-osmotic swelling test, three embryos were available for transfer in six women whereas only three women in the group treated before the introduction of hypo-osmotic swelling test had three embryos for transfer. As a result, the cumulative embryo score was significantly higher in the group in which sperm were selected using the hypo-osmotic swelling test. In this preliminary study, three of eight couples conceived after ICSI using these hypo-osmotic swelling test-selected sperm. One of these pregnancies resulted in the delivery of healthy twin boys. Unfortunately, the other two pregnancies were lost as spontaneous abortions at 8 and 14 weeks gestation, after fetal heart activity had been observed in both cases. We suggest that the sperm are not damaged by the hypo-osmotic swelling test procedure based on the similar percentage of high quality embryos in both groups and on the occurrence of pregnancy after the hypo-osmotic swelling test.

The hypo-osmotic swelling test is extremely simple to perform. The curled tails of the sperm with intact membranes are easy to identify. These sperm can be aspirated individually from the hypo-osmotic solution and resume their normal size and shape once placed in the usual culture medium before injection. Approximately 30% of the sperm were identified as having intact membranes by the hypo-osmotic swelling test, which was similar to the percentage identified as viable using Eosin Y staining. It is extremely likely, therefore, that the hypo-osmotic swelling test is able to identify viable sperm. We believe these results are extremely promising and provide a simple method to select immotile but viable sperm for ICSI.

REFERENCES

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2. Greenblatt EM, Meriano JS, Casper RF. Type of stimulation protocol affects oocyte maturity, fertilization rate and cleavage rate after intracytoplasmic sperm injection. Fertil Steril 1995;64:557-63.
3. Eliasson R, Treichl L. Supravital staining of human spermatozoa. Fertil Steril 1971; 22:134-7.
4. Van Steirteghem AC, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et ai. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993; 8:1061-6.
5. Steer CV, Mills CL, Tan SL, Campbell S, Edwards RG. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer program. Hum Reprod 1992;7:117-9.
6. Veeck L. Atlas of human oocyte and early conceptus. Vol. 2. Baltimore: Williams and Wilkins, 1991.