1 Department of Urology and 2 Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, School of Medicine, 2330 Post Street, 6th Floor, San Francisco, California 941151695, USA
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Abstract |
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Key words: cryopreservation/epididymis/hypo-osmotic swelling/sperm viability/testis
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Introduction |
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The published experience with frozenthawed epididymal spermatozoa and ICSI suggests that fertilization and pregnancy success rates are comparable to those achieved with freshly retrieved spermatozoa (Nagy et al., 1995; Oates et al., 1996
; Holden et al., 1997
; Hutchon et al., 1998
). It seems that comparable success may be achieved with frozenthawed and fresh testis spermatozoa (Friedler et al., 1997b
; Liu et al., 1997
; Oates et al., 1997
; DeCroo et al., 1998
; Marmar 1998
; Perraguin-Jayot et al., 1998
). In summary, these data suggest that it is certainly possible to use frozenthawed testicular and epididymal spermatozoa for ICSI. However, there is scarce information on whether it is, in fact, feasible to use frozenthawed spermatozoa for ICSI. Questions relative to this issue include: how well do extracted testicular or epididymal spermatozoa recover from the process of freeze and thaw? How does the cryopreservation process affect the viability of extracted spermatozoa? The goal of this study was to define the cryobiological behaviour of frozenthawed spermatozoa from several sites within the male reproductive tract. This information may help determine the feasibility of using frozenthawed spermatozoa for ICSI.
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Materials and methods |
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Patient groups
Testis spermatozoa
In five men with non-obstructive azoospermia, testicular sperm extraction (TESE) was performed for IVF and ICSI. Tissue was placed in 1.0 ml of Earle's medium (Gibco BRL, Grand Island, NY, USA), supplemented with 4.0 mmol/l sodium bicarbonate (Fisher Scientific, Santa Clara, CA, USA), 21 mmol/l HEPES (Calbiochem, La Joya, CA, USA), 0.47 mmol/l pyruvate (Sigma, St Louis, MO, USA), 100 U/ml penicillin, 50 µg/ml streptomycin sulphate (Sigma) and 10% v/v synthetic serum substitute (SSS; Irvine Scientific, Santa Ana, CA, USA). Testis tissue was maintained at 37°C in a 2.5 cm Petri dish (Falcon 3001®; Becton Dickinson, Lincoln Park, NJ, USA) and lightly macerated to provide fresh testis spermatozoa for the study.
Epididymal spermatozoa
In eight patients with obstructive azoospermia (n = 6 prior vasectomy; n = 2 congenital absence of the vas deferens), microscopic epididymal sperm aspiration was performed for IVF and ICSI (Nudell et al., 1998). Epididymal fluid was aspirated with a 24 gauge catheter and 1.0 ml syringe prefilled with 100 µl of Earle's medium (as described above) and expelled into 1.0 ml of similar medium maintained at 37°C. Excess fresh epididymal spermatozoa were used for the study.
Vasal spermatozoa
Before vas deferens ligation in five fertile men undergoing vasectomy, the testicular end of the transected vas deferens was irrigated with 100 µl of Earle's medium (as above) and aspirated with a 24 gauge catheter and 1.0 ml syringe. The vasal aspirate was expelled into 1.0 ml of similar medium maintained at 37°C to provide fresh spermatozoa for the study.
Sperm cryopreservation and thaw protocol
Aspirated spermatozoa from each patient were divided into equal aliquots for fresh analysis and cryopreservation. Aspirated sperm fractions designated for cryopreservation were washed twice (centrifuged at 170 g, 10 min) in phosphate buffered saline (Gibco) with 2% v/v SSS. The final pellet was resuspended in 0.5 ml of Earle's medium. Specimens were cryopreserved in a 1:1 dilution of sperm suspension to test yolk buffer (Irvine Scientific) in 1.0 ml vials by the slow freeze method of Mahadevan and Trouson (Mahadevan and Trouson, 1983) in a Planar Biomed controlled rate freezer (T.S. Scientific, Perkasie, PA, USA). After a minimum of 24 h freezing, vials were thawed by incubation at 37°C for 10 min. The spermatozoa were then gradually reintroduced into isotonic medium by adding an equal volume of Earle's medium to the vial over a 5 min period. This diluted specimen was centrifuged (170 g, 10 min). This dilution and wash procedure was repeated twice and the final pellet resuspended in 0.5 ml of Earle's buffered medium for assessment of motility and viability parameters.
Assessment of sperm motility and viability
The following viability analyses were performed on both fresh and frozenthawed spermatozoa from each patient.
Motility
Motility was scored by WHO standards under light microscopy (WHO, 1992). Ten microlitres of specimen was placed on a microcell slide (Conception Technologies, San Diego, CA, USA) at 37°C and the total number and motile fraction of spermatozoa assessed and recorded. A total of 250 spermatozoa were assessed.
Vital stains
A combination of two vital stains was used to evaluate sperm viability. Viable cells stain green with carboxyfluoroscein diacetate (CFDA; Sigma) (Garner et al., 1986); non-viable cells stain red with propidium iodide (PI; Sigma) (Garner et al., 1994
). These stains were applied simultaneously with dual fluorescence microscopy for analysis of sperm viability, as originally described by Garner et al. (Garner et al., 1986
).
Initially, all aspirated spermatozoa specimens were washed to eliminate background plasma esterase activity that could react with CFDA. One hundred microliters of aspirated fluid was centrifuged (2040 g, 2 min) and resuspended twice in an equal volume of phosphate buffered saline (0.9%) with 2% SSS. Ten microlitres of a stock solution of CFDA (4 mg CFDA/ml in dimethyl sulphoxide) was added to the washed aspirate and mixed well. After 15 min incubation, 10 µl of PI stock solution (20 mg/ml in phosphate buffered saline) was added. After 5 min incubation, 10 µl of the final mixture was placed on a glass microscope slide (Fisher Scientific, Pittsburgh, PA, USA), and illuminated under fluorescence microscopy using filters specific for fluorescein and propidium iodide respectively.
A viable spermatozoon was defined as a cell that exhibited both intact membranes (exclusion of PI stain) and active cytoplasmic esterase activity (assessed as inclusion of CFDA stain). A total of 250 spermatozoa was assessed. Approximately 1% of the spermatozoa stained with both CFDA and PI, and 1% demonstrated a complete absence of staining. Overall sperm viability by vital stain was defined as the mean viable fraction from both two stains.
Hypo-osmotic swelling assay (HOS)
This assay is based upon the principle that viable cells swell when placed in a hypo-osmotic solution, as previously described (Casper et al., 1996; Esteves et al., 1996
; Oosterhuis et al., 1996
; Smikle and Turek, 1997
). In this procedure, 100 µl aspirate was centrifuged (2040 g, 5 min) and resuspended in either 0.9 ml of 75 mmol/l fructose and 25 mmol/l sodium-citrate solution or isotonic phosphate buffered saline. After a 10 min incubation, 10 µl of each solution was placed on a glass microscope and assessed with light microscopy for the presence of coiled tails. The fraction of viable spermatozoa in each sample was determined by subtracting the percentage of spermatozoa with coiled tails in saline from that observed in HOS solution.
Frozenthawed spermatozoa were similarly assessed. Mean values for fresh and post-thaw viability in each group of patients were compared. Unpaired t-tests were used to assess whether observed differences exhibited statistical significance.
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Results |
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Hypo-osmotic swelling assay (HOS)
As a clinically useful measure of sperm viability, the HOS assay revealed that fresh vasal spermatozoa were the most viable of the three types of spermatozoa tested by a significant margin (P < 0.001 relative to epididymal spermatozoa and P = 0.013 relative to testicular spermatozoa) (Table I). When compared to vital stain as a measure of fresh cell viability, the HOS assay was not as sensitive: for all sperm categories, a mean of 75% of vital stain-viable spermatozoa were detected by HOS assay. However, the HOS assay captured more viable spermatozoa among a fresh population of cells than did motility in all categories of spermatozoa studied (Table I
). This difference in detection was particularly evident with testicular spermatozoa, in which a mean of 5% exhibited motility, but 60% demonstrated HOS viability. When compared to motility results after freezethaw, HOS identified a similar fraction of viable vasal spermatozoa (39 versus 38%), but a significantly higher fraction of viable epididymal (P = 0.03) and testicular (P < 0.001) spermatozoa. As with the vital stain viability results after freezethaw, the HOS assay demonstrated that 4352% of fresh spermatozoa retained viability (4256% with vital stain). In fact, viable spermatozoa were identified by HOS in every aspirated sperm specimen studied, fresh or frozenthawed, regardless of reproductive tract source (data not shown).
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Discussion |
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The data suggest several observations about the viability of fresh, aspirated spermatozoa. Similar to other studies that have assessed the quality of aspirated vasal spermatozoa during vasectomy (Hovatta and von Smitten, 1993; Wen et al., 1993
), we show that vasal spermatozoa from fertile men exhibit motility and viability close to that found in the normal ejaculate. The motility values reported here for epididymal and testicular spermatozoa are also in agreement with those of other investigators (Nagy et al., 1995
; Angelopoulos et al., 1999
). Compared to vasal spermatozoa, testicular spermatozoa from men with non-obstructive azoospermia demonstrate similarly high viability by vital stain and HOS, despite a much lower motile fraction. In fact, these data suggest that the vast majority of spermatozoa from the testis are viable on extraction and usable for ICSI, despite the lack of motility. It also begs the physiological question of whether or not spermatozoa actually die where they are made. Does sperm death largely occur after spermatozoa leave the testis, during traversal of other reproductive tract organs?
If vital staining is taken as the gold standard measure of cell viability, then the data also demonstrate that the HOS assay captures more viable spermatozoa within a population than does the motility test. This was true of all three types of spermatozoa studied. The use of this or another functional assessment of sperm viability may have value in cases of fresh epididymal or testis sperm aspiration procedures in which cell motility is absent. A viability-based selection method is even more critical for cases of frozenthawed aspirated spermatozoa, as cryopreservation further compromises sperm viability.
Aspirated spermatozoa from all three biological reservoirs also showed similar recovery of viability after cryopreservation. This is true when viability is defined by either vital stain or HOS assay. In general, ~4050% of the viable population of fresh aspirated spermatozoa remained viable after freezethaw, similar to the recovery of ejaculated spermatozoa after cryopreservation. This suggests that post-spermiogenetic maturation events that occur in the male reproductive tract do not alter the ability of spermatozoa to withstand the rigours of cryopreservation. In the case of frozenthawed aspirated spermatozoa, motility may significantly underestimate the viable population of spermatozoa potentially usable for ICSI. As noted with fresh spermatozoa, the HOS assay appears to be a more sensitive indicator of sperm viability than motility among frozenthawed aspirated spermatozoa.
In summary, it appears feasible to use fresh and frozenthawed aspirated spermatozoa for IVF and ICSI. We observed decreases in viability with epididymal and testicular spermatozoa after freezethaw that approximated those seen with vasal spermatozoa and after cryopreservation of ejaculated spermatozoa. Although the most commonly applied measure of sperm viability, motility was inferior to the HOS assay in the detection of potentially usable spermatozoa from fresh and frozenthawed aspirated specimens. In addition, the HOS assay detected viable spermatozoa in all aspirated specimens studied, among both fresh and frozenthawed sperm populations. Lastly, the viability of fresh, aspirated testis spermatozoa is very high by vital stain, a finding which may justify its use in ICSI procedures without additional selection measures.
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Notes |
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References |
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Submitted on June 9, 1999; accepted on August 25, 1999.