The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation

N.E. Bachtell1, J. Conaghan2 and P.J. Turek1,3

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 94115–1695, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testicular and epididymal spermatozoa are routinely used with in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to achieve pregnancies. In addition, excess cryopreserved spermatozoa can be thawed and used for ICSI. However, information on the recovery of epididymal and testicular spermatozoa after freeze–thaw is lacking. This is important to determine the feasibility of using previously cryopreserved aspirated spermatozoa for ICSI. We prospectively compared the viability of fresh and frozen–thawed spermatozoa from the vas deferens, epididymis and testicle by several measures. Testis spermatozoa were obtained from men with non-obstructive azoospermia (n = 5), epididymal spermatozoa from men with obstructive azoospermia (n = 8), and vasal spermatozoa from fertile men by vasal irrigation at vasectomy (n = 5). The viability of fresh spermatozoa was assessed by motility, two vital stains (carboxyfluorescein, 0.08 mg/ml and propidium iodide, 20 mg/ml) and the hypo-osmotic swelling assay (HOS; 100 mmol/l citrate and fructose). After cryopreservation, spermatozoa were thawed and all viability measures repeated. Although fresh vasal spermatozoa were the most motile, testicular spermatozoa exhibited similar, high viability (91 and 86% respectively) by vital stain. Spermatozoa from testis, epididymis and vas deferens survived cryopreservation equally well by vital stain, but not by motility. As a selection measure, the HOS assay identified significantly more viable epididymal and testicular spermatozoa than did motility in both fresh and frozen–thawed populations. It appears feasible to use frozen–thawed extracted spermatozoa for ICSI when motility and a selection measure such as the HOS assay are used. With fresh testis spermatozoa, selection methods may not be necessary prior to ICSI, as cell viability is high.

Key words: cryopreservation/epididymis/hypo-osmotic swelling/sperm viability/testis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mature, viable spermatozoa retrieved from the male reproductive tract are routinely used for in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). With testicular spermatozoa from either obstructed or non-obstructed testes, excellent oocyte fertilization and pregnancy rates are possible (Hovatta et al., 1995Go; Schoysman et al., 1995; Devroey et al., 1995Go; Tournaye et al., 1996Go; Kahraman et al., 1996Go; Cha et al., 1997Go; Schlegel et al., 1997Go; Friedler et al., 1997aGo; Turek et al., 1999Go). Equal or even greater success has been obtained with aspirated epididymal spermatozoa and ICSI (Silber et al., 1990Go; Schlegel et al., 1994Go; Tournaye et al., 1994Go; Oates et al., 1996Go; Cha et al., 1997Go; Nudell et al., 1998Go). The successful use of freshly aspirated spermatozoa with ICSI led to the use of cryopreserved–thawed spermatozoa. The addition of sperm cryopreservation is valuable as it may reduce the number of procedures required for pregnancy success (Oates et al., 1996Go; Nudell et al., 1998Go) as well as minimize the inconvenience associated with sperm retrieval performed concurrently with ovulation induction (Oates et al., 1996Go).

The published experience with frozen–thawed epididymal spermatozoa and ICSI suggests that fertilization and pregnancy success rates are comparable to those achieved with freshly retrieved spermatozoa (Nagy et al., 1995Go; Oates et al., 1996Go; Holden et al., 1997Go; Hutchon et al., 1998Go). It seems that comparable success may be achieved with frozen–thawed and fresh testis spermatozoa (Friedler et al., 1997bGo; Liu et al., 1997Go; Oates et al., 1997Go; DeCroo et al., 1998Go; Marmar 1998Go; Perraguin-Jayot et al., 1998Go). In summary, these data suggest that it is certainly possible to use frozen–thawed testicular and epididymal spermatozoa for ICSI. However, there is scarce information on whether it is, in fact, feasible to use frozen–thawed 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 frozen–thawed spermatozoa from several sites within the male reproductive tract. This information may help determine the feasibility of using frozen–thawed spermatozoa for ICSI.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aspirated spermatozoa from three groups of patients who received care at UCSF Male Fertility Clinic were evaluated. Spermatozoa were retrieved either for clinical use or for the purpose of the study, and all specimens underwent motility and viability analyses before and after cryopreservation. The study received prior approval from the university institutional review board.

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., 1998Go). 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 frozen–thawed 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., 1986Go); non-viable cells stain red with propidium iodide (PI; Sigma) (Garner et al., 1994Go). These stains were applied simultaneously with dual fluorescence microscopy for analysis of sperm viability, as originally described by Garner et al. (Garner et al., 1986Go).

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., 1996Go; Esteves et al., 1996Go; Oosterhuis et al., 1996Go; Smikle and Turek, 1997Go). 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.

Frozen–thawed 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sperm motility
As expected, the motility of freshly aspirated sperm specimens varied widely, depending on the organ from which it was aspirated (Table IGo). Sperm aspirated from the vas deferens of fertile men showed significantly higher motility (71 ± 16) than that derived from the epididymis of men with obstructive azoospermia (22 ± 18) and from the testis of men with non-obstructed azoospermia (5 ± 3.6) (P < 0.001 and P < 0.001 respectively, unpaired t-test). Interindividual variability in spermatozoa motility was greatest with epididymal spermatozoa derived from patients with obstructive azoospermia and least variable among the testis spermatozoa retrieved from patients with non-obstructive azoospermia. Cryopreservation had a more profound effect on the motility of testicular spermatozoa than on either epididymal or vasal spermatozoa (Table IIGo). The motility of fresh testicular spermatozoa fell by 96% after the cryopreservation, whereas vasal sperm motility decreased 46% after freeze–thaw.


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Table I. Motility and viability of freshly aspirated and frozen–thawed spermatozoa from three sites in the male reproductive tract measured before and after freezing
 

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Table II. Fraction of viable spermatozoa recovered after thaw as assessed by three measures of viability
 
Vital stain viability
Overall, the fraction of freshly aspirated spermatozoa that demonstrated viability by vital stain was quite high, regardless of sperm source (Table IGo). The mean viability of fresh testicular spermatozoa was similar to that of vasal spermatozoa, but significantly higher than that of epididymal spermatozoa (P = 0.001, unpaired t-test). The sperm viability recovered after freeze–thaw was similar among the three types of aspirated spermatozoa, and ranged from 42–56%, as shown in Table IIGo.

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 IGo). 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 IGo). 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 freeze–thaw, 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 freeze–thaw, the HOS assay demonstrated that 43–52% of fresh spermatozoa retained viability (42–56% with vital stain). In fact, viable spermatozoa were identified by HOS in every aspirated sperm specimen studied, fresh or frozen–thawed, regardless of reproductive tract source (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite convincing evidence that aspirated spermatozoa from the epididymis and testicle provide excellent fertilization and pregnancy success with IVF and ICSI, little research has addressed the actual feasibility of using spermatozoa from these sources (Nagy et al., 1995Go). This study attempts to delineate the viability parameters associated with fresh and frozen–thawed aspirated spermatozoa from the epididymis and testis. Aspirated vasal spermatozoa from fertile men were used as control groups in this assessment. Obviously, these data cannot be used to assess how motility changes as spermatozoa progress through the various organs in the male reproductive tract because spermatozoa were aspirated from infertile men who have different aetiologies as a cause of the infertility. However, valuable information about the utility of using these biological reservoirs of spermatozoa can be obtained through such an examination.

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, 1993Go; Wen et al., 1993Go), 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., 1995Go; Angelopoulos et al., 1999Go). 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 frozen–thawed 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, ~40–50% of the viable population of fresh aspirated spermatozoa remained viable after freeze–thaw, 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 frozen–thawed 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 frozen–thawed aspirated spermatozoa.

In summary, it appears feasible to use fresh and frozen–thawed aspirated spermatozoa for IVF and ICSI. We observed decreases in viability with epididymal and testicular spermatozoa after freeze–thaw 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 frozen–thawed aspirated specimens. In addition, the HOS assay detected viable spermatozoa in all aspirated specimens studied, among both fresh and frozen–thawed 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.


    Notes
 
3 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on June 9, 1999; accepted on August 25, 1999.