1 Fertility Clinic and 2 Laboratory of Biology and Psychology of Human Fertility Erasmus Hospital French Speaking Free University of Brussels 808, Brussels, Belgium
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: in-vitro culture/obstructive azoospermia/testicular sperm extraction/testicular sperm cryopreservation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Testicular biopsy preparation
Biopsy samples were placed into a Petri dish and first washed in 20 mmol/l HEPES-buffered modified Earl's balanced salt solution (HEBSS), supplemented with 0.5% human albumin (Belgian Red Cross, Brussels, Belgium), 0.43 mmol/l sodium pyruvate (Sigma Chemical Co., St Louis, MO, USA) and 44 mmol/l sodium lactate (Sigma). The biopsies were mechanically dissociated with two sterile scalpels. The dissociated tissue was emptied on a 40 µm Falcon (Becton-Dickinson & Co., Meylan Cedex, France) cell strainer, placed onto a 50 ml conical Falcon tube and centrifuged at 300 g for 10 min. The pellet was then resuspended in 1 ml of modified Earl's balanced salt solution (EBSS) buffered with 28 mmol/l sodium bicarbonate, supplemented with sodium pyruvate, lactate and 20% inactivated serum (35 min at 56°C), obtained from the same patient. The pellet was filtered through a 35 µm Falcon cell strainer and re-centrifuged. The pellet was resuspended to a final volume of 100 µl.
Testicular biopsy culture
After retrieval, testicular samples from group I patients were immediately frozen, with a quantity of tissue also being retained for research purposes at the same time. Two portions of this tissue were placed in culture, one at 37°C and one at 32°C. Culture was carried out in Nunc (Roskilde, Denmark) four-well dishes containing 1 ml of EBSS and 20% serum, in a humidified atmosphere containing 5% CO2 and 5% O2. The percentage of motile spermatozoa was assessed daily by counting 100200 spermatozoa. At 72 h intervals, 0.5 ml of the top layer of the medium was removed and replaced with the same volume of fresh EBSS.
Testicular biopsy freezing
The wells containing testicular spermatozoa were each emptied into a 5 ml Falcon tube and washed with extra EBSS to remove remaining cells. The tube was centrifuged for 10 min at 300 g, and the pellet resuspended in a small volume of EBSS. The same volume of 15% glycerol (Sigma)20% egg yolk (Difco, Grayson, GA, USA) buffer, which is used routinely for the cryopreservation of ejaculated spermatozoa was added. After 10 min of equilibration, 0.15 ml IMV straws (L'Aigle, France) were charged and cryopreserved (using a Kryo 10 Planer, Middlesex, UK), according to the following curve: from 22°C to 5°C at 1°C/min, from 5°C to 80°C at 10°C/ min, and from 80°C to 130°C at 25°C/min; the straws were then plunged in liquid nitrogen. One portion of the specimens retained for research purposes was thawed by leaving the straws at room temperature for a few minutes. The cryoprotectant was removed by a centrifugation step in EBSS, after which the samples were placed in culture at 37°C in a humidified atmosphere under 5% CO2 and 5% O2 for 24 h. At least 100 spermatozoa were counted on the day of thawing, and 24 h later the percentage of motile spermatozoa was calculated.
In group II, one portion of each sample was frozen on the day of the biopsy (day 0), and one portion after being cultured for 72 h (day 3) at 37°C. In both cases the percentage motility was determined before freezing. Some of the samples retained for research (frozen using both strategies) were thawed, and post-thaw motility and recovery rate were determined. The percentage recovery rate was defined as the ratio of motile spermatozoa after thawing to motile spermatozoa in the fresh sample. Pre-freezing and post-thaw motility and the recovery rates in samples frozen on day 0 and day 3 were compared.
Statistical analysis
Statistical analysis was performed using linear regression, Student's t-test to compare means in the case of normal distributions, and the MannWhitney test to compare means in the case of non-normal distributions. A SPSS software package was used. Statistical significance was defined as P < 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism implicated in the observed improvement of testicular sperm motility after a short culture period remains unexplained, but it is probably mediated by a complex interaction between somatic and germinal compartments (Jutte et al., 1985; Dym, 1994
; Foucault et al., 1994
; Albanesi et al., 1996
; Chen et al., 1997
; Cudicini et al., 1997
). In a recent study (Tesarik et al., 1998
), it was shown that developmental changes could occur in culture of human testicular samples where germ cells lack a direct contact with Sertoli cells, indicating that the interaction is mediated by humoral factors. This necessary interaction could also explain the lack of improvement in the motility of cultured epididymal spermatozoa (Edirisinghe et al., 1996
; our unpublished data). The similar motility improvement of cultured spermatozoa induced by two different temperatures suggests that it is not the temperature change that is the principal factor inducing this improvement in sperm motility, but rather the dilution of one or more inhibitory factors that occurs during the washing of samples for processing before culture.
Several authors have demonstrated that performing ICSI with fresh or frozen spermatozoa gives similar results (Gil-Salom et al., 1996; Friedler et al., 1997
; Liu et al., 1997
; Oates et al., 1997
), and that freezing does not affect the spermatozoa (Lin et al., 1998
). Consequently, some groups have attempted to improve the protocols for testicular sperm freezing (Allan and Cotman, 1997
; Crabbe et al., 1999
; Gianaroli et al., 1999
). When testicular spermatozoa were cultured in vitro after thawing, either a slight increase in the total number of motile spermatozoa was observed (Edirisinghe et al., 1996
), or a significant improvement in the number of progressively motile spermatozoa was reported after 72 h of culture (Liu et al., 1997
). In the first part of this study, thawed testicular spermatozoa frozen at retrieval were cultured for a 24 h period, and a decrease in motility was observed thereafter. The reason for this might be the destruction of somatic testicular cells. Consequently, the substitution of a glycerol-based protocolwhich is designed for ejaculated spermatozoawith a protocol adapted for somatic cell freezing may provide better results.
A previous attempt has been made to culture testicular samples for a few hours before freezing (Aslam and Fishel, 1998). In our case, the period of culture was extended to 72 h, the time at which we observed the peak in percentage of motile spermatozoa. The percentage of motile spermatozoa in samples frozen after 72 h of culture was significantly higher than that in samples frozen at retrieval and, moreover, a significantly higher recovery rate was obtained in the specimens cultured for 72 h prior to freezing. Amongst in-vitro-matured frozen spermatozoa, 27% were in fact still motile after thawing, compared with the 15% observed from non-matured frozen samples (P < 0.01), indicating a stronger resistance of cultured spermatozoa to freezing. A prospective randomized study, with a larger series of patients, will be necessary to compare the clinical outcome of the two freezing strategies.
In conclusion, these data indicate that a more rational management of testicular samples, in the case of obstructive azoospermia, consists of improving sperm motility of fresh samples by 3 days of culture, before ICSI or before freezing. This would allow the biopsy to be taken 23 days before oocyte retrieval, it would ease the selection of motile spermatozoa for ICSI, and it would reduce the volume of material necessary per cycle, offering the possibility for several further attempts with one single sample.
![]() |
Notes |
---|
3 To whom correspondence should be addressed at: Fertility Clinic, Department of Obstetrics and Gynaecology, Erasmus Hospital, Route de Lennik 808, 1070 Brussels, Belgium. E-mail: semilian{at}ulb.ac.be
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Allan, J. and Cotman, A. (1997) A new method for freezing testicular biopsy sperm: three pregnancies with sperm extracted from cryopreserved sections of seminiferous tubule. Fertil. Steril., 68, 741744.[ISI][Medline]
Angelopoulos, T., Adler, A., Krey, L. et al. (1999) Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue. Fertil. Steril., 71, 240243.[ISI][Medline]
Aslam, I. and Fishel, S. (1998) Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception. Hum. Reprod., 13, 634638.[Abstract]
Balaban, B., Bulent, U., Sertac, A. et al. (1999) In vitro culture of spermatozoa induces motility and increases implantation and pregnancy rates after testicular sperm extraction and intracytoplasmic sperm injection. Hum. Reprod., 14, 28082811.
Chen, Y., Dicou, E. and Djakiew, D. (1997) Characterisation of nerve growth factor precursor protein expression in fat round spermatid and the tropic effects of nerve growth factor in the maintenance of Sertoli cell viability. Mol. Cell. Endocrinol., 127, 129136.[ISI][Medline]
Crabbe, E., Verheyen, G., Tournaye, H. and Van Steirteghem, A. (1999) Freezing of testicular tissue as a minced suspension preserves sperm quality better than whole-biopsy freezing when glycerol is used as cryoprotectant. Int. J. Androl., 22, 4348.[ISI][Medline]
Craft, I., Tsirigotis, M. and Zhu, J. (1995) In vitro culture of testicular sperm [letter]. Lancet, 346, 1438.
Cudicini, C., Lejeune, H., Gomez, E. et al. (1997) Human Leydig cells and Sertoli cells are producers of interleukins-1 and -6. J. Clin. Endocrinol. Metab., 82, 14261433.
Dym, M. (1994) Spermatogonial stem cells of the testis. Proc. Natl Acad. Sci. USA, 91, 1128711289.
Edirisinghe, W.R., Junk, S.M., Matson, P.L. et al. (1996) Case report: changes in motility pattern during in-vitro culture of fresh and frozen thawed testicular and epididymal spermatozoa: implications for planning treatment by intracytoplasmic sperm injection. Hum. Reprod., 11, 24742476.[Abstract]
Foucault, P., Drosdowsky, M.A. and Carreaun, S. (1994) Germ cell and Sertoli cell interactions in human testis: evidence for stimulatory and inhibitory effects. Hum. Reprod., 11, 20622068.
Friedler, S., Raziel, A., Soffer, Y. et al. (1997) Intracytoplasmic sperm injection of fresh and cryopreserved testicular spermatozoa in patients with non-obstructive azoospermia a comparative study. Fertil. Steril., 68, 892897.[ISI][Medline]
Gianaroli, L., Magli, M.C., Selman, H.A. et al. (1999) Diagnostic testicular biopsy and cryopreservation of testicular tissue as an alternative to repeated surgical openings in the treatment of azoospermic men. Hum. Reprod., 14, 10341038.
Gil-Salom, M., Romero, J., Minguez, Y. et al. (1996) Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Hum. Reprod., 11, 13091313.[Abstract]
Hu, Y., Maxon, W.S., Hoffman, D.I. et al. (1999) Clinical application of intracytoplasmic sperm injection using in-vitro cultured testicular spermatozoa obtained in the day before egg retrieval. Fertil. Steril., 72, 666669.[ISI][Medline]
Jutte, N., Jansen, J.R., Grootegoed, J. et al. (1985) Regulation of survival of rat pachytene spermatocytes by lactate supply from Sertoli cells. J. Reprod. Fertil., 62, 399405.
Lacham-Kaplan, O. and Trounson, A. (1993) The effects of the sperm motility activators 2-deoxyadenosine and pentoxifylline used for sperm microinjection on mouse and human embryo development. Hum. Reprod., 8, 945952.[Abstract]
Lin, M.H., Morshedi, M., Srisombut, C. et al. (1998) Plasma membrane integrity of cryopreserved human sperm: an investigation of the results of the hypoosmotic swelling test, the water test, and eosin-Y staining. Fertil. Steril., 70, 11481155.[ISI][Medline]
Liu, J., Garcia, E. and Baramki, A. (1996) The difference in outcome of in-vitro culture of human testicular spermatozoa between obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 15871588.
Liu, J., Tsai, Y. and Katz, E. (1997) Outcome of in-vitro culture of fresh and frozen thawed human testicular spermatozoa. Hum. Reprod., 12, 16671672.[Abstract]
Oates, D.R., Mulhall, J., Burgess, C. et al. (1997) Fertilisation and pregnancy using intentionally cryopreserved testicular tissue as the sperm source for intracytoplasmic sperm injection in 10 men with non-obstructive azoospermia. Hum. Reprod., 12, 734739.[Abstract]
Tesarik, J., Greco, E., Rienzi, L. et al. (1998) Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum. Reprod., 13, 27722781.
Urman, B., Alatas, C. and Aksoy, S. (1998) Performing testicular or epididymal sperm retrieval prior to the injection of hCG. J. Assist. Reprod. Genet., 15, 125128.[ISI][Medline]
Zhu, J., Tsirigotis, M., Pelekanos, M. et al. (1996) In-vitro maturation of human testicular spermatozoa. Hum. Reprod., 11, 231232.[ISI][Medline]
Submitted on May 8, 2000; accepted on July 28, 2000.