1 The Center for Reproductive Medicine and Infertility and 2 The James Buchanan Brady Foundation, Department of Urology, The New York HospitalCornell Medical Center, and 3 The Population Council, New York, NY 10021, USA
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: azoospermia/intracytoplasmic sperm injection/microsurgical epididymal sperm aspiration/testicular sperm extraction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Original concerns about ICSI relating to its aggressiveness and arbitrary sperm selection have been eased by reports on the outcome and follow-up of the ICSI newborns (Bonduelle et al., 1994; Palermo et al., 1996a
). The presence of a significant frequency of Y chromosome microdeletions and karyotypic anomalies (de Kretser et al., 1972
; Martin and Rademaker, 1988
; Rucker et al., 1998
) in men with non-obstructive azoospermia has raised concerns about the risks of treatment for these men.
The aetiology of azoospermia can be divided into obstructive and non-obstructive categories. The former is characterized by normal sperm production and is often caused by congenital maldevelopment of the vas deferens, a condition known to be associated with cystic fibrosis gene mutations. The treatment for obstructive azoospermia is microsurgical epididymal sperm aspiration (MESA), and when epididymal access is lacking is testicular sampling appropriate. Non-obstructive azoospermia is characterized by a varying degree of spermatogenic failure and at times is associated with an increased number of chromosomal abnormalities (Reijo et al., 1996; Girardi et al., 1997
). The only method to retrieve spermatozoa from this form of azoospermia is by direct extraction of spermatozoa or germ cells from the testis.
The present study analyses the outcome of 308 consecutive ICSI cycles with spermatozoa surgically retrieved from azoospermic patients, and any possible relationship between the collection method, the aetiology of the azoospermia, and the ICSI outcome. The genetic profile of the patients presenting with obstructive azoospermia was recorded. To assess the safety of the ICSI procedure, the incidence of chromosomal abnormality in the embryos and fetuses and the occurrence of neonatal malformations were evaluated.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Genetic screening
All couples with non-obstructive azoospermia were offered genetic screening with molecular analysis for Y chromosome deletions as well as standard peripheral karyotype analysis. High resolution G-banding was performed on mitotically arrested lymphocytes isolated from peripheral blood (Seabright, 1971; Rucker et al., 1998
). An assessment for Yq microdeletions was performed on DNA of peripheral leukocytes by multiplex PCR (Girardi et al., 1997
).
Men with idiopathic or congenital obstructive azoospermia, including those with congenital bilateral absence of the vas deferens and their female partners were recommended to have cystic fibrosis gene mutation analysis prior to treatment (Schlegel et al., 1995a). Genetic counselling was offered to all couples. Cytogenetic analysis of amniotic fluid was performed by direct culture of cytotrophoblast cells overnight and harvest of chromosomes within 24 h, or after long-term culture of mesenchymal cells with a harvest of chromosomes within 7 days. Giemsa banding (trypsin) staining procedure was subsequently performed on both direct and long-term cultures (Wolf, 1974
).
Semen collection and analysis
Epididymal sperm retrieval
Men with irreparable obstructive azoospermia were offered the choice of MESA (Schlegel et al., 1994; 1995b) or percutaneous testicular retrieval of spermatozoa (Sheynkin and Schlegel, 1997
). For MESA procedures, 15 µl of fluid was aspirated from the lumen of an individual epididymal tubule in the midportion of the obstructed epididymis. Initial puncture with a 300350 µm glass pipette was secured; this fluid was then diluted with 300 µl of human tubal fluid (HTF) medium. Additional proximal punctures of the epididymis were performed until enough spermatozoa of optimum quality were obtained. Because spermatozoa are highly concentrated (often over 1x106/µl) in epididymal fluid, only microlitre quantities are generally required. During 131 attempts at MESA simultaneously with ICSI for obstructive azoospermia, epididymal or vasal spermatozoa were retrieved in 129 procedures (98.5%). Testicular sperm retrieval was performed in one case because of prior removal of the epididymis from a scrotal surgical procedure, and testicular sperm retrieval was performed in a second case to allow microsurgical cross-over vasal reconstruction in the presence of a contralateral non-functioning testis with ipsilateral vasal obstruction. ICSI cases using fresh or cryopreserved epididymal spermatozoa were considered congenital (n = 138) or acquired (n = 103) according to the aetiology of the obstruction. Epididymal fluid was diluted, analysed, and processed by two- or three-layer density gradient centrifugation. After removal of the density gradient medium, a 1 µl aliquot of the final suspension at ~1x106/ml was placed in the injection dish (Palermo et al., 1993
, 1995
).
Cryopreservation of epididymal spermatozoa
Excess epididymal spermatozoa were cryopreserved (n = 112) for later use, thus avoiding the need for repeated microsurgery (Verheyen et al., 1993). An average of 5.5 vials of spermatozoa were obtained per MESA procedure (Brandell et al., 1998a
). The sperm suspension (at a concentration of ~30x106/ml) was diluted v/v with an equal amount of cryopreservation medium (freezing medium: testyolk buffer with glycerol; Irvine Scientific, Irvine, CA, USA). Up to 1 ml aliquots of the final solution were placed in 1 ml cryogenic vials (Nalgene® Brand Products, Rochester, NY, USA). The vials were kept at 20°C for 35 min, exposed to liquid nitrogen vapour at 70°C for 10 min, and then plunged in liquid N2 at 196°C. When required, vials were brought to room temperature to allow thawing. Epididymal samples were processed similarly to fresh semen and when necessary were exposed to a motility enhancer (3.5 mM pentoxifylline) to allow selection of viable spermatozoa (Palermo et al., 1996b
).
Testicular sampling
For men with non-obstructive azoospermia, spermatozoa were retrieved directly from the testis because they do not collect in the epididymis. Typically, multiple biopsies are needed to find the rare spermatogenic foci that are present within the testicles of men with non-obstructive azoospermia (Figure 1). A single open biopsy was obtained from the testis with better histology on diagnostic biopsy, or from the larger testis when a diagnostic biopsy result was not available. Biopsies were performed under optical magnification to identify and preserve the subtunical testicular blood supply, and biopsy incisions were placed in avascular regions. Each biopsy was dispersed and analysed as described below (Schlegel et al., 1997
). If spermatozoa were not retrieved, subsequent biopsies were obtained until either sperm cells were found or no avascular regions of the testis were left. Up to 14 biopsies were required to first identify spermatozoa (Ostad et al., 1998
).
|
|
|
Analysis of retrieved sperm specimens
Sperm concentration and motility were assessed in a sperm counting chamber. The morphology parameters were evaluated and classified according to strict criteria (Kruger et al., 1986).
Ovarian stimulation and oocyte preparation
Oocyte retrieval was performed after pituitary desensitization with gonadotrophin-releasing hormone agonist and ovulation induction with gonadotrophins (Palermo et al., 1995, 1996a
,b
) and the oocytes were exposed to hyaluronidase to remove cumuluscorona cells (Palermo et al., 1995
).
Microinjection procedure and embryological evaluation
The micromanipulation setting, the characteristics of the microtools as well as the details of injection procedure have been previously described (Palermo et al., 1995, 1996b
), as have the selection of the spermatozoon and its immobilizationpermeabilization method (Palermo et al., 1996a
). When no spermatozoa were available, an attempt to retrieve round spermatids was made according to the criteria that we have previously described (Colombero et al., 1997a
). Oocytes were examined 1217 h after the injection procedure for fertilization, defined by the presence of two distinct pronuclei and two clear polar bodies. The evaluation for early embryonic cleavage was performed after an additional 24 h. Morphologically good quality embryos were transferred into the uterine cavity on the third day after the microinjection procedure.
Pregnancy assessment and therapeutic implantation support
Starting on the day of oocyte retrieval, methylprednisolone (16 mg/day) and tetracycline (250 mg every 6 h) were administered for 4 days to all patients. Progesterone administration (2550 mg i.m./day) was started on day 3 after human chorionic gonadotrophin administration and was continued until the establishment of pregnancy.
Statistical analysis
Statistical comparison was carried out by 2 analysis, two-tailed at 5% level of significance using the Statistical Analysis System (SAS Institute, Cary, NC, USA) to evaluate all hypotheses. Where appropriate, Fisher-type adjustments were made to ensure no violations because of small cell counts in
2 procedures. Student's t-test was used to compare the age in the patients where surgically retrieved spermatozoa were used according to the aetiology of the azoospermia and whether samples used were fresh or cryopreserved.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
Evolution of ICSI pregnancies with surgically retrieved spermatozoa
No differences in the pregnancy loss nor in the ongoing pregnancy and delivery rates were observed as a function of the origin of the sample and the aetiology of the azoospermia when the use of fresh and frozen spermatozoa was considered together (Tables VII and VIII). The incidence of miscarriages with surgically retrieved spermatozoa was similar to that noted with the use of ejaculated spermatozoa during a concurrent period of ICSI treatments. None of the fetuses conceived with surgically retrieved spermatozoa was available for cytogenetic analysis, and when this was performed on the amniotic fluid, in 12% (18/150) of such cases, only one abnormal karyotype was observed. Similarly, the incidence of congenital malformation did not vary in relation to the sample origin or the cause of azoospermia (Table VII
). A comparison of the influence of cryopreservation in the epididymal and testicular groups showed no significant difference in terms of pregnancy losses and ongoing pregnancies (Table VIII
). The difference in clinical pregnancy rate observed in the epididymal group was lost when deliveries and ongoing pregnancies were considered (Table VIII
). In both epididymal and testicular spermatozoa, neonatal abnormalities were observed when the fresh sample was used (Table VIII
). In the couples where round cells were injected (n = 14), no normal fertilization was observed. In the four cycles with necrozoospermia, the fertilization rate was 55.2% (16/29). Supravital staining performed on spermatozoa present in the ejaculates of these patients indicated 0% viability. Two clinical pregnancies were established and three healthy newborns were delivered from the two pregnancies.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Non-obstructive azoospermia is associated with a significant risk of failure to retrieve spermatozoa and a variety of genetic risks (Devroey et al., 1995, 1996
; Kahraman et al., 1996
; Friedler et al., 1997
). In our experience, ~40% of men with carefully documented non-obstructive azoospermia were unable to have spermatozoa extracted from the testis despite extensive biopsies of testicular tissue. For these men, simultaneous attempts at sperm retrieval during an IVF cycle may result in retrieved oocytes without spermatozoa to inject. Couples with this risk must consider the options of donor insemination or adoption prior to entering an attempt of sperm retrieval with ICSI.
It has been clearly demonstrated that men with impaired sperm production have a higher prevalence of chromosomal abnormalities (de Kretser et al., 1972). Similarly in this study, we observed chromosomal abnormalities in 20.0% of treated men with non-obstructive azoospermia. The majority were sex chromosomal abnormalities. Sex chromosomal abnormalities were also detectable at a molecular level in the form of Yq microdeletions for 7.8% of men with non-obstructive azoospermia. Although these abnormalities were assessed in leukocytes, chromosomal defects have also been seen in the spermatozoa of infertile men (Martin and Rademaker, 1988
; Colombero et al., 1997a
,b
); thus there is a demonstrated need for careful genetic screening. We have previously noted that the detection of genetic abnormalities has several important roles in the treatment of men with non-obstructive azoospermia (Rucker et al., 1998
). First of all, detection of chromosomal abnormalities allows the performance of preimplantation genetic diagnosis. Secondly, identification of a genetic abnormality provides men with reassurance of the aetiology for low sperm production and eases concerns about personal culpability for the subfertile condition. In addition, 21% of couples where the man has an identifiable genetic abnormality will choose to proceed with a different approach to parenting, including donor insemination or adoption.
In order to most accurately reflect the chances of success for a couple with azoospermia, we have presented the results for initial simultaneous attempts at sperm retrieval with assisted reproduction, as well as the overall results of treatment. Couples were typically treated with simultaneous sperm retrieval and ICSI whether cryopreserved spermatozoa were generally retrieved after a previous ICSI failure. Therefore, results for simultaneous collection, i.e. spermatozoa and oocytes, cannot be fairly compared to cycles using cryopreserved spermatozoa, regardless of sperm source, as a bias toward worse results may occur for frozen cycles due to our treatment approach. Female age may also affect treatment results and can be a confounding factor for results of treatment of male factor infertility. For the series presented here, men with acquired obstructive azoospermia are older than men with congenital obstructive azoospermia at the time of treatment. Therefore, the female partners were older, as reflected by the difference in maternal ages noted for men with obstructive azoospermia (Tables VII and VIII).
Where epididymal spermatozoa were used for ICSI in obstructive azoospermia, the fertilization and pregnancy rates were at least comparable to those obtained contemporarily with ejaculated spermatozoa. Moreover, the origin of the obstruction, congenital or acquired, did not influence ICSI outcome. Cryopreservation had a deleterious effect on sperm motility in this obstructive group, and although it did not affect the fertilization rate presumably it impaired pregnancy outcome.
Fertilization after ICSI with testicular spermatozoa was higher in the obstructive group without affecting the clinical pregnancy rate. Obstructive patients were biopsied because of the failure of epididymal aspiration or the absence of the epididymis. The complete absence of morphologically normal testicular spermatozoa is related to the particular characteristics of these `immature' sperm cells that do not fit with the conventional criteria (Kruger et al., 1986). The good performance of the cryopreserved testicular spermatozoa, although presented here in a small series, may be related to the membrane characteristics of these cells which enable them to sustain the stress of the freezing process (Palermo et al., 1996b
).
Pregnancy loss in couples with surgically retrieved spermatozoa ranged between 4.0 and 15.4%, comparable to that seen with ejaculated spermatozoa. The aetiology of azoospermia did not affect the live birth rate nor was there any relationship to neonatal malformations (Van Steirteghem et al., 1998). The cryopreservation of epididymal and testicular spermatozoa did not increase pregnancy loss nor the ongoing pregnancies and deliveries.
The similarly high pregnancy outcome with surgically retrieved spermatozoa, whether fresh or cryopreserved in men with obstructive azoospermia, suggests that such cryopreservation be used as a routine clinical practice for subsequent attempts. It has been clearly demonstrated that repeating a testicular biopsy within 6 months may decrease the chance of sperm retrieval and increase the risk of permanent testicular devascularization areas (Schlegel et al., 1997). When ejaculated spermatozoa are used, the origin of the sample as well as the semen parameters appeared irrelevant to the ICSI outcome, and this also seems to be true for azoospermic men.
Earlier concerns related to ICSI may now apply to the treatment of azoospermia where spermatozoa are obtained from men with severely abnormal sperm production. Nonetheless, in spite of being associated with a higher frequency of genetic abnormalities, the present results suggest that these conditions can be successfully treated with ICSI without a significant increase in adverse outcomes for progeny. The only factor that clearly depresses the pregnancy rate with ICSI independently of the origin of the spermatozoon is maternal age (Silber et al., 1996).
Even men with Klinefelter's syndrome can produce some fully developed spermatozoa and contribute to successful pregnancies using testicular sperm extraction with ICSI (Palermo et al., 1998). On the other hand, the need for careful genetic screening of patients with severely compromised spermatozoa or azoospermia prior to ICSI treatment is still necessary, as is the monitoring of such pregnancies. In addition, newborns should be carefully evaluated at birth and in the early years of development. With a continued careful approach to follow-up of pregnancy results and offspring, we can thus far confirm the safety of ICSI.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brandell, R.A., Mielnik, A., Liotta, D. et al. (1998a) AZFb deletions predict the absence of sperm with testicular sperm extraction: Preliminary report of a prognostic genetic test. Hum. Reprod., 13, 28122815.
Brandell, R.A., Rosenwaks, Z. and Schlegel, P.N. (1998b) Microsurgical epididymal sperm aspiration (MESA): An effective and successful procedure for obstructive azoospermia. [Abstr. no. P-625.] Fertil. Steril. (Program Supplement) 70, 5323.
Colombero, L.T., Hariprashad, J., Moy, F. et al. (1997a) Impact of abnormal semen parameters on the genetic status of human spermatozoa. [Abstr. no. 11.] J. Andrology (Jan/Feb Suppl.) p. 27.
Colombero, L.T., Hariprashad, J., Moy, F. et al. (1997b) Non-disjunction rates of 18, 21, X and Y chromosomes as a function of ejaculate semen quality. [Abstr. O-170.] Hum. Reprod., 12 (Suppl. 1) 83.[Medline]
de Kretser, D.M., Burger, H.G.G., Fortune, D. et al. (1972) Hormonal, histological, and chromosomal studies in adult males with testicular disorders. J. Clin. Endocrinol. Metab., 35, 392401.[ISI][Medline]
Devroey, P., Liu, J., Nagy, Z., Goossens, A. et al. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum. Reprod., 10, 14571460.[Abstract]
Devroey, P., Nagy, Z., Tournaye, H. et al. (1996) Outcome of intracytoplasmic sperm injection with testicular spermatozoa in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 10151018.[Abstract]
Friedler, S., Raziel, A., Strassburger, D. et al. (1997) Testicular sperm retrieval by percutaneous fine needle aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum. Reprod., 12, 14881493.[Abstract]
Girardi, S.K., Mielnik, A. and Schlegel, P.N. (1997) Submicroscopic deletions in the Y chromosome of infertile men. Hum. Reprod., 12, 16351641.[Abstract]
Kahraman, S., Ozgur, S., Alatas, C. et al. (1996) Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non obstructive azoospermic men. Hum. Reprod., 11, 756760.[Abstract]
Kruger, T.F., Menkveld, R., Stander, F.S.H. et al. (1986) Sperm morphologic features as a prognostic factor in vitro fertilization. Fertil. Steril., 46, 11181123.[ISI][Medline]
Mak, V. and Jarvi, K.A. (1996) The genetics of male infertility. J. Urol., 156, 12451256; discussion 12561257.[ISI][Medline]
Martin, R.H. and Rademaker, A. (1988) The relationship between sperm chromosomal abnormalities and sperm morphology in humans. Mutat. Res., 207, 159164.[ISI][Medline]
Nagy, Z., Liu, J., Cecile, J. et al. (1995) Using ejaculated, fresh, and frozenthawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil. Steril., 63, 808815.[ISI][Medline]
Ostad, M., Liotta, D., Ye, Z. and Schlegel, P.N. (1998) Testicular sperm extraction (TESE) for non-obstructive azoospermia: results of a multibiopsy approach with optimized time dispersion. Urology, 52, 692697.[ISI][Medline]
Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic sperm injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Palermo, G., Joris, H., Derde, M.-P. et al. (1993) Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil. Steril., 59, 826835.[ISI][Medline]
Palermo, G.D., Cohen, J., Alikani, M. et al. (1995) Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil. Steril., 63, 12311240.[ISI][Medline]
Palermo, G.D., Colombero, L.T., Schattman, G.L. et al. (1996a) Evolution of pregnancies and initial follow-up of newborns delivered after intracytoplasmic sperm injection. J. Am. Med. Assoc., 276, 18931897.[Abstract]
Palermo, G.D., Schlegel, P.N., Colombero, L.T. et al. (1996b) Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum. Reprod., 11, 10231029.[Abstract]
Palermo, G.D., Schlegel, P.N., Sills, E.S. et al. (1998) Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter's syndrome. N. Engl. J. Med., 338, 588590.
Pavlovich, C.P. and Schlegel, P.N. (1997) Fertility options after vasectomy: a cost-effectiveness analysis. Fertil. Steril., 67, 133141.[ISI][Medline]
Reijo, R., Alagappan, R.K., Patrizio, P. et al. (1996) Severe oligospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet, 347, 12901293.[ISI][Medline]
Rucker, G.B., Mielnik, A., King, P. et al. (1998) Pre-operative screening for genetic abnormalities in men with non-obstructive azoospermia prior to testicular sperm extraction. J. Urol., 160, 20682071.[ISI][Medline]
Schlegel, P.N. and Su, L.-M. (1997) Physiologic consequences of testicular sperm extraction. Hum. Reprod., 12, 16881692.[Abstract]
Schlegel, P., Berkeley, A., Goldstein, M. et al. (1994) Epididymal micropuncture with in vitro fertilization and oocyte micromanipulation for the treatment of unreconstructable obstructive azoospermia. Fertil. Steril., 61, 895901.[ISI][Medline]
Schlegel, P.N., Cohen, J., Goldstein, M. et al. (1995a) Cystic fibrosis gene mutations do not affect sperm function during in vitro fertilization with micromanipulation for men with bilateral congenital absence of vas deferens. Fertil. Steril., 64, 421426.[ISI][Medline]
Schlegel, P.N., Palermo, G. D., Alikani, M. et al. (1995b) Micropuncture retrieval of epididymal sperm with IVF: importance of in vitro micromanipulation techniques. Urology, 46, 238241.[ISI][Medline]
Schlegel, P.N., Palermo, G.D., Goldstein, M. et al. (1997) Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia. Urology, 49, 435440.[ISI][Medline]
Schlegel, P.N., Su, L.-M. and Li, P.S. (1998) Gonadal sperm retrieval: potential for testicular damage in non-obstructive azoospermia. In Filicori, M. and Flamigni, C. (eds), Treatment of Infertility: The New Frontiers. Communications Media for Education, New Jersey, pp. 383392.
Schoysman, R., Vanderzwalmen, P., Nijs, M. et al. (1993) Pregnancy after fertilisation with human testicular spermatozoa. Lancet, 342, 1237.
Seabright, M. (1971) A rapid banding techniques for human chromosomes. Lancet, 731, 971972.
Sheynkin, Y.R. and Schlegel, P.N. (1997) Sperm retrieval for assisted reproductive technologies. Contemp. Urol., 9, 2136.
Silber, S.J., Van Steirteghem, A., Nagy, Z. et al. (1996) Normal pregnancies resulting from testicular sperm extraction and intracytoplasmic sperm injection for azoopsermia due to maturation arrest. Fertil. Steril., 66, 110117.[ISI][Medline]
Sinclair, A.H., Berta, P., Palmer, M.S. et al. (1990) A gene from the human sex-determining region encoded a protein with homology to a conserved DNA-binding motif. Nature, 346, 240244.[ISI][Medline]
Tournaye, H., Devroey, P., Liu, J. et al. (1994) Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil. Stertil., 61, 10451051.[ISI][Medline]
Van Steirteghem, A.C., Liu, J., Joris, H. et al. (1993) Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum. Reprod., 8, 10551060.[Abstract]
Van Steirteghem, A., Bonduelle, M., Hamberger, L. et al. (1998) Assisted reproduction by intracytoplasmic sperm injection: a survey on the clinical experience in 1994 and the children born after ICSI, carried out until 31 December 1993. Hum. Reprod., 13, 17371746.[Abstract]
Verheyen, G., Pletinck, I., Van Steirteghem, A.C. (1993) Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm. Hum. Reprod., 8, 16781684.[Abstract]
Wolf, U. (1974) Culture and preparation of cells from amniotic fluid. In Schwarzacher, H.G. and Wolf, U. (eds), Cytogenics. Springer, Berlin, pp. 6769.
Submitted on May 5, 1998; accepted on November 24, 1998.