In-vitro fertilization treatment for severe male factor: the fertilization potential of immotile spermatozoa obtained by testicular extraction

Adrian Shulman1,3, Baruch Feldman1, Igael Madgar2, Jacob Levron1, Shlomo Mashiach1 and Jehoshua Dor1

1 The IVF Unit, Department of Obstetrics and Gynecology and 2 The Andrology Unit, Department of Urology, The Chaim Sheba Medical Center, Tel Hashomer (affiliated with Sackler Faculty of Medicine, Tel Aviv University), Israel


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A retrospective analysis in 50 couples of 53 cycles of intracytoplasmic sperm injection (ICSI) with immotile spermatozoa from testicular-retrieved spermatozoa was performed to evaluate whether total immotile spermatozoa achieved after testicular sperm extraction could fertilize ova and result in pregnancies. We assessed the efficacy of ICSI with totally immotile testicular spermatozoa extracted from the testes of azoospermic patients with severe spermatogenic failure (group 1) and compared these results with those from spermatozoa which were recovered after several hours of incubation and were motile (group 2) at the time of injection. In 19 cycles, only totally immotile spermatozoa were injected at the time of ICSI. For the remaining 34 cycles, at least one motile spermatozoon was found for injection. The oocyte fertilization rates were 51% for group 1 and 62% for group 2 (P < 0.02). Eighteen of 19 cycles in group 1 (90%) and all 34 (100%) cycles in group 2 had embryos for replacement. The mean number of embryos per cycle was 5.2 ± 0.8 and 7.5 ± 0.9 in groups 1 and 2 respectively; this and the embryo quality (cumulative embryo scoring = 40 ± 8 for group 1 and 50 ± 7 for group 2), and clinical pregnancy rates (15.8% per oocyte retrieval in group 1 and 23.5% in group 2) were not significantly different between groups. Fertilization, cleavage and pregnancy can be achieved with intracytoplasmic testicular sperm injection from patients with immotile spermatozoa, at levels comparable with those of ICSI using motile spermatozoa.

Key words: infertility/intracytoplasmic sperm injection/in-vitro fertilization/male factor/testicular-retrieved spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Initially, intracytoplasmic sperm injection (ICSI) was performed in couples with repeated failed fertilization (Palermo et al., 1992Go), following standard in-vitro fertilization (IVF). It soon became clear that ICSI could also be used for couples in which the male partner has a limited number of spermatozoa in the ejaculate, or for epididymal or testicular spermatozoa (Madgar et al., 1996Go). Moreover, it is now clear that none of the conventional semen parameters has any influence on the results of ICSI from ejaculated spermatozoa, except when totally immotile spermatozoa are available for the microinjection procedure (Nagy et al., 1995Go). Less is known about the features of testicular-retrieved spermatozoa and ICSI outcome.

In this study, we evaluated the performance of testicular immotile spermatozoa by comparing their results after ICSI with those of testicular motile spermatozoa.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ICSI was introduced into our IVF programme in April 1993 and, since 1994, we have used the technique after testicular biopsies in patients suffering from non-obstructive azoospermia. In this study, we included all the testicular retrieval procedures from January 1995 in which immotile spermatozoa were found at the initial screening, because by this time the laboratory procedures were well established and beyond the learning curve.

The female patients received ovarian stimulation with gonadotrophin-releasing hormone analogue (GnRHa; Decapeptyl Depot 3.75 mg, C.R.Ferring, Malmo, Sweden) long protocol (injected either at mid luteal phase or on the first day of the menstrual cycle), human menopausal gonadotrophin (HMG; Pergonal 225 IU, Teva Pharmaceuticals Ltd, Petach Tikva, Israel) or follicle stimulating hormone (FSH; Teva Pharmaceuticals Ltd), and luteinizing hormone (LH; Teva Pharmaceuticals Ltd), commencing 15 days after GnRH administration (Dor et al., 1990Go). The cycle monitoring has also been described by Dor et al. (1990).

The testicular sperm retrievals were always performed on the day of ovum retrieval under general anaesthesia, anticipating the female procedure. The surgical technique for testicular sperm retrieval was performed as described by Silber et al. (1995). A horizontal incision measuring 2 cm was made in the scrotal skin, and carried through the peritoneal tunica vaginalis. The testis was withdrawn and a small incision albuginea was made. A small piece of extruding testicular tissue was excised, and placed in a Petri dish containing IVF medium (human tubal fluid; Irvine Scientific, Santa Ana, CA, USA). If no spermatozoa were found in the first specimen of testicular tissue, biopsy was repeated in other areas of the testis. A maximum of four biopsies was performed on each testis to prevent vascular damage.

Testicular specimens obtained were gently crushed under direct microscopy. The number of retrieved spermatozoa and their motility status were recorded, as well as the motility status at the time of injection into the oocyte.

The ICSI procedure was performed according to the method described by Palermo et al. (1992), and was virtually identical with that of the injection of motile spermatozoa, including the step of immobilization. Fertilization status of the oocyte was assessed 16–18 h after the injection. Fertilization was considered normal when two pronuclei were present. Up to five embryos were placed in the uterine cavity 44–48 h after the microinjection procedure. Numbers of blastomeres and embryo morphological quality were noted. Any additional embryos of good morphological quality were cryopreserved for possible later use. Pregnancy was confirmed by detecting rising ß-HCG concentrations (on two or more occasions) at least 12 days after embryo replacement. Clinical pregnancies were diagnosed only when a gestational sac and fetal pole with a beating heart were noted at ultrasonography.

Statistical analysis
Statistical significance was tested using Student's t-test, {chi}2 test and Fisher's exact test, as appropriate. Statistical differences were considered significant at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study comprised a total of 50 patients from 53 treatment cycles. In 19 cycles, all the injected spermatozoa were totally immotile at the time of ICSI (group 1). For the remaining 34 cycles (group 2), at least one motile spermatozoon was injected.

The relevant baseline parameters of couples in the two groups are summarized in Table IGo. The only significant difference was the higher FSH concentrations among males in the immotile spermatozoa group (group 1) (P = 0.02). No significant differences were noted between the two groups with respect to ovarian stimulation and number of eggs recovered (Table IIGo).


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Table I. Baseline parameters of the couples from the two groupsa
 

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Table II. Details of ovarian stimulation cycles and azoospermia
 
Although a higher number of embryos appeared to be available in group 2 couples, the difference did not achieve significance (Table IIIGo). Furthermore, the transferred embryos were of comparable morphology.


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Table III. ICSI procedure resultsa
 
The clinical pregnancy rates were not significantly different at 15.8% per transferred cycle for the immotile group of patients and 23.5% for the motile group (Table IVGo). This was after a statistically higher fertilization rate was obtained when motile spermatozoa were injected (P = 0.02). In several cycles where embryos were derived from motile spermatozoa, embryos were also obtained after injection of immotile spermatozoa. Whenever the total number of embryos obtained from motile spermatozoa was low (one or two maximum), and if for the same patient we had also achieved embryos from immotile spermatozoa, then some were transferred (in addition). Therefore, after analysing these data, we proceeded to a further dichotomization of the cycles, in which group 2 patients (motile sperm) was further divided into two subgroups (Table VGo). The first subgroup (group 3) contained cycles where mixtures of motile and immotile spermatozoa were available for injection, whereas the second subgroup (group 4) included cycles in which only motile spermatozoa were injected. The cycle characteristics and the outcomes with these new groupings are presented in Table VGo. There were no significant differences in the rates of fertilization, embryo implantation or pregnancy between patients in group 1 and those in the two subgroups (3 and 4).


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Table IV. Cycle results
 

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Table V. Cycle results for patient subgroups
 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
ICSI has now been accepted by many clinicians as the method of choice to overcome the infertility of patients with severe male factor. Since Devroey et al. (1995) demonstrated the presence of spermatozoa in patients with Sertoli cell-only or tubal sclerosis, it is logical to attempt sperm retrieval in patients with germinal failure and azoospermia, even in the presence of high FSH concentrations. Patients with immotile spermatozoa (group 1) had significantly higher FSH concentrations (Table IGo) than those with motile spermatozoa (group 2). This may be indicative of a higher degree of testicular failure, leading to the production of morphologically abnormal spermatozoa. The purpose of this study was to evaluate fertilization and implantation potential of the resultant embryos from immotile spermatozoa which had been released from the Sertoli cells. Kahraman et al. (1997) were the first to report a pregnancy and delivery of a healthy baby after injection of testicular immotile spermatozoa. Like Nagy et al. (1998), our results showed that although the fertilization rate was higher for motile spermatozoa (Table IVGo), the number of embryos available for transfer and the implantation potential of the resulting embryos did not differ significantly between the two groups. The implantation rate of embryos resulting from the injection of motile spermatozoa (group 4, Table VGo) was 8.8%, which we feel is acceptable for testicular sperm extraction cycles. The apparently lower implantation rate achieved by embryos resulting from the injection of immotile spermatozoa (group 1, Table VGo) may be due to a higher incidence of chromosomal abnormalities. The comparable implantation rates per embryo in both groups, and the quality of embryos as assessed by the embryo scoring system, indicated that immotile testicular spermatozoa could be used successfully.

We have found, like others (Dozortsev et al., 1995Go; Parrington et al., 1996Go), that tail `hitting' is essential in all ICSI procedures, including when using immotile spermatozoa. This may be due to the effect of the procedure on destabilization of the plasma membrane, with the release of an activating factor into the ooplasm. Nijs et al. (1996) have reported on pregnancies after ICSI with totally immotile spermatozoa. Although they obtained fertilization with immotile spermatozoa of various origins, ongoing pregnancies were obtained only with immotile spermatozoa originating from the testis, and not from the ejaculate. Although there was a trend for higher fertilization rates, embryo implantation and pregnancy rates for cycles where embryos from motile-only spermatozoa (group 4) were used than for cycles where immotile-only spermatozoa (group 1) were used, the numbers were small and no statistical differences were found (Table VGo). For comparative purposes, our routine success rates are 24% and 17.7% for ongoing pregnancies per embryo transfer in patients with obstructive and non-obstructive azoospermia respectively (Madgar et al., 1998Go). At the time of this study, we did not use the controversial hypo-osmotic swelling test (Barros et al., 1997Go) since many live spermatozoa were known to become non-viable after 30 min of incubation in the hypo-osmotic solution (Tsai et al., 1997Go). In addition, a low fertilization rate was reported by Casper et al. (1996) after using the hypo-osmotic swelling test protocol for ICSI. Recently, Liu et al. (1997) reported on a `promising' modified hypo-osmotic swelling test using 150 mOsm NaCl solution.

We conclude that immotile testicular spermatozoa recently released from the Sertoli cells can successfully fertilize mature oocytes, resulting in a normal pregnancy. In cases with totally immotile spermatozoa from the ejaculate, we recommend performing a testicular sperm retrieval procedure. However, if only immotile spermatozoa are still found, then those retrieved from the testis should be injected.


    Notes
 
3 To whom correspondence should be addressed Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Barros, A., Sousa, M., Angelopoulos, T. and Tesarik, J. (1997) Efficient modification of intracytoplasmic sperm injection technique for cases with total lack of sperm movement. Hum. Reprod., 12, 1227–1229.[ISI][Medline]

Casper, R.F., Meriano, J.S., Jarvi, K.A. et al. (1996) The hypo-osmotic swelling test for selection of viable sperm injection in men with complete asthenospermia. Fertil. Steril., 65, 972–976.[ISI][Medline]

Devroey, P., Liu, J., Nagy, Z. et al. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm extraction in non-obstructive azoospermia. Hum. Reprod., 10, 1457–1460.[Abstract]

Dor, J., Ben Shlomo, S. and Lipitz, S. (1990) Ovarian stimulation with gonadotropin releasing hormone (GnRH) analog improves the in vitro fertilization (IVF) pregnancy rate with both transvaginal and laparoscopic oocyte recovery. J. In Vitro Fertil. Embryo Transfer, 7, 351–354.[ISI][Medline]

Dozortsev, D., Rybouchkin, A., De Sutter, P. et al. (1995) Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum. Reprod., 10, 403–407.[Abstract]

Kahraman, S., Isik, A.Z., Vicdan, K. et al. (1997) A healthy birth after sperm injection by using immotile testicular spermatozoa in a case with totally immotile ejaculated spermatozoa before and after Percoll gradients. Hum. Reprod., 12, 292–293.[Abstract]

Liu, J., Tsai, Y.-L., Katz, E. et al. (1997) High fertilization rate obtained after intracytoplasmic sperm injection with 100% nonmotile spermatozoa selected by using a simple modified hypo-osmotic swelling test. Fertil. Steril., 68, 373–375.[ISI][Medline]

Madgar, I., Seidman, D.S., Levran, D. et al. (1996) Micromanipulation improves in vitro fertilization results after epididymal or testicular sperm aspiration in patients with congenital absence of the vas deferens. Hum. Reprod., 11, 2151–2154.[Abstract]

Madgar, I., Hourvitz, A., Levron, J. et al. (1998) Outcome of in vitro fertilization and intracytoplasmic injection of epididymal and testicular sperm extracted from patients with obstructive and nonobstructive azoospermia. Fertil. Steril., 69, 680–684.

Nagy, Z.P., Liu, J., Joris, H. et al. (1995) The results of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum. Reprod., 10, 1123–1129.[Abstract]

Nagy, Z.P., Joris, H., Verheyen, G. et al. (1998) Correlation between motility of testicular spermatozoa, testicular histology and the outcome of intracytoplasmic sperm injection. Hum. Reprod., 13, 890–895.[Abstract]

Nijs, M., Vanderzwalmen, P., Vandamme, B. et al. (1996) Fertilizing ability of immotile spermatozoa after intracytoplasmic sperm injection. Hum. Reprod., 11, 2180–2185.[Abstract]

Palermo, G., Joris, H., Devroey, P. and Van Steirteghem, A.C. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 17–18.[ISI][Medline]

Parrington, J., Swann, K., Shevchenko, V. et al. (1996) Calcium oscillations in mammalian eggs triggered by a soluble sperm protein. Nature, 379, 364–368.[ISI][Medline]

Silber, S.J., Van Steirteghem, A.C., Liu, J. et al. (1995) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum. Reprod., 10, 148–152.[Abstract]

Tsai, Y.-L., Liu, J., Garcia, J.-E. et al. (1997) Establishment of optimal hypo-osmotic swelling test by examining single spermatozoa from different hypo-osmotic solutions. Hum. Reprod., 12, 111–113.

Submitted on July 6, 1998; accepted on November 17, 1998.