Pregnancies achieved after frozen–thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen–thawed testicular tissues from non-obstructive azoospermic men

S. Al-Hasani1,3, L.C. Demirel2, B. Schöpper1, M. Bals-Pratsch1, N. Nikolettos1, W. Küpker1, M. Ugur1, R. Sturm1 and K. Diedrich1

1 Department of Obstetrics and Gynaecology, Medical University of Lübeck, Lübeck, 23538 Germany and 2 Department of Obstetrics and Gynaecology, Medical University of Ankara, Ankara, 06100, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of frozen–thawed testicular tissue as a source of spermatozoa for intracytoplasmic sperm injection (ICSI) in non-obstructive azoospermia yields favourable fertilization and pregnancy rates while avoiding both repetitive biopsies and unexpected cycle cancellations. Spermatozoa were obtained from frozen–thawed testicular biopsy specimens from 67 non-obstructive azoospermic men. Following fertilization, supernumerary two pronuclear (2PN) oocytes were frozen. After thawing, 17 cycles of embryo transfer were carried out with a mean number of 2.7 embryos and a mean cumulative embryo score (CES) of 18.3 per transfer. The clinical pregnancy and implantation rates per transfer in these cycles (23.5 and 8.3% respectively) were comparable to those of fresh embryo transfers (35.7 and 12.7% respectively) with a mean number of 2.7 embryos and a mean CES of 28.7 per transfer. Abortion rates, although higher with cryopreserved 2PN oocytes were not significantly different. With this approach, cryopreservation of supernumerary 2PN oocytes can be used to improve the cumulative pregnancy rates in a severely defective spermatogenetic population. To our knowledge, these are the first pregnancies reported which have been obtained by the transfer of cryopreserved pronuclear oocytes obtained from ICSI using cryopreserved testicular spermatozoa.

Key words: cryopreservation/ICSI/non-obstructive azoospermia/pregnancy/testicular spermatozoa


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Intracytoplasmic sperm injection (ICSI) with spermatozoa extracted from testicular tissue has proved to yield favourable fertilization and pregnancy rates in patients with non-obstructive azoospermia (Devroey et al., 1995Go). Since ~30% of these patients may not have any spermatozoa in their testicular biopsy samples (Mulhall et al., 1996Go; Oates et al., 1997Go; Tournaye et al., 1997Go), it becomes imperative to know if any spermatozoa will be available at the time of ICSI. Hence, the cryopreservation of any testicular tissue found to contain spermatozoa during a diagnostic testicular biopsy or a therapeutic testicular sperm extraction (TESE) attempt conducted before an anticipated ICSI cycle becomes a novel approach. The availability of multiple vials of frozen testicular tissue also avoids repetitive testicular biopsy procedures for successive ICSI cycles in this group of patients.

The use of frozen–thawed testicular spermatozoa for ICSI in non-obstructive azoospermia patients has already yielded satisfactory fertilization and pregnancy rates (Gil-Salom et al., 1996Go; Romero et al., 1996Go; Friedler et al., 1997Go; Oates et al., 1997Go; Perraguin-Jayot et al., 1997Go). Although spermatozoa can be cryopreserved within the intact testicular tissue, a minimal processing of the testicular tissue (Hovatta et al., 1996Go; Friedler et al., 1997Go; Khalifeh et al., 1997Go; Oates et al., 1997Go; Perraguin-Jayot et al., 1997Go) or extraction and purification of the spermatozoa from the tissue before freezing can also be performed (Gil-Salom et al., 1996Go; Romero et al., 1996Go; Verheyen et al., 1997Go).

Here we report the use of frozen–thawed testicular tissue as a source of spermatozoa for ICSI in patients with non-obstructive azoospermia, yielding not only favourable fertilization and pregnancy rates but also many supernumerary two pronuclear (2PN) oocytes which, with further cryopreservation, improved the cumulative pregnancy rates for this group of patients.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
From December 1995 to October 1998, testicular biopsy specimens from 67 non-obstructive azoospermic men were frozen once they had been shown to contain spermatozoa during either a diagnostic testicular biopsy for infertility work-up or a therapeutic TESE attempt conducted before an ICSI cycle. The diagnosis of non-obstructive azoospermia was verified by the concomitant histological evaluation of the harvested testicular tissue. In our programme, no freshly harvested testicular spermatozoa are used for an ICSI cycle. The initial diagnostic work-up of these males included karyotype analysis and screening for microdeletions in the Y chromosome. No chromosomal aberrations were found. The routine infertility work-up of the female partners did not reveal any abnormalities.

Technique of testicular biopsy
Under general anaesthesia, the entire testis was delivered out through a median raphe incision. The tunica albuginea was incised and a good piece of testicular parenchyma was harvested from cranial and caudal sides on each testis with scissors. The pieces of tissue thus obtained were placed into Ham's F10 medium in a Petri dish. A piece from each harvested tissue was then minced using a sterile surgical blade. The fragmented tissue was assessed for the presence of motile spermatozoa under the phase contrast microscope at x200–400 magnification. A part of each testicular biopsy sample was sent for histological analysis.

Cryopreservation and thawing of testicular biopsies
Once the wet preparation revealed spermatozoa in harvested testicular tissue, 3–4 mm tissue pieces were placed separately in cryovials (Nunc, Wiesbaden, Germany) containing 0.5 ml of HEPES-buffered medium (SpermFreeze, Medicult, Hamburg, Germany) consisting of modified Earle's balanced salt solution with 0.4% human serum albumin and 15% glycerol as cryoprotectant. The tissues were frozen using a Planner Kryo10 III apparatus (Messer Griesheim, Germany). The cooling procedure was performed in liquid nitrogen vapour down to –30°C within the first 5 min and exponentially to –150°C in the next 55 min. The tissues were then stored in liquid nitrogen.

Early in the morning of oocyte retrieval, a vial of frozen testicular tissue was thawed in a 37°C water bath for 3 min. The sample was washed twice in Ham's F10 medium and then minced into fine pieces using two surgical blades. An aliquot of medium was checked for the presence of spermatozoa under the microscope. If no spermatozoa were visible, a second vial was thawed for sperm retrieval, a situation which we encountered very rarely. The supernatant was then incubated for 5 h in Ham's F10 medium in a humidified atmosphere with 5% CO2 at 37°C, put into 2 ml Eppendorf tubes and centrifuged at 500 g for 1 min. The pellet was resuspended with 3 µl Ham's F10. A 1 µl aliquot of this suspension was transferred into a Petri dish containing droplets of Ham's F10 medium and 5 µl droplets of PVP (polyvinylpyrrolidone: Medicult, Hamburg, Germany). Immobilization of each single spermatozoon was in a PVP droplet.

After thawing, most spermatozoa were initially immotile but eventually resumed motility after incubation, mostly in the form of tail twitching. In all cases, sufficient motile spermatozoa were present in at least one biopsy specimen to inject all the available metaphase II (MII) oocytes.

Ovarian stimulation
Ovarian stimulation was achieved mainly using the gonadotrophin-releasing hormone agonist (GnRHa) triptorelin (DecapeptylGyn-Depot®, Ferring, Kiel, Germany) for pituitary suppression and human menopausal gonadotrophins (HMG) (Menogon, Ferring, Kiel, Germany) in a long protocol. The GnRHa Cetrorelix (Asta-Medica, Frankfurt upon Maine, Germany) and Ganirelix (Organon, Oss, The Netherlands) with HMG was occasionally used in the Lübeck protocol (Diedrich et al., 1994Go). Vaginal ultrasound guided follicle puncture was carried out 36 h after the injection of 10 000 IU of HCG.

Oocyte preparation and ICSI
The cumulus and corona radiata cells were removed mechanically with two hypodermic needles under stereomicroscopy after exposure to 0.5% hyaluronidase (type I, Sigma, Heidelberg, Germany) for 30 s. Only MII oocytes were used for ICSI. ICSI was performed as previously described (Al-Hasani et al., 1995Go). Briefly, a motile spermatozoon was picked up head first from 5 µl of Ham's F10 culture medium using an injection pipette filled with a small amount of PVP and transferred into the PVP droplet. The tail of each spermatozoon was crushed with the injection pipette and then loaded tail first for the microinjection. The whole procedure was performed on the heated stage of an inverted microscope (Diaphot, Nikon Corporation, Tokyo, Japan) at x200 magnification using a phase contrast optic system, equipped with two coarse positioning and two three dimensional hydraulic micromanipulators (Narishige, Tokyo, Japan). After 18 h of incubation in 100 µl microdrops of medium under mineral oil at 37°C in a humidified atmosphere with 5% CO2, fertilization was confirmed from the presence of two or more pronuclei. Supernumerary 2PN oocytes were cryopreserved. Up to three cleaving embryos were transferred into the uterine cavity after an overnight culture. The luteal phase was supported either with HCG injections every 3 days or with 600 mg of intravaginally administered micronized progesterone (Utrogestan: Besins-Iscovesco, Paris, France) for 14 days (Ludwig et al., 1997Go). Serum ßHCG was measured 15 days after embryo transfer. Clinical pregnancy was defined by the detection of fetal heart beats by sonography at 6–7 weeks of gestation.

Cryopreservation and thawing of supernumerary two pronuclear oocytes
Cryopreservation was performed as previously described (Al-Hasani et al., 1996Go) in Ham's F10 (Biochrom Company, Berlin, Germany) containing 20% human umbilical cord serum with 1,2-propanediol (PROH, 1.5 M) and sucrose (0.1 M) as cryoprotectants. Up to three 2PN oocytes were transferred to each ministraw (CTE Company, Erlangen, Germany). An open freezing system, the CTE-880 appliance (Cryo-Technik, Erlangen, Germany), was used for cryopreservation with a self seeding procedure. The straws were cooled from room temperature to –33°C slowly, held at this temperature for 30 min, and then plunged into liquid nitrogen. The ministraws were thawed in a water bath at 30°C and the cryoprotectants were then removed in four steps, using 1.0 M PROH and 0.2 M sucrose, 0.5 M PROH and 0.2 M sucrose, 0.2 M sucrose, and finally Ham's F 10 medium alone, each for 5 min. After overnight culture, up to three early cleaving embryos were transferred into the uterine cavity.

Nine replacement cycles were clomiphene citrate stimulated and eight were artificially prepared with transdermal 17-ß oestradiol (straderm TTS 100®, Novartis, Wehr, Germany; 100 µg per patch) and vaginal progesterone gel (Crinone 8%®, Weyth, Münster, Germany) without prior down regulation with a GnRHa. Briefly, patients started transdermal oestradiol treatment on cycle day 1 with one patch and the dosage was gradually increased up to four patches at a time. After the confirmation of satisfactory endometrial development and exclusion of ovulation by transvaginal sonography and hormonal assessment, patients received 90 mg vaginal progesterone in gel form daily, from day 15 to day 29. Embryo transfer was scheduled for the third day of progesterone treatment. If the pregnancy test was positive on day 29, oestradiol and progesterone supplementation was continued for 8 weeks.

Assessment of embryo quality
The early cleaving embryos were graded as 1 (poor), 2 (good) or 3 (ideal) (modified grading according to Veeck, 1991Go) according to the degree of fragmentation and regularity of blastomeres. The grade of each embryo was multiplied by its number of blastomeres to produce a quality score. The total score of all embryos transferred in a particular cycle was defined as the cumulative embryo score (CES) (Steer et al., 1992Go)

Statistical analysis
Fisher's exact test and unpaired t-test were used for the comparisons of the rates and means between the groups. Welch's correction was performed for unpaired t-test if the group variances to be compared were significantly different. P < 0.05 was considered as statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eighty-four cycles of fresh embryo transfer were performed after ICSI with spermatozoa extracted from frozen–thawed testicular biopsies of 67 patients with non-obstructive azoospermia. There were 17 embryo replacement cycles of the supernumerary frozen–thawed 2PN oocytes thus obtained. Table IGo presents the characteristics of fresh and frozen–thawed embryo transfer cycles. After thawing, 68% of frozen 2PN oocytes were found to have survived the freezing process. Though the mean number of embryos transferred per cycle was 2.7 in each group, the mean CES per transfer was significantly higher with fresh embryo transfers (P < 0.0001) A total of 30 pregnancies, four of which were twins, was achieved with fresh embryo transfers, while four singleton pregnancies were obtained from cryopreserved 2PN oocytes. There were no significant differences between the fresh and frozen–thawed embryo transfer cycles, embryo cleavage, implantation rate or clinical pregnancy rates. Abortion rates, although higher with cryopreserved 2PN oocytes, were not significantly different (25 versus 16.7%). ICSI cycles with frozen–thawed testicular spermatozoa which offered supernumerary 2PN oocytes for cryopreservation had higher normal fertilization rates than those which did not (68.3 versus 54.6%) (P = 0.0002, Fisher's exact test). Table IIGo shows the histological diagnoses of frozen–thawed testicular tissues and the number of pregnancies achieved with the use of spermatozoa extracted from them. In all, 147 cryopreserved 2PN oocytes were still available for use in future replacement cycles.


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Table I. Characteristics of fresh and frozen–thawed embryo transfer cycles after ICSI with spermatozoa extracted from the frozen–thawed testicular tissues of patients with non-obstructive azoospermia
 

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Table II. The histological diagnosis of frozen–thawed testicular tissues in patients with non-obstructive azoospermia and the number of pregnancies achieved following ICSI with thawed spermatozoa extracted from each condition
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of testicular spermatozoa for ICSI in cases of obstructive azoospermia when microsurgery is not feasible, has previously failed or no spermatozoa can be obtained from the epididymis, and in cases of severely impaired spermatogenesis, has resulted in several pregnancies (Schoysman et al., 1993Go; Devroey et al., 1995Go; Gil-Salom et al., 1995Go; Nagy et al., 1995Go; Silber et al., 1995Go). The sperm recovery rate for ICSI treatment is ~60–70% in patients with non-obstructive azoospermia (Oates et al., 1997Go; Jezek et al., 1998Go). Therefore there is always a chance that spermatozoa for ICSI may not be available on the day of oocyte retrieval. There are also no clear-cut parameters to predict the success of sperm recovery in patients with non-obstructive azoospermia. For instance, serum follicle stimulating hormone (FSH) concentrations and testicular volume measurements have been found not to be predictive for successful sperm recovery in patients with Sertoli-cell-only syndrome and maturation arrest (Tournaye et al., 1997Go).

Cryopreserving the testicular spermatozoa at the time of a diagnostic testicular biopsy or a TESE attempt for use in future ICSI cycles would avoid both unnecessary ovarian stimulation of the female partner in case of sperm non-availability and the need for repetitive biopsies for successive ICSI cycles. Repetitive biopsies threaten the testicular function of patients with non-obstructive azoospermia (Schlegel, 1996Go). Cryopreserving testicular spermatozoa from subjects with non-obstructive azoospermia has been shown to be feasible (Gil-Salom et al., 1996Go; Oates et al., 1997Go; Perraguin-Jayot et al., 1997Go). Testicular spermatozoa can be frozen within the intact testicular tissue; with a minimal processing of the testicular tissue or after being extracted and purified from the tissue. Since the number of motile spermatozoa within the testicular biopsies is very limited in cases of non-obstructive azoospermia, the preferred method should be to freeze the tissue intact, as this may allow better post-thaw survival and motility. Glycerol, with or without yolk association, is the recommended cryoprotectant. Vials can be held in liquid nitrogen vapour for 30 min before being plunged into the liquid nitrogen or they may be frozen in a computer controlled freezing unit utilizing a slow controlled freezing program (Sheikh et al., 1998Go).

Fertilization rates of 44–63% (Gil-Salom et al., 1996Go; Romero et al., 1996Go; Friedler et al., 1997Go; Oates et al., 1997Go), embryo cleavage rates of 89–92% (Gil-Salom et al., 1996Go; Friedler et al., 1997Go), implantation rates of 8–11% (Friedler et al., 1997Go; Oates et al., 1997Go) and clinical pregnancy rates/embryo transfer of ~27% (Friedler et al., 1997Go) have been reported following ICSI using frozen–thawed testicular spermatozoa from patients with non-obstructive azoospermia. These figures are comparable with our results. Fertilization rates of 17–69% (Schoysman et al., 1993Go; Nagy et al., 1995Go; Silber et al., 1995Go; Watkins et al., 1997Go), embryo cleavage rates of 94% (Friedler et al., 1997Go), implantation rates of 9–11% (Friedler et al., 1997Go; Watkins et al., 1997Go) and clinical pregnancy rates/embryo transfer of 21–36% (Devroey et al., 1995Go; Friedler et al., 1997Go; Nagy et al., 1995Go; Watkins et al., 1997Go; Ghazzawi et al., 1998Go) have been reported following ICSI with fresh testicular spermatozoa, supporting the safety and efficacy of the cryopreservation of testicular spermatozoa. Two studies have indicated that embryos obtained from fresh or cryopreserved testicular spermatozoa are similar in quality (Gil-Salom et al., 1996Go; Friedler et al., 1997Go). An abortion rate of 16.7% in our study suggests that the embryos obtained by ICSI with frozen–thawed testicular spermatozoa are developmentally competent.

In an overall review of 5 years of ICSI practice (Van Steirteghem et al., 1998Go), a comparison was made between the fertilization rates following ICSI and the outcome of embryo transfers with different types of spermatozoa. Fresh testicular spermatozoa had a possibly lower fertilization rate than ejaculated and fresh epididymal spermatozoa (53.4, 64.8 and 58.5% respectively), although no difference was noted for pregnancy rates (33.6–44.3%). Though it is not our aim to compare frozen–thawed testicular spermatozoa and spermatozoa of other origins, our data suggest that frozen–thawed testicular spermatozoa are at least as efficient as fresh testicular spermatozoa.

After thawing, extended culture of the testicular sprematozoa allows the development of motility and, as long as motile spermatozoa are retrieved, fertilization rates can be expected to be high. A recent study (Verheyen et al., 1997Go) showed that the mean recovery rates of motility and vitality immediately after the thawing of cryopreserved testicular spermatozoa fractions were 26 and 32% respectively.

In our group of patients with non-obstructive azoospermia, frozen–thawed testicular tissue not only served as a source of spermatozoa for multiple attempts of ICSI, but also provided many supernumerary 2PN oocytes which, when cryopreserved, improved the overall pregnancy rates per oocyte collection and testicular tissue retrieval. The clinical pregnancy rate per embryo transfer of 23.5% using frozen–thawed 2PN oocytes was quite satisfactory when compared with the expected rates for cryopreserved embryo transfers. The post-thaw survival (68%) and implantation (8.3%) rates of the cryopreserved 2PN oocytes implied that 100 frozen 2PN oocytes would still be available out of the remaining 147 after thawing for transfer, allowing an additional eight implantations to occur. Many cryovials of frozen testicular tissue remain which serve as a source for a future series of attempts.

To our knowledge, these are the first pregnancies reported following transfer of frozen–thawed pronuclear oocytes obtained by ICSI with spermatozoa extracted from frozen–thawed testicular tissues from patients with non-obstructive azoospermia.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on February 1, 1999; accepted on April 21, 1999.