Increased Fertilization Rates after In Vitro Culture of Frozen-Thawed Testicular Immotile Sperm in Nonobstructive Azoospermic Patients

Objective. To optimise the use of freeze/thaw testicular immotile spermatozoa from nonobstructive azoospermia patients and to analyse the outcome of intracytoplasmic sperm injection (ICSI) of such spermatozoa. Methods. Testicular specimens were retrieved and cryopreserved from forty patients with nonobstructive azoospermia and underwent one cycle with thawed spermatozoa (Group I) that led to pregnancy in sixteen cases. Twenty-four patients of group I underwent treatment with the same batch of thawed spermatozoa (Group II). For the first ICSI attempt, injection was performed when motile spermatozoa were found. In group II, injection was performed when maximum motility was reached. We compared mean of fertilization rate, embryo quality, clinical pregnancy rate and embryo implantation rate. Results. The mean percentage of motility was significantly higher in the group II than in the group I (18, 6 versus 8, 2). Group I showed a significant decrease in fertilization rates when compared with cryopreserved testicular spermatozoa in group II (54% versus 72%, P < 0.05). No difference was noted between the cleavage rate, embryo quality, clinical pregnancy rates and implantation rates among group II and I. Conclusion. Fecundation rate can be significantly improved after in-vitro culture and sperm selection of frozen-thawed immotile testicular spermatozoa in patients with nonobstructive azoospermia.


Introduction
It has been shown that the combination of human testicular sperm biopsy and intracytoplasmatic sperm injection (ICSI) is an efficient method for the treatment of male-factor infertility caused by azoospermia [1][2][3][4]. The successful use of testicular sperm extraction (TESE) procedures 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, and repeated testicular biopsies can then be avoided [5]. It seems that comparable success may be achieved with frozen-thawed and from testis spermatozoa [6][7][8]. It has been also reported that the motility of testicular spermatozoa can be improved after "in vitro" culture [7,9,10], although it is not known whether implantation and pregnancy rates are affected from such a practice [11]. Furthermore, it has also been observed [7] that the number of progressively motile spermatozoa was significantly increased after 72 h of culture, even if the total number of motile spermatozoa remained constant. Konc et al. [12] showed that condition of injected testicular spermatozoa has influence to embryo development, and even frozen/immotile testicular spermatozoa are able to induce/support fertilization and early embryo development. However, only limited data available connected with the efficacy of ICSI carried out with frozen nonmotile testicular spermatozoa.
The aim of this study was to optimise the use of freeze/ thaw testicular immotile spermatozoa from nonobstructive azoospermia (NOA) patients and to analyse the outcome of intracytoplasmic sperm injection (ICSI-TESE) of such spermatozoa. The outcome measures studied and compared were fertilization rate, embryo cleavage rate, clinical pregnancy rate and implantation rate.

Patients.
The data of sixty-four TESE-ICSI cycles performed in our centre in patients with NOA were evaluated prospectively. We studied forty couples in which the husband was diagnosed as having nonobstructive azoospermia (NOA) and only immotile testicular sperm cells were isolated by testicular sperm extraction (TESE). Testicular specimens were retrieved and cryopreserved and each couple underwent an ICSI cycle with thawed spermatozoa (Group I) that led to pregnancy in sixteen cases. Twenty-four patients of Group I subsequently underwent a consecutive cycle, which used frozen-thawed sperm that were retrieved in the original TESE procedure but were cryopreserved and stored until use (Group II).
For the first ICSI attempt (Group I), injection was performed when motile spermatozoa were found. In most cases, viable sperm began to twitch after 2-4 hours in culture, and these cells were selected for ICSI. In all the twenty-four samples of Group II, one portion of the specimen was thawed the day before ICSI, and changes in postthaw motility after 2, 4, 6, 8, 12, and 24 h of culture at 37 • C were recorded. In Group II, injection was performed when maximum motility was reached.

Testicular Biopsy Preparation.
The procedure of testicular sperm biopsy was similar to what has been already described [13] and the preparation of testicular spermatozoa was similar to the one reported by Liu et al. [14]. Briefly, the testicular tissue was placed in a Petri dish containing 2 mL sperm medium (Origio). After extraction of the connective tissue surrounding the seminiferous tubules with glass slides, an inverted microscope was used to detect the presence of spermatozoa at ×200 and ×400 magnifications (Diaphot, Nilkon). If a few spermatozoa were found, the contents of the Petri dish were transferred to a 15 mL Falcon tube. Prior to centrifugation, the larger debris of testicular tissue was removed from the tube, and the tube containing sperm suspension was centrifuged for 5 min at 300 g. After centrifugation, the supernatant was removed and the pellet was resuspended in about 100 : l IVF medium (Origio). A 15 : l aliquot of the sperm suspension was taken and put on a glass slide for examination of the initial motility of these spermatozoa. Motility of the spermatozoa was assessed and graded as nonmotile, weakly shaking, and progressively motile spermatozoa.

Ovarian Stimulation and Retrieval of Oocytes Used for
ICSI. All female partners were stimulated using gonadotropin releasing hormone agonist or antagonist, human menopausal gonadotropin, and human FSH. Human chorionic gonadotropin was administered when optimal follicle development was achieved, as evaluated by serial transvaginal ultrasound and estrogen determinations. Oocyte retrieval was performed via a transvaginal approach with sonographic guidance 36 hours after human chorionic gonadotropin injection [7]. After oocyte retrieval, maturity of the oocytes was evaluated under the inverted microscope at ×400.
Cumulus-oocyte complexes were collected in IVF medium (COOK) and cultured for another 2 hours in the incubator before careful denudation using hyaluronidase (Origio). Immediately after this process, ICSI was started.

Embryo Assessment and Transfer.
Fertilization was checked 16-18 h after ICSI. The fertilized oocytes were transferred and cultured in 50 mL droplets of Cleavage (COOK) medium covered by mineral oil (COOK), at 37 • C in 6% CO 2 , 5% O 2 and 89% N 2 with maximal humidity in air. Cleavage was assessed after 48-72 h and embryo quality was evaluated prior to transfer. Embryo quality was evaluated on the basis of morphology and developmental stage. Embryos/oocytes were classified into five categories: (1) excellent, (2) good, (3) fair, (4) poor, and (5) degenerate. Embryo with normal morphology, which was being in a developmental stage appropriate for its age, was classified as transferable. Only category 1, 2, and 3 embryos were used for transfer and the maximum number of embryos transferred was three.
Pregnancy was defined as a spontaneous rise in a βCG concentration at least 10 days after transfer. Clinical pregnancy implied the presence of an intrauterine gestational sac and fetal heart beat on an ultrasound performed at 7 weeks of gestation.

In Vitro Culture of Frozen-Thawed Testicular
Spermatozoa. IVF medium (Origio) without additional supplements was used for culture of testicular spermatozoa. A 5 : l aliquot of testicular sperm suspension was added to each 10 : l droplet of IVF medium covered under mineral oil (COOK) and incubated at 37 • C in an atmosphere of 6% CO 2 . In vitro culture of testicular spermatozoa was normally carried out without change of culture medium.

Motility of Fresh and Frozen-Thawed Spermatozoa.
Washed spermatozoa were cultured in microdroplets under paraffin oil using bicarbonate-buffered medium containing 10% human serum in a 6% CO 2 incubator at 37 • C.
Motility of frozen-thawed testicular spermatozoa was assessed under an inverted microscope (Nikon Diaphot) at ×400 magnification. One hundred testicular spermatozoa were counted in culture droplets for each sample. The percentage of nonmotile, weakly shaking, and progressively motile spermatozoa were recorded in both groups. Motility of frozen-thawed testicular spermatozoa in Group II was assessed at different intervals (1,4,6,8,12, and 24 h), and injection was performed when maximum motility was reached.

Cryopreservation and Thawing of Spermatozoa.
After preparation of testicular spermatozoa, the same volume of cryoprotectant solution (TEST yolk buffer with glycerol, CRIOSPERM) was added to the testicular sperm suspension (volume/volume) and was gently mixed. This mixture was kept at 4 • C for 45 minutes (in a refrigerator) and then the pellet of dry ice (Snowpack, MG Gas Products) was prepared. This is a modified cryopreservation method of Nagase and Niva [15], adapted to human spermatozoa [16,17]. After the refrigeration, we prepared the mixture pills of 10 : l and placed then on dry ice for 2 minutes. Each pill was placed in 1.0 mL vials and plugged in liquid nitrogen. For thawing, the vials containing testicular spermatozoa were taken out from liquid nitrogen, and some of them were transferred into a 10 mL test tube and kept in a 37 • C water bath for 3-5 minutes. About 5 mL medium (IVF, Origio) were added into the tube. After gentle mixing, the tube was centrifuged at 300 g for 5 min. After centrifugation, the supernatant was removed and the pellet was resuspended in about 50 : l IVF medium. The procedures of the in vitro culture of the frozen-thawed testicular spermatozoa and the examination of their motility were the same as the one described above for fresh testicular spermatozoa.

Statistic.
A paired t-test was carried out to analyse the changes of motility before and after culture of fresh and frozen-thawed testicular spermatozoa. The chi-square test was used to compare the outcome of ICSI with fresh and frozen-thawed testicular spermatozoa. Probabilities <5% were considered significant.

Results
The average ages of the female and male patients were 35 + 2.9 (range 24-41) and 37.5 + 6.3 years (range 26-51). The average ages of the female and male patients were similar in the different treatment groups. A total of 40 men underwent 40 TESE procedures.The result of the histology of testicular tissue indicated spermatogenic arrest in 80% of the cases. The spermatogenic arrest occurred at a different level of spermatogenesis (primary spermatocytes, 23%; spermatid, 77%). Sertoli-only syndrome was diagnosed in 20% of the patients. Male serum levels of FSH and testosterone were 23.4 + 12.1 mIU/mL and 11.7 + 7.6 nmol/L (mean + S.D.).
The main results of the treatment outcome are summarized in Table 1. No differences were found between the two groups in the days of gonadotrophin stimulation, number of ampoules/cycle, endometrial thickness, and estradiol level on the day of HCG administration. The number of oocytes retrieved did not differ among Groups I-II. We found motile spermatozoa in both sperm groups. The mean percentage of motility was significantly higher in Group II (after culture, with the peaked) than in Group I (when spermatozoa reached some kind of motility) (18,6 ± 2,5 and 8,2 ± 3,2, resp.) (P < 0.01). In Group II, the motility improved markedly on the sixth hour of culture (Table 2).
Group I showed a significant decrease in fertilization rates when compared with cryopreserved testicular spermatozoa in Group II (54% versus 72%, P < 0.05, resp.).
No differences were found in the embryo formation between oocytes injected with frozen-thawed testicular spermatozoa when reached some kind of motility (Group I) versus frozen-thawed testicular spermatozoa after culture, (94% versus 96%) (Table 3), neither quality of embryos between both groups (2,6 versus 2,7).
No difference was noted between the clinical pregnancy rates (40% versus 41,6%). There was no difference in implantation rates (25 versus 20,8) among Groups II and I (Table 3).

Discussion
Several authors have demonstrated that performing ICSI with fresh or frozen spermatozoa produces similar results [7,8,18,19], and that freezing does not affect the spermatozoa [20].
Schlegel et al. observed in NOA patients, that in 30-38% of the cases the TESE procedure is unsuccessful. Thus, they and others underline the importance of the cryopreservation of the obtained testicular tissue in the clinical application of TESE.
Considering these results, repeated testicular biopsies are avoided in subsequent ICSI treatment cycles by cryopreservation of testicular spermatozoa in patients with NOA. Consequently, some groups have attempted to improve the protocols for testicular sperm freezing [5,21,22].
Our results support the observations of others that testicular tissue may be cryopreserved successfully without markedly reducing subsequent fertilization and implantation rates.
In our attempt to develop more efficient strategies, including freezing of testicular spermatozoa, we compared fertilization rates, cleavage rates, clinical pregnancy rates and implantation rates resulting from ICSI of frozen-thawed immotile testicular spermatozoa incubated 2-4 hours in a simple culture medium after thawing versus frozen-thawed immotile testicular spermatozoa incubated 2-24 hours in the same simple culture medium after thawing.
Our results showed that after a few hours of culture of fresh and frozen-thawed testicular sperm samples obtained from patients with NOA, even without motile spermatozoa, the motility was significantly improved. The motility of cultured testicular spermatozoa reached a peak around 6 hours without the need for special media or treatment. The changes in motility patterns during in vitro culture of testicular spermatozoa has been observed previously [7], and practical implications of using in vitro matured testicular spermatozoa for ICSI were also described [10,23]. The motility of cultured testicular spermatozoa in these cases reached a peak around 72 hours, and the results reveal that cryopreservation had an adverse effect on the survival rate of testicular spermatozoa, since the percentage of nonmotile spermatozoa significantly increased after cryopreservation [7]. These differences may be related to the procedures used in this study for in vitro culture and for cryopreservation of testicular spermatozoa.
In order to improve the outcome of ICSI carried out with nonmotile spermatozoa, various methods have been described for the selection of the immotile but viable spermatozoa. These include the addition of pentoxifylline (PF), the laser touch technique, mechanical touch, and performing the hypo-osmotic swelling (HOS) test [24][25][26][27]. However, it has been argued, that using HOS test many live spermatozoa are to become nonviable after 30 min of incubation in the hypo-osmotic solution [27]. In addition, Casper et al. [27] reported lower fertilization rate after using HOS test protocol for ICSI. On the other hand, some studies showed that cAMP phosphodiesterase inhibitors are not beneficial in enhancing fertilization rates [28] or can have a negative effect on animal oocytes and embryos. They can cause meiotic arrest in mammalian oocytes [29], poorer development of mouse embryos [30,31], 2-cell embryo block in some mouse strains [32,33], or parthenogenetic activation of mouse oocytes [34]. On the basis of the findings that PF is not beneficial in enhancing fertilization rates and can be toxic for oocytes, it was suggested that PF should be used only in strictly selected patients [30,31].
Conversely, it has never been described the influence of the effect of frozen-thawed human previously immotile testicular spermatozoa incubation time on intracytoplasmic sperm injection (ICSI) outcome.
In our study, frozen-thawed testicular nonmotile spermatozoa cultured for a period of 6-8 h resulted in a greater fertilization compared with testicular nonmotile spermatozoa without culture. No differences were found in embryo cleavage and clinical pregnancy outcomes between the two groups.
The present findings are in agreement with other studies [35] that reported the motility of testicular spermatozoa was enhanced or initiated after in vitro culture and testicular biopsy culture may be an alternative to the use of motility stimulants to obtain motile spermatozoa for intracytoplasmic sperm injection. These results contrast with other reports that indicate that superior results are obtained with fresh TESE sperm rather than with TESE sperm cryopreservation [36,37]. One possibility to account for these discrepancies is that the cryopreservation methods are variable between these groups and that freeze-thaw techniques for TESE sperm can significantly affect ICSI outcomes. Comparative studies to optimise tissue preparation, freezing protocols, and freezing media are rare and few attempts have been made to define the effects of cryopreservation on testicular spermatozoa.
These data suggest that fecundation rate can be significantly improved after in vitro culture and sperm selection of frozen-thawed immotile testicular spermatozoa in patients with nonobstructive azoospermia. After all, a more rational management of testicular samples, in the case of frozen testicular spermatozoa, even though nonmotile spermatozoa, consists of improving sperm motility of samples by several hours of culture, after thawing and before ICSI. Higher fertilization rates with this method will produce more embryos. Therefore, it will be better embryo selection for transfer and embryo cryopreservation, offering the possibility for further attempts with one single sample.
We conclude that, although cryopreservation does impair motility, incubation of frozen/thawed testicular immotile sperm in cases of nonobstructive azoospermia appears to be beneficial.