IVF and Infertility Unit, Department of Obstetrics and Gynecology, Assaf Harofeh Medical Center, Zerifin 70300, Israel
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: epididymal sperm/ICSI/non-obstructive azoospermia/obstructive azoospermia/testicular sperm
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we aimed to examine variable parameters that might be predictors of success following ICSI, in patients with obstructive and non-obstructive azoospermia (OA and NOA). Male partner's age, serum FSH, testicular histology as well as the non-ejaculated sperm source, status as fresh or cryopreservedthawed along with the female partner's age and ovarian reserve were analysed. The results might contribute to the ability to inform patients and establish clinical management policy in such cases.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The andrological evaluation of the male partners included intake of their familial and personal history (i.e. familial male infertility, undescended testicles in childhood, orchiepididymitis or mumps orchitis), physical examination of their genitalia as well as testicular and transrectal sonography to determine existence of normal anatomy of seminal vesicles, prostate, distal vasa deferentia and ejaculatory ducts and the ruling out of suspicious testicular findings. Hormonal profile and karyotype in all patients were assessed. Those presenting with congenital bilateral absence of vas deferens (CBAVD) were also screened for cystic fibrosis mutations and underwent genetic counselling. Extensive workup by the extended sperm preparation method (ESP) (Ron-El et al., 1997) of several ejaculates prior to surgical sperm retrieval and on the day of the oocyte retrieval revealed no sperm in any of the ejaculates provided. Diagnosis of obstructive or non-obstructive azoospermia was based upon histological report, taken previously or during the current procedure. Evidently, prior testicular biopsy was available only in some patients diagnosed of suffering from testicular failure. Aetiology for the obstruction included CBAVD, orchiepididymitis, vas deferens laceration and vasectomy reversal failure. In contrast with normal spermatogenesis seen in cases with OA the non-obstructive patients had tubular sclerosis, severe hypospermatogenesis, complete maturation arrest, germ cell aplasia or a mixed picture of germ cell aplasia in some tubules in conjuction with immature sperm cells in other tubules as the main histological diagnosis on their testicular biopsy.
Sperm retrieval and preparation
The technique of surgical sperm aspiration in patients with azoospermia, the preparation and ICSI techniques have been described in detail elsewhere (Friedler et al., 1997a,b
, 1998
). In patients with OA, PESA was performed using a 23-gauge butterfly needle attached to a 20 ml plastic syringe serving as an aspiration device. Patients with NOA underwent TESE. Following ICSI using either epididymal or testicular sperm, excessive epididymal sperm or the remaining testicular tissue extract was cryopreserved using a standard freezing protocol. The sperm-containing extract was diluted dropwise 1:1 with the test yolk buffer freezing medium (Irvine Scientific, Santa Anna, CA, USA) and finally sealed in commercially available freezing straws (0.5 ml; Instruments de Medicine Veterinaire-IMV, l'Aigle, France) (Friedler et al., 1997a
,b
, 1998
). The straws were rapidly cooled by immersion into a nitrogen vapour-containing chamber (cooling rate of 10°C/min), stabilized at 80°C for 20 min and stored finally in liquid nitrogen at 196°C. Thawing was performed rapidly, simply by removing the straws from liquid nitrogen and exposing it to room temperature. Dilution of the cryoprotectant-containing sperm mixture was performed with insemination medium, centrifuged at 300 g for 7 min followed by examination of the pellet in multiple droplets.
Ovulation induction and oocyte retrieval was performed using a routine long protocol of GnRH agonist suppression, followed by hMG for ovarian stimulation. Oocytes were retrieved 36 h after administration of 500010000 IU hCG by vaginal ultrasound-guided follicular puncture.
Sperm collection and ICSI procedure
When sperm could be identified and isolated, ICSI was performed as described (Van Steirteghem et al., 1993). Following the oocyte's denudation of the surrounding cumulus and corona cells, nuclear maturation assessment was performed using an inverted microscope, to allow injection of metaphase II oocytes only. Fertilization was assessed on the following day, 1618 h post sperm injection, and confirmed if two distinct pronuclei could be observed.
Embryo transfer, luteal support and pregnancy evaluation
After assessment of fertilization, embryonic cleavage and morphological quality ~24 h later, embryo transfer was performed. Luteal support included i.m. injections of hCG (Chorigon; Teva; Petach Tikva, Israel), 2500 IU on days of embryo transfer, +3, +6, +9 or of micronized progesterone (300 mg/day, intravaginally; Utrogestan; Laboratoires Basins-Iscovesco, Paris), from day +1 until serum ß-hCG measurement 14 days following embryo transfer. Only clinical pregnancies including sonographic demonstration of a gestational sac were counted. Outcome was compared between the groups and evaluated in cycles using fresh and cryopreservedthawed sperm.
Statistical analysis
Statistical evaluation was performed using Student's t-test, 2-test and Fisher's exact test, where appropriate. Differences were considered significant at P < 0.05. The predictors of success were evaluated by performing uni- and multifactorial analysis using logistic and stepwise statistical analysis, including separate analysis of only the first trial of the patients (52 PESA and 123 TESEICSI cycles). Comparative statistics were performed including all ICSI cycles. The JMP v 3.2.2 statistical package (SAS Institute, Cary, NC, USA) was used.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Concurring with previous reports, our results found that patient's age or serum FSH level failed to predict presence or absence of testicular sperm after TESE. The strongest indicator for finding sperm for ICSI was testicular histopathology, which was significantly higher in cases with severe hypospermatogenesis, in agreement with some of the reports in the literature (Tournaye et al., 1996, 1997
; Mercan et al., 2000
, Vicari et al., 2001
). The success rate of retrieving mature sperm cells in OA (100%) and NOA (41%) patients in our series is in the range reported in the literature by other investigators (Kahraman et al., 1996
; Westlander et al., 1999
).
The outcome of ICSI using non-ejaculated sperm may be influenced by various factors, including the aetiology of azoospermia and the surgically retrieved sperm source, sperm status being fresh or after cryopreservation and thawing and factors related to the female partner such as age and ovarian reserve.
Aetiology of azoospermia
The underlying aetiology of azoospermia could influence the viability of the retrieved sperm cells as well as determinein most casestheir source, epididymal or testicular, in OA or NOA respectively, thus influencing their maturational status. Viability of sperm, at least as judged by motility, may be influenced by their source (Bachtell et al., 1999). Comparison of the outcome of ICSI performed in the same institution, in patients with OA and NOA, may contribute to the clarification of this issue. Regarding outcome of ICSI in our study, the 2PN fertilization rate was similar between patients with OA and NOA. Clinical pregnancy rate, early miscarriage rate and the ongoing pregnancy/delivery rates, although higher in patients with OA, did not reach a statistically significant difference.
Among patients with NOA, main testicular histology was not correlated significantly with the chance of achieving pregnancy.
The ESHRE ICSI Task Force reported their experience for the year 1995 (Tarlatzis and Bili, 1998) and found a tendency for lower fertilization rates following ICSI in NOA compared with OA patients. A total of 339 ICSI cycles performed in 298 patients with OA and 287 ICSI cycles performed in 247 patients with NOA resulted in a 2PN fertilization rate of 54.7 and 46.7% respectively, compared with 56 and 51% in our study. No significant difference was noted in the pregnancy rate, delivery rate and perinatal outcome between the two groups of patients. Silber et al. (1997)
reported data concerning outcome of ICSI in 186 patients with OA and 163 patients with NOA (Silber et al., 1997
). Neither the pathology, nor the sperm source, quality or the quantity had any effect on fertilization or pregnancy rates. Comparing outcome of 30 and 39 ICSI cycles in OA and NOA patients respectively, Vicari et al. reported similar fertilization and pregnancy rates in OA, significantly higher abortion rates for NOA, resulting in a significantly higher ongoing/delivery rates for OA (Vicari et al., 2001
).
Our data do not concur with those of Palermo et al. who reported a significantly higher fertilization rate of 73% in 241 cycles of OAacquired and congenitalusing epididymal sperm compared with 57% in 53 cycles of NOA as well as a significantly higher clinical pregnancy rate of 56% (135/241) compared with 49.1% (26/53) for OA and NOA cases, respectively (Palermo et al., 1999). Our results differ also with those reported by Mansour et al. who compared the outcome of ICSI using epididymal sperm from patients with OA (44 cycles) the use of testicular sperm in NOA (106 cycles), and found a significantly lower fertilization rate in the latter group (59.5 versus 39%), significantly lower clinical pregnancy rate per cycle (27.3 versus 11.3%) and lower pregnancy rate per embryo transfer (27.3 versus 15.2%) (Mansour et al., 1997
). Other authors also reported higher pregnancy rates after ICSI in OA compared with NOA (Kahraman et al., 1996
; Aboulghar et al., 1997
; Mansour et al., 1997
; Ghazzawi et al., 1998
). Ubaldi et al. compared outcome of ICSI using testicular sperm from OA (33 cycles), NOA (29 cycles) and patients with ejaculated sperm (Ubaldi et al., 1999
). They found a significantly lower implantation rate in the NOA group (13.4 versus 24.5%, for NOA and OA respectively, P < 0.05). However, the 2PN fertilization rate, clinical pregnancy rate (37.9 versus 51.5% for NOA and OA respectively) and clinical abortion rates (6.2 versus 17.6% for NOA and OA respectively) were not significantly different. Finally, the ongoing/delivered pregnancy rates were lower, but not significantly, for NOA (34.4 and 42.4% for NOA and OA respectively). Therefore, testicular pathology resulted in a decreased implantation rate without affecting fertilization or early preimplantation development (Ubaldi et al., 1999
).
In a retrospective analysis involving 139 patients with OA and 54 with NOA who underwent ICSI using testicular sperm, fertilization rates were significantly lower in patients with NOA. However, pregnancy and embryo implantation rates were similar (De Croo et al., 2000). In patients with NOA, pregnancy rates were lowest for maturation arrest (20%), but not significantly different from Sertoli cell-only or hypoplasia. Implantation rate was lowest in germ cell hypoplasia (15.8%).
Sperm cryopreservation
Proving the efficiency of ICSI using non-ejaculated sperm after cryopreservation is of paramount importance. Cryopreservation of non-ejaculated sperm following surgical sperm retrieval allows us to perform further ICSI cycles, thus avoiding the repetition of surgical sperm retrieval. Furthermore, efficient non-ejaculated sperm preservation allows the independence of the surgical sperm retrieval from the controlled ovarian stimulation and oocyte retrieval (Oates et al., 1996; Nudell et al., 1998
; Ben-Yosef et al., 1999
; Janzen et al., 2000
). Thus, unnecessary ovarian stimulation is prevented in cases where no sperm can be retrieved, and oocyte retrieval planning is possible in the others. Although in OA a greater number of motile sperm may usually be aspirated and cryopreserved than in cases of NOA, only a small number of sperm are identified in the testicular biopsies, and remain available for cryopreservation.
The efficacy of non-ejaculated sperm cryopreservation was examined in our programme by comparing the outcome of ICSI using fresh or cryopreservedthawed sperm, in both groups of patients suffering from OA and NOA.
Although in the OA group, fertilization rate following thawing was affected significantly, neither the implantation rate nor the clinical or ongoing pregnancy rates achieved following embryo transfer were different comparing fresh with cryopreservedthawed sperm, in either the OA or NOA groups. Our results concur with those reported by others concerning successful cryopreservation of epididymal sperm in OA patients (Devroey et al., 1995; Nagy et al., 1995
; Oates et al., 1996
; Holden et al., 1997
; Silber et al., 1997
; Madgar et al., 1998
; Palermo et al., 1999
; Tournaye et al., 1999
; Janzen et al., 2000
; Cayan et al., 2001
). Also, successful testicular sperm cryopreservation has been reported (Fischer et al., 1996
; Gil-Salom et al., 1996
; Hovatta et al., 1996
; Romero et al., 1996
; Liu et al., 1997
; Oates et al., 1997
; DeCroo et al., 1998
; Marmar, 1998
; Perraguin-Jayot et al., 1998
; Ben Yosef et al., 1999
; Tuuri et al., 1999
; Gil-Salom et al., 2000
).
Examining outcome of ICSI in patients suffering from OA, using fresh epididymal sperm (75 cycles) and frozen epidydimal sperm (27 cycles), Silber et al. found no significant differences in 2PN fertilization (58 versus 48%), implantation rate (20 versus 14%), clinical pregnancy rate/embryo transfer (49 versus 41%) and delivery rate/embryo transfer (36 versus 37%) (Silber et al., 1997). Comparing use of fresh or cryopreservedthawed epididymal sperm in patients undergoing their first ICSI cycle (108 fresh and 33 cryopreserved), Janzen et al. reported similar fertilization rates (78.2 and 76.9%), clinical pregnancy rates (67 and 61%) and delivery rates (57 and 51% respectively) (Janzen et al., 2000
). Van Steirteghem et al. summarized the 5 year experience of the Brussels group performing ICSI, and reported no significant differences in the fertilization rate, embryo cleavage rate using fresh epididymal, cryopreserved epididymal or testicular sperm (Van Steirteghem et al., 1998
). Comparing outcome of 145 ICSI cycles with fresh and 77 frozenthawed epididymal sperm, the delivery rate of per embryo transfer was 31.3 and 27.2% respectively. Several studies aiming to control for oocyte factor by comparing outcome of ICSI in the same couples, found no significant differences in fertilization or clinical pregnancy rates when using fresh or frozenthawed epididymal sperm (Friedler et al., 1998
; Tournaye et al., 1999
; Cayan et al., 2001
). Palermo et al. reported no difference in fertilization rate after cryopreservation using epididymal sperm (72 versus 74%, fresh versus thawed respectively) but a significant decrease in the clinical pregnancy rate (64.3 versus 46.4% accordingly) (Palermo et al., 1999
).
Using testicular sperm no difference was noted in fertilization or clinical pregnancy rate following cryopreservation. Our results concur also with those reported by Ben-Yosef et al. with no significant difference in 2PN fertilization, pregnancy or implantation rates (59 versus 61%, 26.7 versus 21.7% and 12.5 versus 8.5%, using fresh or cryopreservedthawed testicular sperm respectively) in patients with NOA (Ben-Josef et al., 1999). Also Habermann et al. reported no differences in fertilization rate, clinical pregnancy rate, implantation rate, comparing ICSI using fresh or frozenthawed epididymal or testicular sperm, in OA or NOA patients (Habermann et al., 2000
).
In our patients, when using cryopreserved sperm, early abortion rate was high in the NOA group and low in the OA group; however, the significance of these observations may be limited because of the small size of the groups compared.
The cryopreservation methodology used in our unit is quite simple (Friedler et al., 1997a,b
, 1998
). It seems that epididymal as well as testicular sperm may be successfully cryopreserved, irrespective of the specific histological diagnosis of the biopsy.
We may conclude that in view of better fertilization rates with fresh epidydimal sperm in OA patients, ICSI should be planned in conjunction with surgical sperm retrieval. In contrast, in patients with NOA, as sperm retrieval rate is lower, but performance of ICSI using thawed sperm is similar to that using fresh, elective surgical sperm retrieval may be offered to patients prior to ovarian stimulation of their partners, especially when donor back-up is not an alternative for the couple. The main benefit of this policy would be the avoidance of ovarian hyperstimulation in cases where no sperm are found for ICSI following sperm retrieval. However, in some men only very few sperm may be found in their testicular biopsy for cryopreservation, or in others the risk of repeated surgeryin case the sperm cells do not survive cryopreservationis unacceptable. In these cases elective sperm cryopreservation may not serve the patient's best interest.
Factors related to the female partner
Parameters determined by the female partner such as age and number of oocytes available for ICSI may also have a significant contribution to the success of ICSI in these patients (Silber et al., 1997). According to our study, once non-ejaculated sperm is available for ICSI, female factors bear significant impact upon clinical success rate. The chances of becoming pregnant for female partners aged >38 years and/or with limited ovarian reserve with four or fewer mature oocytes for injection, are significantly hampered. Our findings concur with those reported by Silber et al. (1997)
). They analysed the outcome of ICSI in patients with OA and found that in 102 ICSI cycles performed, for women <37 years old compared with those aged
37 years, implantation rates were 20 versus 7% and the delivery rate per cycle was 39 versus 17%, demonstrating the effect of the female partner's age on the clinical outcome (Silber et al., 1997
). Regarding the female partner's ovarian reserve, ovarian response producing less than eight 2PN, mature oocytes available for ICSI, dramatically diminished the chances of achieving successful implantation and a viable pregnancy in patients with NOA (Silber et al., 1997
). In patients with OA this trend did not reach statistical significance. Also in our study the chances of achieving a clinical pregnancy was significantly correlated with the number of oocytes retrieved and injected. However, in our study the 10th quantile of oocytes retrieved and injected was five and four, numbers that correspond more to the conventional definitions of diminished ovarian reserve, than the limit of eight oocytes reported by Silber et al. (1997)
.
All the options and relevant parameters should be discussed with the couple and clinical decisions made based on the reported experience.
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ballesca, J.L., Balasch, J., Calafell, J.M., Alvarez, R., Fabregues, F., Martinez de Osaba, M.J., Ascaso, C. and Vanrell, J.A. (2000) Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia. Hum. Reprod., 15, 17341738.
Bachtell, N.E., Conaghan, J. and Turek, P.J. (1999) The relative viability of human spermatozoa from the vas deferens, epididymis and testis before and after cryopreservation. Hum. Reprod., 14, 30483051.
Ben-Yosef, D., Yogev, L., Hauser, R., Yavetz, H., Azem, F., Yovel, I., Lessing, J.B. and Amit, A. (1999) Testicular sperm retrieval and cryopreservation prior to initiating ovarian stimulation as the first line approach in patients with azoospermia. Hum. Reprod., 14, 17941801.
Brugo-Olmedo, S., De Vincentiis, S., Calamera, J.C., Urrutia, F., Nodar, F. and Acosta, A. (2001) Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia. Fertil. Steril., 76, 11241129.[ISI][Medline]
Cayan, S.L.D., Conaghan, J., Givens, C.A., Ryan, I.P., Schriock, E.D. and Turek, P.J. (2001) A comparison of ICSI outcomes with fresh and cryopreserved epididymal spermatozoa from the dame couples. Hum. Reprod., 16, 495499.
DeCroo, I., Van der Elst, J., Everaert, K., De Sutter, P. and Dhont, M. (1998) Fertilization, pregnancy and embryo implantation rates after ICSI with fresh or frozen thawed testicular spermatozoa. Hum. Reprod., 13, 18931897.[Abstract]
DeCroo, I., Van der Elst, J., Everaert, K., De Sutter, P. and Dhont, M. (2000) Fertilization, pregnancy and embryo implantation rates after ICSI in cases of obstructive and non-obstructive azoospermia. Hum. Reprod., 15, 13831388.
Devroey, P., Silber, S., Nagy, Z., Liu, J., Tournaye, H., Joris, H., Verheyen, G. and Van Steirteghem, A. (1995) Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozenthawed epididymal spermatozoa. Hum. Reprod., 10, 903906.[Abstract]
Friedler, S., Raziel, A., Strassburger, D., Soffer, Y., Komaroversusky, D. and Ron-El, R. (1997a) Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum. Reprod., 12, 14881493.[Abstract]
Friedler, S., Raziel, A., Soffer, Y., Strassburger, D., Komaroversusky, D. and Ron-El, R. (1997b) Intracytoplasmic injection of fresh and cryopreserved testicular spermatozoa in patients with non-obstructive azoospermiaa comparative study. Fertil. Steril., 68, 892897.[ISI][Medline]
Friedler, S., Raziel, A., Soffer, Y., Strassburger, D., Komaroversusky, D. and Ron-El, R. (1998) The outcome of intracytoplasmic injection of fresh and cryopreserved epididymal spermatozoa in patients with obstructive azoospermiaa comparative study. Hum. Reprod., 13, 18721877.[Abstract]
Fischer, R., Baukloh, V., Naether, O.G., Schultze, W., Salzbrunn, A. and Benson, D.M. (1996) Case report: pregnancy after sperm injection of spermatozoa extracted from the frozen thawed testicular biopsy. Hum. Reprod., 11, 21972199.[Abstract]
Ghazzawi, I.M., Saraf, M.G., Taher, M.R. and Khalifa, F.A. (1998) Comparison of fertilizing capability of spermatozoa from ejaculates, epididymal aspirates and testicular biopsies using intracytoplasmic sperm injection. Hum. Reprod., 13, 348352.[Medline]
Gil-Salom, M., Romero, J., Minguez, Y., Rubio, C., De los Santos, M.J., Remohi, J. and Pellicer, A. (1996) Pregnancies after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Hum. Reprod., 11, 13091313.[Abstract]
Gil-Salom, M., Romero, J., Minguez, Y., Molero, M.D., Remohi, J. and Pellicer, A. (1998) Testicular sperm extraction and intracytoplasmic sperm injection: a chance of fertility in non-obstructive azoospermia. J. Urol., 160, 20632067.[ISI][Medline]
Gil-Salom, M., Romero, J., Rubio, C., Ruiz, A., Remohi, J. and Pellicer, A. (2000) Intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Mol. Cell. Endocrinol., 169, 1519.[ISI][Medline]
Habermann, H., Seo, R., Cieslak, J. Niederberger, C., Prins, G.S. and Ross, L. (2000) In vitro fertilization outcomes after after intracytoplasmic sperm injection with fresh or frozenthawed testicular spermatozoa. Fertil. Steril., 73, 955960.[ISI][Medline]
Holden, C.A., Fuscaldo, G.F., Lackson, P., Cato, A., Southwick, G.J., Hauser, R., Temple-Smith, P.D. and McLachlan R.I. (1997) Frozen-thawed epididymal spermatozoa for intracytoplasmic spern injection. Fertil. Steril., 67, 8187.[ISI][Medline]
Hovatta, O., Foudila, T., Siegberg, R., Johansason, K., Von Smitten, K., Reima, I. (1996) Pregnancy resulting from intracytoplasmic injection of spermatozoa from a frozen-thawed testicular biopsy specimen. Hum. Reprod., 11, 24722473.[Abstract]
Janzen, N., Goldstein, M., Schlegel, P.N., Palermo, G.D. and Rozenwaks, Z. (2000) Use of electively cryopreserved microsurgically aspirated epididymal sperm with IVF and intracytoplasmic sperm injection for obstructive azoospermia. Fertil. Steril., 74, 696701.[ISI][Medline]
Kahraman, S., Ozgur, S., Alatas, C., Aksoy, S., Balaban, B., Evrenkaya, T., Nuhoglu, A., Tasdemir, M., Biberoglu, K., Schoysman, R. et al. (1996) High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 673676.[Abstract]
Liu, J., Tsai, YL., Katz, E. Compton, G., Garcia, J.E. and Baramki, T.A. (1997) Outcome of in-vitro culture of fresh and frozen-thawed human testicular spermatozoa. Hum. Reprod., 12, 16671672.[Abstract]
Madgar, I., Hourvitz, A., Levron, J., Seidman, D., Shulman, A., Raviv, G., Levran, D., Bider, D., Maschiach, S. and Dor, J. (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, 10801084.[ISI][Medline]
Mansour, R.T., Kamal, A., Fahmy, I., Tawab, N., Serour, G.I. and Aboulghar, M.A. (1997) Intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 12, 19741979.[Abstract]
Marmar, J.L. (1998) The emergence of specialized procedures for the acquisition, processing and cryopreservation of epididymal and testicular sperm in connection with intracytoplasmic sperm injection. J. Androl., 19, 517526.
Mercan, R., Urman, B., Alatas, C. Aksoy, S., Nuhoglu, A., Isiklar, A. and Balaban, B. (2000) Outcome of testicular sperm retrieval procedures in non-obstructive azoospermia: precutaneous aspiration versus open biopsy. Hum. Reprod., 15, 15481551.
Mulhall, J.P., Burgess, C.M., Cunningham, D., Carson, R., Harris, D. and Oates, R.D. (1997) Presence of mature sperm in testicular parenchyma of men with non-obstructive azoospermia: prevalence and predictive factors. Urology, 49, 9196.[ISI][Medline]
Nagy, Z., Liu. J., Janssenwillen, C., Silber, S., Devroey, P. and Van Steirteghem, A.C. (1995) Using ejaculated, fresh and frozen-thawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil. Steril., 63, 808815.[ISI][Medline]
Nudell, D.M., Conaghan, J., Pedersen, R.A. Givens, C.R., Schriock, E.D. and Turek, P.J. (1998) The mini-microepididymal sperm aspiration for sperm retrieval: a study of urological outcomes. Hum. Reprod., 13, 12601265.[Abstract]
Oates, R.D., Lobel, S.M., Harris, D.H., Pang, S., Burgess, C.M. and Carson, R.S. (1996) Efficacy of intracytoplasmic sperm injection using intentionally cryopreserved epididymal spermatozoa. Hum. Reprod., 11, 133138.[Abstract]
Oates, R.D., Mulhall, J., Burgess, C., Cunninham, D. and Carson, R. (1997) Fertilization 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]
Palermo, G.D., Schlegel, P.N., Hariprashad, J.J., Ergun, B., Mielnik, A., Zaninovic, N., Veeck, L.L. and Rozenwaks, Z. (1999) Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum. Reprod., 14, 741748.
Perraguin-Jayot, S., Audebert, A., Emperaire, J.C. and Parneix, I. (1998) Ongoing pregnancies after intracytoplasmic injection using cryopreserved testicular spermatozoa. Hum. Reprod., 12, 27062709.[Abstract]
Romero, J., Remohi, J., Minguez, Y., Rubio, C., Pellicer, A. and Gil-Salom, M. (1996) Fertilization after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Fertil. Steril., 65, 877879.[ISI][Medline]
Ron-El, R., Strassburger, D., Friedler, S., Komaroversusky, D., Bern, O., Soffer, Y. and Raziel, A. (1997) Extended sperm preparation: an alternative to testicular sperm extraction in non-obstructive azoospermia. Hum. Reprod., 12, 12221226.[ISI][Medline]
Silber, S.J., Nagy, Z., Devroey, P., Camus, M. and Van Steirteghem, A.C. (1997) The effect of female age and ovarian reserve on pregnancy rate in male infertility: treatment of azoospermia with sperm retrieval and intracytoplasmic sperm injection. Hum. Reprod., 12, 26932700.[Abstract]
Tarlatzis, B. and Bili, H. (1998) Survey on intracytoplasmic sperm injection: report from the ESHRE ICSI Task Force. Hum. Reprod., 13 (Suppl. 1), 165177.[Abstract]
Tournaye, H., Liu, J., Nagy, P.Z. Camus, M., Goossens, A., Silber, S., Van Steirteghem, A. and Devroey, P. (1996) Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum. Reprod., 11, 127132.[Abstract]
Tournaye, H., Verheyen, G., Nagy, P., Ubaldi, F., Goossens, A., Silber, S., Van Steirteghem, A.C. and Devroey, P. (1997) Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum. Reprod., 12, 8086.[ISI][Medline]
Tournaye, H., Merdad, T., Silber, S., Joris, H., Verheyen, G., Devroey, P. and Van Steirteghem, A.C. (1999) No differences in outcome after intracytoplasmic sperm injection with fresh or with frozenthawed epididymal spermatozoa. Hum. Reprod., 14, 9095.
Tuuri, T., Moilanen, J., Kaukoranta, S., Makinen, S., Kotola, S. and Hovatta, O. (1999) Testicular biopty gun needle biopsy in collecting spermatozoa for intracytoplasmic injection, cryopreservation and histology. Hum. Reprod., 14, 12741278.
Ubaldi, F., Nagy, Z.P., Rienzi, L., Tesarik, J., Anniballo, R., Franco, G., Menchini-Fabris, F. and Greco, E. (1999) Reproductive capacity of spermatozoa from men with testicular failure. Hum. Reprod., 14, 27962780.
Van Steirteghem, A.C., Nagy, Z., Joris, H., Liu, J., Staessen, C., Smitz, J., Wisanto, A. and Devroey, P. (1993) High fertilization and implantation rates after intracytoplasmic sperm insemination. Hum. Reprod., 8, 10611066.[Abstract]
Van Steirteghem, A., Nagy, P., Joris, H., Janssenswillen, C., Staessen, C., Verheyen, G., Caus, M., Tournaye, H. and Devroey, P. (1998) Results of intracytoplasmic sperm injection with ejaculated, fresh and frozenthawed epididymal and testicular spermatozoa. Hum. Reprod., 13 (Suppl. 1), 134142.[ISI][Medline]
Vernaeve, V., Tournaye, H., Schiettecatte, J., Verheyen, G., Van Steirteghem, A. and Devroey, P. (2002) Serum inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive azoospermia. Hum. Reprod., 17, 971976.
Vicari, E., Grazioso, C., Burrello, N., Cannizzaro, M., D'Agata, R. and Calogero, A.E. (2001) Epididymal and testicular sperm retrieval in azoospermic patients and the outcome of intracytoplasmic sperm injection in relation to the etiology of azoospermia. Fertil. Steril., 75, 215216.[ISI][Medline]
Von Eckardstein, S., Simoni, M., Bergmann, M., Weinbauer, G.F., Gassner, P., Schepers, A.G. and Niesschlag, E. (1999) Serum inhibin B in combination with serum follicle-stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men, but cannot predict the presence of sperm in testicular tissue samples. J. Clin. Endocrinol. Metab., 84, 24962501.
Westlander, G., Hamberger, L., Hanson, C., Lundin, K., Nilsson L., Söderlund, B.,Werner, C. and Bergh, C. (1999) Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men. Fertil. Steril., 14, 118122.
Submitted on May 23, 2002; accepted on September 9, 2002.