1 Department of Urology, University of Giessen and 2 Department of Urology, University of Cologne and 3 Outpatient Department of Obstetrics and Gynaecology, Bad Münder and 4 Outpatient Department for Reproductive Medicine, Cologne and 5 Clinic for Reproductive Medicine GmbH, Bad Münder, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: assisted reproductive techniques/infertility/microsurgical epididymal sperm aspiration/obstructive azoospermia
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MESA combined with intracytoplasmic sperm injection (ICSI) has increased the number of pregnancies (Silber et al., 1994). The fertilization rate is not affected by the kind of obstruction (congenital or acquired) and can be as high as 90% per cycle (Silber et al., 1995
). At present, pregnancy rates of between 14 and 66% per cycle are achieved when MESA is combined with ICSI, while the live birth rate is as high as 36% per cycle (Belker et al., 1994
; Silber et al., 1995
; Ubaldi et al., 1995
; Zumbé et al., 1996
; Bispink et al., 1997
; Schroeder-Printzen et al., 1997
).
The synchronized combination of MESA and ICSI requires a great number of logistic facilities and involves the hormonal stimulation of the female partner pre-operatively, even though at this point it is not certain whether spermatozoa will be retrievable at all. When epididymal aspiration with subsequent cryopreservation is dissociated from ICSI, hormonal stimulation of the female is started after it has been clarified that enough motile spermatozoa are available for ICSI (Gips et al., 1996). There are no data in the literature which show a significant difference in clinical pregnancy rates when using fresh or cryopreserved epididymal spermatozoa (Oates et al., 1996
; Chang et al., 1999
; Tournaye et al., 1999
). Thus damage to the spermatozoa caused by cryopreservation seems to be tolerable (Holden et al., 1997
; Devroey et al., 1995
).
Successful sperm retrieval does not depend on the different variations of the microsurgical technique (Matthews and Goldstein, 1996; Oates et al., 1996
; Holden et al., 1997
). It seems clear that the greatest numbers of motile spermatozoa (mean 413%) are to be found in the caput of the epididymis (Silber et al., 1995
; Matthews and Goldstein, 1996
; Oates et al., 1996
; Holden et al., 1997
). Aspiration is normally not difficult in this compartment (Devroey et al., 1995
) and successful retrieval can be as high as 100%. The aspirate fluid which is not used for an intentional ICSI is cryopreserved (Silber et al., 1997
). There are only limited data on the number of vials taken for ICSI and the quality of the cryopreserved spermatozoa in the above-mentioned literature. So far only Oates et al. have reported on the number of cryopreserved vials retrieved (Oates et al., 1996
). In their study they were able to obtain 310 cryopreserved vials when sperm harvesting was done on a different day to the ICSI procedure, while the number of vials was no more than three when both procedures were performed on the same day (Oates et al., 1996
).
In this study the day of sperm retrieval was separated from the day of ICSI procedure with a view to analysing the amount and quality of the spermatozoa aspirated under these conditions using a standardized MESA technique (Köhn et al., 1996; Schroeder-Printzen et al., 1996
). In particular, the motility after thawing and the amount of cryopreservable straws were evaluated. Furthermore, the overall outcome, including the pregnancy and take home baby rate of ICSI cycles, using the cryopreserved epididymal spermatozoa, were analysed.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The predominant indications for MESA were CBAVD and failed microsurgical reconstruction (Table I). These two indications were analysed separately with regard to the patient's age, sperm concentration and global motility in the aspirate, motility after thawing and number of cryopreserved straws.
|
Fifty-five patients were hospitalized for 1 day. Thirty-eight patients who were operated on outpatient conditions were allowed to leave 4 h after the end of the operation. They were examined the next day for haematoma or infection.
MESA was performed according to the standards of the German MESA/testicular sperm extraction (TESE) group (Zumbé et al., 1996). This standard has already been described (Schroeder-Printzen et al., 1996
). MESA was performed under general anaesthesia. After scrotal exploration, spermatozoa were sampled from the microsurgically opened epididymal tubule with a micropipette or a 24-gauge cannula using a binocular microscope (magnification x15). One drop (10 µl) was filled into a haemocytometer (Makler® chamber) and was immediately examined for the presence of motile spermatozoa using a phase-contrast microscope (magnification x400). This procedure started at the cauda epididymis. If no motile spermatozoa were found, another tubule was opened 0.5 cm above the first one, until motile spermatozoa were aspirated. Sometimes it was necessary to open the ductuli efferentes. The opened tubules were closed by single stitches using 110 sutures. After examination, the samples were placed into a pre-warmed and pre-filled Eppendorf chamber®. The medium used for pre-filling the chamber was either buffered Earle's medium (Devroey et al., 1995
), human tubular fluid medium [HTFM (Hovatta et al., 1995
)] plus 1% serum albumin (Behringer, Marburg, Germany) or HEPES-buffered Ham's F-10 (Sigma, Deisenhofen, Germany). Afterwards these probes were taken to the laboratory using an aluminium chamber which had been pre-heated to 37°C. Sperm concentration and motility were examined. The examination was followed by sperm preparation using the migration-sedimentation technique (Tea et al., 1983
). When sperm quality was >2x106 motile spermatozoa, a sperm preparation was not performed (Bispink et al., 1997
). After mixing the sperm suspension with equal volumes of a cryoprotectant, the samples were filled in straws (Minitüb®; Landshut, Germany) each with a volume of 250 µl (Devroey et al., 1995
). In the case of high amounts of motile spermatozoa, the aspirated fluid was diluted in order to increase the number of straws. The concentration was adjusted to 15x106 motile spermatozoa. The filled straws were stored in a computer-controlled freezer (Kryo 10 Serie III®; Messer Griesheim, Krefeld, Germany) and automatically frozen at 10°C/min.
Samples in the straws were thawed for straw examination and ICSI cycles at room temperature. Sperm density and motility were examined for a second time. No straws were thawed for examination in cases where there were four straws or fewer. At the beginning of the study, the gynaecologists decided in some cases to use fresh spermatozoa only, and therefore the semen was not cryopreserved. The remaining straws were counted and divided into four groups (no straws, 4 straws, 510 straws, >10 straws).
Standard protocols for ovarian stimulation were used (Gips et al., 1996). Oocytes were aspirated by transvaginal, ultrasound-guided follicle puncture. Adherent cumulus cells were removed by exposure to hyaluronidase (80 IU/ml) for 1 min. The remaining parts of the cumulus were removed mechanically. ICSI procedure was carried out under direct visualization with phase-contrast optics and the assistance of electromechanical micromanipulators. Injected oocytes were maintained in drops of HTF supplement with human plasma proteins (Baxter, Unterschleißheim, Germany) and examined for the formation of a second pronucleus at 1622 h. Up to three pronuclei were allowed to undergo further development during the ensuing 48 h. The surplus pronuclei were frozen or aborted. A flexible catheter was used for transcervical transfer.
For statistical analysis the Statistical Package for Social Science (SPSS for windows, Release 7.0; SPSS Inc., Chicago, IL, USA) and the MannWhitney U-test were used.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The decisive question for this procedure is how many vials can be cryopreserved after sperm preparation? Up to now, only Oates et al. (1996) have published data concerning cases involving an asynchronous procedure. Similar to this study, they analysed how many vials are available when MESA and ICSI are not performed simultaneously. They obtained 6.4 vials/patient, compared with 7.6 vials in the current study. The same group harvested only 2.8 vials during MESA, when the operation was carried out under the condition that hormonal stimulation and oocyte harvesting of the female partner were performed on the same day, thus making it necessary to use the freshly harvested spermatozoa immediately (Oates et al., 1996). These data provide evidence that during asynchronous epididymal sperm aspiration, as already claimed by Oates et al. (1996), a high number of vials is always available, thus giving additional arguments for separating MESA from the oocyte retrieval procedure from an andrological-surgical point of view.
Using cryopreserved epididymal spermatozoa, the pregnancy rate of 42.4% is within the range given in the current literature (Silber et al., 1995; Oates et al., 1996
), thus underlining the comparative efficiency of using cryopreserved spermatozoa in comparison to fresh spermatozoa for ICSI procedures (Devroey et al., 1995
; Silber et al., 1995
; Oates et al., 1996
).
In conclusion, the results of this study demonstrate MESA as a successful procedure for retrieving spermatozoa in 94% of all men with obstructed azoospermia. Using this method, enough vials can be cryopreserved for a high number of consecutive ICSI cycles. As a high number of cryopreserved vials is available, a second operation becomes unnecessary in most of the patients. It is suggested that this constitutes a definite advantage in comparison to percutaneous retrieval methods (Collins et al., 1996), since the possibility of collecting spermatozoa for cryopreservation is successful in only 3354% of the cases (Levine and Lisek, 1998
; Rosenlund et al., 1998
), so that further PESA procedures will become necessary. Even when percutaneous epididymal sperm aspiration is successful in further attempts (Meniru et al., 1998
), there is an increasing risk for post-surgical complications. The recently suggested `mini-MESA technique' in sedoanalgesia, which has a success rate in cryopreservation of 92%, may be an alternative for the future (Nudell et al., 1998
). This technique would combine the advantages of the MESA method (a predictably high number of preservable sperm) with the low invasiveness of the percutaneous method.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
* Members of the MESA/TESE GROUP GIESSEN in alphabetical order: H.J.G.Herrero, F.Köhn, W.Künzel, W.-B.Schill, I.Schroeder-Printzen, R.Stalf, W.Weidner.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bispink, L., Schröter, D., Schroeder-Printzen, I. et al. (1997) Intracytoplasmatische Spermatozoeninjektion mit operativ gewonnenen Spermatozoen. Geburtsh. u. Frauenheilk., 57, 6265.[ISI]
Chang, H.C., Hsieh, J.T., Liu, S.P. et al. (1999) Fertilization capability of frozen epididymal sperm for intracytoplasmatic sperm injection. J. Formos. Med. Assoc., 98, 171174.[ISI][Medline]
Chen, C.S., Chu, S.H., Soong, Y.K. et al. (1995) Epididymal sperm aspiration with assisted reproductive techniques: difference between congenital and acquired obstructive azoospermia. Hum. Reprod., 10, 11041108.[Abstract]
Collins, G.N., Critchlow, J.D., Lau, M.W.M. et al. (1996) Open versus closed epididymal sperm retrieval in men with secondarily obstructed vasal systems | a preliminary report. Br. J. Urol., 78, 437439.[ISI][Medline]
Deutsche Gesellschaft für Gynäkologie und Geburtshilfe/Arbeitsgemeinschaft für Gynäkologische Endokrinologie und Fortpflanzungsmedizin (1995) Empfehlungen zur Durchführung der intrazytoplasmatischen Spermieninjektion (ICSI) als Zusatzmaßnahme bei IVF-ET-Therapie. Frauenarzt, 36, 818819.
Devroey, P., Silber, S.J., Nagy, Z.P. et al. (1995) Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen-thawed epididymal spermatozoa. Hum. Reprod., 10, 903906.[Abstract]
Gips, H., Hormel, P. and Hinz, V. (1996) Ovarian stimulation in assisted reproduction. Andrologia, 28 (Suppl. 1), 37.
Girardi, S.K. and Schlegel, P.N. (1996) Microsurgical epididymal sperm aspiration. Urol. Clin. North. Am. (Atlas), 4, 5566.
Holden, C., Fuscaldo, G.F., Jackson, P. et al. (1997) Frozen-thawed epididymal spermatozoa for intracytoplasmatic sperm injection. Fertil. Steril., 67, 8187.[ISI][Medline]
Hovatta, O., Moilanen, J., von Smitten, K. et al. (1995) Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmatic sperm injection in obstructive azoospermia. Hum. Reprod., 10, 25952599.[Abstract]
Köhn, F.M., Schroeder-Printzen, I., El Mulla, K.F. et al. (1996) Andrological work up of patients undergoing microsurgical epididymal sperm aspiration or testicular sperm extraction. Andrologia, 28 (Suppl. 1), 7781.[ISI][Medline]
Levine, L.A. and Lisek, E.W. (1998) Successful sperm retrieval by percutaneous epididymal and testicular sperm aspiration. J. Urol., 159, 437440.[ISI][Medline]
Matthews, G.J. and Goldstein, M. (1996) A simplified method of epididymal sperm aspiration. Urology, 47, 123125.[ISI][Medline]
Meniru, G., Gorgy, A., Batha, S. et al. (1998) Studies of percutaneous epididymal aspiration (PESA) and intracytoplasmatic sperm injection. Hum. Reprod. Update, 4, 5771.
Nudell, D.M., Conaghan, J., Pedersen, R.A. et al. (1998) The mini-micro-epididymal sperm aspiration for sperm retrieval: a study of urological outcomes. Hum. Reprod., 13, 12601265.[Abstract]
Oates, R.D., Lobel, S.M., Harris, D.H. et al. (1996) Efficacy of intracytoplasmatic sperm injection using intentionally cryopreserved epididymal spermatozoa. Hum. Reprod., 11, 133138.[Abstract]
Rosenlund, B., Westlander, G., Wood, M. et al. (1998) Sperm retrieval and fertilization in repeated percutaneous epididymal sperm aspiration. Hum. Reprod., 13, 28052807.
Schroeder-Printzen I., Köhn, F.-M., Ludwig, M. et al. (1996) Mikrochirurgische Spermatozoenaspiration (MESA) und TEstikuläre Spermatozoen Extraktion (TESE). Aktuel. Urol., 27, Operative Techniken 6.33.
Schroeder-Printzen I., Köhn, F.-M., Ludwig, M. et al. (1997) Mikrochirurgische Spermatozoenaspiration (MESA) und TEstikuläre Spermatozoen Extraktion (TESE) eine Übersicht Aktuel. Urol., 28, 251259.[ISI]
Silber, S.J., Nagy, Z.P., Devroey, P. et al. (1997) The effect of female age and ovarian reserve on pregnancy rates in male infertility: treatment of azoospermia with sperm retrieval and intracytoplasmatic sperm injection. Hum. Reprod., 12, 26932700.[Abstract]
Silber S.J., Nagy, Z.P., Liu, J. et al. (1994) Conventional in-vitro fertilization versus intracytoplasmatic sperm injection for patients requiring microsurgical sperm aspiration. Hum. Reprod., 9, 17051709.[Abstract]
Silber S.J., Nagy, Z.P., Liu, J. et al. (1995) The use of epididymal and testicular spermatozoa for intracytoplasmatic sperm injection: the genetic implications for male infertility. Hum. Reprod., 10, 20312043.[Abstract]
Tea, N.T., Jonded, M. and Scholler, R. (1983) A migration-gravity sedimentation method for collecting human motile spermatozoa from human semen. In Harrison, R.F., Bonnaraud, J. and Thompson, W. (eds), In Vitro Fertilization, Embryo Transfer and Early Pregnancy. MTP Press, pp. 117120.
Tournaye, H., Merdad, T., Silber, S. et al. (1999) No differences in outcome after intracytoplasmatic sperm injection with fresh or with frozen-thawed epididymal spermatozoa. Hum. Reprod., 14, 9095
Ubaldi, F., Liu, J., Nagy, Z.P. et al. (1995) Indications for and results of intracytoplasmatic sperm injection (ICSI). Int. J. Androl., 18 (Suppl. 2), 8890.
Zumbé, J., Beintker, M., Denil, J. et al. (1996) Experiences of the German Section for Urological Microsurgery. Andrologia, 28 (Suppl. 1), 8992.
Submitted on December 8, 1999; accepted on September 15, 2000.