The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study

Advance Access publication August The use of the modified hypo-osmotic swelling test for the selection of immotile testicular spermatozoa in patients treated with ICSI: a randomized controlled study Hassan N.Sallam 0 2 Ashraf Farrag 0 2 Abdel-Fattah Agameya 0 2 Yehia El-Garem 0 1 Fathy Ezzeldin 0 0 Alexandria Fertility Centre , Alexandria , Egypt 1 Andrology, the University of Alexandria and the 2 Obstetrics and Gynaecology BACKGROUND: Various methods for the selection of viable sperm from among immotile testicular spermatozoa for use in ICSI have been described in non-randomized studies. We have conducted a randomized controlled study to compare the use of the modified hypo-osmotic swelling (HOS) test (50% culture medium + 50% Milli-Q grade water) with that of sperm selection on the basis of their morphology alone. METHODS: A total of 79 couples with immotile testicular spermatozoa treated with ICSI were randomly assigned into two groups. In the first group, spermatozoa used for injection were selected using the modified HOS test, while in the second group spermatozoa were selected on the basis of their morphology. RESULTS: The fertilization rate was significantly higher in the HOS test group (43.6%) compared with the no-HOS test group (28.2%) [odds ratio (OR) 2.494; 95% confidence interval (CI) 1.606-3.872]. The pregnancy and ongoing pregnancy rates were also higher in the HOS test group (27.3% versus 20.5%) compared with the no-HOS test group (5.7% versus 2.9%) (OR 6.188, 95% CI 1.282-29.860; and OR 8.743, 95% CI 1.050-72.783, respectively). CONCLUSIONS: The use of the modified HOS test for the selection of viable sperm from among immotile testicular spermatozoa for ICSI results in higher fertilization, pregnancy and ongoing pregnancy rates compared with morphological selection. azoospermia/hypo-osmotic swelling test/ICSI/immotile spermatozoa/total asthenospermia Introduction ICSI using fresh or frozenthawed testicular sperm is now an established method for the treatment of male infertility due to azoospermia (Palermo et al., 1999; Schlegel, 2004; Verheyen et al., 2004). In these cases, fresh or frozenthawed testicular spermatozoa used for the injection of oocytes are selected on the basis of their morphology and motility. However, in some cases of azoospermia, all testicular spermatozoa are immotile, making this selection difficult. Numerous publications have reported that the haphazard selection of immotile testicular or ejaculated spermatozoa for use in ICSI or their selection on the sole basis of morphology is associated with significantly diminished pregnancy and fertilization rates (Kahraman et al., 1996; Nijs et al., 1996; Vandervorst et al., 1997; Nagy et al., 1998; Abu-Musa et al., 1999; Shibahara et al., 1999a; b; Shulman et al., 1999). In order to improve the outcome of ICSI in these cases, various methods have been described for the selection of the immotile but viable spermatozoa. These include the addition of pentoxifyllin (Tasdemir et al., 1998; Terriou et al., 2000), the mechanical touch technique (de Oliveira et al., 2004), the laser touch technique (Aktan et al., 2004) and performing the hypo-osmotic swelling (HOS) test (Jeyendran and Zaneveld, 1986; Jeyendran et al., 1992) using the original Jeyendran solution (Casper et al., 1996; Ved et al., 1997; Peeraer et al., 2004). As the Jeyendran solution has not been adequately tested on human spermatozoa and is meant to be used in vitro, we have recently reported the successful use of the modified HOS test proposed by Verheyen in an in-vitro study, for the selection of immotile but viable testicular spermatozoa (Sallam et al., 2001). The Verheyen hypo-osmotic solution consists of a mixture of 50% culture medium and 50% Milli-Q grade water (Verheyen et al., 1997a; b). However, no previous observations, including our own work, have been confirmed by randomized controlled trials (RCTs). The aim of the present work was to conduct a RCT to evaluate the use of the modified HOS test for the selection of immotile but viable spermatozoa in cases of ICSI using testicular sperm (TeSE-ICSI). Materials and methods Patients All couples treated in our centre between January 2002 and August 2004 by ICSI using testicular sperm due to azoospermia (n = 316) The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. 3435 For Permissions, please email: were eligible for the study (71 with obstructive azoospermia and 245 with non-obstructive azoopsermia). Of those, the 79 couples whose testicular biopsy revealed spermatozoa with total absence of motility were included in the study in their first cycle of treatment (Figure 1). All 79 male partners were diagnosed with non-obstructive azoospermia. Patients whose testicular biopsies revealed motile (n = 156) or no (n = 81) spermatozoa were excluded from the study. The couples were randomly assigned at the time of ICSI using a closed envelope system to one of two groups. In patients belonging to the first group (HOS test group, n = 44, consisting of 25 fresh and 19 frozen testicular sperm), the modified HOS was performed on the immotile spermatozoa and only reactive spermatozoa were used for the injection, while in the second group (noHOS test group, n = 35, consisting of 20 fresh and 15 frozen testicular sperm), spermatozoa used for injection were selected on the basis of morphology (i.e. normal head and intact tail). The envelopes were drawn by the patients, who were blinded to the type of intervention. We calculated that in order to show an improvement in our clinical pregnancy rate from 10% to 30%, accepting an 80% probability of detecting a true difference and taking 5% as the level of significance, a minimum of 34 couples would have to be studied in each arm of the trial. The mean age ( SD) of the female partners was 31.4 ( 6.0) years in the HOS test group compared with 32.3 ( 5.4) in the noHOS test group (P = 0.48). The mean age ( SD) of the male partners was 39.8 ( 6.6) years in the HOS test group compared with 41.3 ( 8.4) in the no-HOS test group (P = 0.39). The duration of infertility ( SD) was 8.6 ( 5.7) years in the HOS test group compared with 8.6 ( 6.2) years in the no-HOS test group (P = 0.99). Informed consent was obtained from all couples and the study was approved by the Ethical Committee of the Alexandria Fertility Center. Randomised (n = 79) Discontinued (n = 0) Analysed (n = 44) Excluded (n = 237) Not meeting inclusion criteria (n = 237) Refused (n = 0) Other reasons (n = 0) Discontinued (n = 0) Analysed (n = 35) Stimulation protocol and oocyte retrieval All female partners were stimulated using the short down-regulation protocol and HMG was administered by daily intramuscular injection. The mean ( SD) number of HMG ampoules was 31.9 8.9 in the HOS test group compared with 32.3 ( 7.0) in the no-HOS test group (P = 0.83). Monitoring of the ovarian fol (...truncated)