The use of a modified hypo-osmotic swelling test for the selection of viable ejaculated and testicular immotile spermatozoa in ICSI

Human Reproduction The use of a modified hypo-osmotic swelling test for the selection of viable ejaculated and testicular immotile spermatozoa in ICSI H.N.Sallam 0 1 A.Farrag 0 A.F.Agameya 0 1 F.Ezzeldin 0 A.Eid 0 A.Sallam 0 1 0 The Alexandria Fertility Centre , Alexandria , Egypt 1 Department of Obstetrics and Gynaecology, the University of Alexandria A modified hypo-osmotic solution was used to select viable ejaculated and testicular spermatozoa to perform intracytoplasmic sperm injection (ICSI) in 27 treatment cycles from patients with total absence of sperm motility. The treatment cycles consisted of 15 cycles in which ejaculated spermatozoa were used and 12 cycles in which testicular spermatozoa were used. The hypo-osmotic solution consisted of 50% culture medium and 50% deionized water and was shown in previous in-vitro studies to be superior to the original solution used in the classical hypo-osmotic swelling test. Fertilization was achieved in 37.3% of the oocytes injected. Embryos were replaced in 70.4% of the cycles with a mean of 2.0 embryos per cycle. There were no statistically significant differences between the ejaculated sperm group and the testicular sperm group in the fertilization rate (42.7 versus 30.1%) or in the cleavage rate (92.7 versus 77.3%). Four pregnancies resulted, two in the ejaculated sperm group and two in the testicular sperm group, a pregnancy rate of 14.8%. All pregnancies were singletons but one pregnancy in each group had an early miscarriage. There were no statistically significant differences between both groups in the pregnancy rates (13.3 versus 16.7%), in the implantation rates (5.3 versus 11.8%) or in the delivery/ongoing pregnancy rates (6.7 versus 8.3%). It is concluded that the use of this solution to select viable but immotile spermatozoa for ICSI is a simple and practical method and is associated with acceptable fertilization and pregnancy rates. azoospermia/hypo-osmotic swelling test/immotile spermatozoa/intracytoplasmic sperm injection/testicular spermatozoa - Intracytoplasmic sperm injection (ICSI) is now an established method for the treatment of male infertility in cases with oligoasthenozoospermia (Bonduelle et al., 1999) and even azoospermia (Silber et al., 1996; Aboulghar et al., 1997; Palermo et al., 1999, Bonduelle et al., 1999). However, in some cases, no motile spermatozoa can be found in the ejaculate or in the testicular sperm preparation. In these cases, fertilization and pregnancies have been reported after the injection of immotile ejaculated spermatozoa (Nijs et al., 1996; Barros et al., 1997; Vandervorst et al., 1997; Ved et al., 1997; Wang et al., 1997; Nagy et al., 1998) and even testicular spermatozoa (Nijs et al., 1996; Kahraman et al., 1996; Shulman et al., 1999). However, the fertilization and pregnancy rates are lower when compared to the injection of motile spermatozoa (Nagy et al., 1998; Shulman et al., 1999). In order to differentiate dead spermatozoa from viable but immotile spermatozoa, different techniques have been suggested to select the spermatozoa used for the ICSI procedure. For example, Tasdemir et al. suggested the addition of pentoxifylline to the testicular sperm preparation (Tasdemir et al., 1998). Alternatively, the hypo-osmotic swelling test was used to select immotile but viable spermatozoa from the ejaculated processed semen sample (Casper et al., 1996; Ved et al., 1997; Wang et al., 1997) but, to our knowledge, no reports are available on the use of the test for the selection of testicular immotile spermatozoa. The original Jeyendran solution, consisting of a mixture of 75 mmol/l fructose and 25 mmol/l sodium citrate dehydrate, was originally used as a sperm function test to evaluate the integrity of the sperm membrane (Jeyendran and Zaneveld, 1986; Jeyendran et al., 1992; WHO, 1992). It was later used in ICSI procedures to select immotile spermatozoa from the ejaculate but its effect on the fertilized oocyte or resulting embryos has not been evaluated. A different hypoosmotic solution containing 150 mOsm NaCl was suggested by Tsai et al. and Liu et al. used it for the selection of immotile ejaculated spermatozoa used in ICSI and reported one pregnancy (Tsai et al., 1997; Liu et al., 1997). In 1997, Verheyen et al. performed an in-vitro study and compared three hypo-osmotic solutions: the original Jeyendran solution, deionized-grade water and a solution consisting of Stimulation protocol and oocyte retrieval 50% culture medium and 50% deionized-grade water (Verheyen et al., 1997). They found that although the three solutions resulted in swelling and tail-curling in the immotile but viable spermatozoa, the delayed harmful effects on sperm vitality were least with the 50/50 solution. In this study, we report our experience in using this solution to select immotile but viable spermatozoa from ejaculated and testicular sperm preparations for use in our ICSI programme. Materials and methods From 28 March 1999 until 27 March 2000, a total of 27 couples underwent ICSI procedures in our centre using totally immotile spermatozoa. During the same period a total of 426 couples with male factor infertility were treated with ICSI. The study population consisted of 15 couples undergoing ICSI from ejaculated spermatozoa and 12 couples undergoing ICSI from testicular spermatozoa (TESEICSI). In all cases no motile spermatozoa were found in the ejaculate or in the testicular sperm preparation even after a short period of incubation (30 min). The mean age ( SD) of the female partners was 32.6 6.1 years (33.1 years 5.8 in the ejaculated sperm group compared to 31.9 5.8 in the testicular sperm group). This difference was not statistically significant. The mean age of the male partners was 40.0 6.9 years (38.2 years 7.6 in the ejaculated sperm group compared to 44.5 7.5 in the testicular sperm group). This difference was also not statistically significant. These data are summarized in Table 1. In the ejaculated sperm group (n 15), the median sperm concentration was 1.6 106 spermatozoa/ml (range 0.137 106/ml). Five of the patients had sperm counts of less than 1 106 and strict morphology could not be assessed. In the remaining 10 patients, the median percentage of strict morphology was 3% (range 15%). Two patients were treated for initial leukospermia with appropriate antibiotics after culture and sensitivity tests but the sperm motility did not improve. All patients were offered the possibility of testicular sperm extraction, as suggested by Tournaye et al. (1996), but all declined the offer. In the testicular sperm group (n 12), all patients were suffering from non-obstructive azoospermia. Initial testicular biopsies showed spermatogenic arrest in seven patients, hypospermatogenesis in four patients and Sertoli cells only in one patient. In one couple undergoing TESEICSI, immotile spermatozoa were obtained from a fresh testicular biopsy while in the other 11 couples, the spermatozo (...truncated)