Human Reproduction vol.7 no.2 pp.255-26O, 1992
Differential sperm performance as judged by the zonafree hamster egg penetration test relative to differing sperm penetration techniques
S.Y.W.Chan1 and M.J.Tucker2
Introduction
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA 90048 and 2In-Vitro Fertilization Program, Reproductive Biology Associates, Atlanta, GA 30342, USA
The zona-free hamster egg penetration test (HEPT) has been used in many laboratories to predict male fertility (Blasco, 1984; Prasad, 1984; Yanagimachi, 1984; Barros et al, 1988; Margalioth et al., 1989; Gwatkin et al., 1990), to measure human sperm function after fertility or contraception therapy (Mygatt et al., 1982; Rogers, 1985; Plymate et al., 1987; Wu and Aitken, 1989) and to assess human sperm function for assisted reproductive technology (Rogers, 1986; Aitken, 1988; Kremer and Jager, 1990; Vazquez-Levin et al. (1990). The procedure of HEPT originally involved multiple washing of the semen sample in culture medium by centrifugation to remove the seminal plasma, before sperm capacitation and co-incubation with zona-free hamster eggs (Yanagimachi et al., 1976). Later, this procedure was modified to include a swim-up step after the initial washing to recover the motile spermatozoa (Wolf and Sokoloski, 1982). Recently, Mortimer (1991) reviewed the literature on human sperm separation techniques and in-vitro fertilization (TVF) of human eggs or zona-free hamster eggs; the evidence was summarized to show diat human sperm performance in HEPT can be improved by using alternative methods of sperm preparation, such as discontinuous Percoll gradient centrifugation. According to Aitken and Clarkson (1988), washing and centrifugal pelleting of unselected sperm populations from human ejaculates can cause production of reactive oxygen species (superoxide and hydroxyl radicals) within the washed sperm pellet, so inducing irreversible damage to die spermatozoa and impairment of their in-vitro fertilizing capacity.
'To whom correspondence should be addressed
A prospective study on 61 unselected semen samples from infertile patients was conducted to evaluate the effects of sperm preparation techniques on the outcomes of the zonafree hamster egg penetration test (HEFT) to assess the in-vitro fertilizing capacity of spermatozoa. Each semen sample was divided into two equal portions before separation of the spermatozoa from seminal plasma either by the single-tube swim-up method, or using a two-layer discontinuous Percoll gradient. Spermatozoa were incubated overnight for initiation of capacitation after which HEPT was performed. The swim-up spermatozoa were further divided into two subgroups before HEPT as follows: with or without (control) treatment for 20 min with 50% (v/v) pooled, human follicular fluid (hFF) which had not been heat-inactivated. It was demonstrated (P < 0.05) that the Percoll-separated spermatozoa exhibited higher penetration scores (percentage penetration rate and penetration index) than the control or the hFF-treated swim-up spermatozoa. A short exposure (20 min) to hFF significantly increased the penetration scores in HEPT for swim-up spermatozoa (P < 0.05) but the average results were still significantly lower (P < 0.05) than those of the Percoll separated spermatozoa, which had received no hFF treatment. Based on these findings, we conclude that the Percoll separation technique is better than the centrifugal pelleting and single-tube swim-up technique for reducing the false-negative results in HEPT. In addition, the use of hFF can significantly improve the performance of the swim-up sperm samples in HEPT. The centrifugal pelleting and swim-up technique might have caused some damage to the swim-up spermatozoa in comparison to the Percoll separated spermatozoa, thus resulting in a poorer performance in the HEPT. This last point might also explain the inability of hFF-treated swim-up spermatozoa to exhibit a comparable performance in HEPT to the Percoll separated spermatozoa, which had not received any hFF treatment.
Key words: human follicularfluid/PercoUgradient/sperm preparation/swim-up/zona-free hamster eggs
Recently, human follicular fluid (hFF) collected from female subjects undergoing induction of superovulation for FVF programmes has been demonstrated significantly to enhance the penetration of zona-free hamster eggs by spermatozoa separated from seminal plasma using conventional washing and swim-up procedures (Yee and Cummings, 1988; Blumenfeld and Nahhas, 1989; Fukuda etal., 1989; Siegel etal., 1990). Apparently, the hFF treatment could be used to reduce the impairment of sperm in-vitro fertilizing capacity caused by the reactive oxygen species generated during the centrifugation and swim-up process (Aitken and Clarkson, 1988; Mortimer, 1991), even though die nature and action mechanisms of the specific stimulatory factors in hFF are still not clearly understood. Modification of the original HEPT procedure to incorporate the hFF treatment step should theoretically increase the sensitivity of the test and minimize falsenegative results obtained using spermatozoa separated by the swim-up method. In a small study (Morales et al., 1991), 12 samples prepared
© Oxford University Press
255
S.Y.W.Chan and MJ.Tucker
either by swim-up or Percoll, showed no significant difference in KEPT results between the two methods. However, there have been no published studies specifically comparing hFF-treated swim-up spermatozoa and spermatozoa separated by other preparation methods, such as the discontinuous Percoll gradient centrifugation, for their performance in HEPT. Thus, we undertook a prospective study to compare HEPT results using Percoll separated spermatozoa and swim-up spermatozoa, which were either treated or not treated with hFF after capacitation.
pellet were washed once in 3 ml Earle's medium containing 0.35% BSA-Earle's medium by centrifugation at 150 g for 10 min to remove the Percoll. After centrifugation, the supernatant was discarded and the sperm pellet resuspended in 0.35% BSA-Earle's medium to give a final motile sperm concentration of 15-20 x 106/ml before overnight (24 h) capacitation in a 5% CO2 humidified incubator, ready for the HEPT procedure on the following day. After capacitation, the motile sperm concentration was adjusted to 3.5 x lO^ml before HEPT.
Materials and methods
Computerized analysis of sperm movement characteristics The movement characteristics of spermatozoa in the original semen samples and post-capacitation Percoll or swim-up (control or hFF-treated) sperm preparations were analysed by the automated CellSoft semen analyser (Cryo Resources Ltd, New York, NY, USA), according to the method previously described (Chan et al., 1989a,b, 1990, 1991). The movement characteristics evaluated by automated CellSoft analysis included the curvilinear velocity (/im/s), linearity (the straightxurvilinear distance ratio x 10), mean amplitude of lateral head displacement (mean ALH; and head beat frequency (Hz; 1/s).
Semen samples A total of 61 semen samples, of which 54 were normozoospermic (sperm concentration >20 x 106/ml) and seven were oligozoospermic (sperm concentration
Differential sperm performance as judged by the zonafree hamster egg penetration test relative to differing sperm penetration techniques
S.Y.W.Chan1 and M.J.Tucker2
Introduction
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA 90048 and 2In-Vitro Fertilization Program, Reproductive Biology Associates, Atlanta, GA 30342, USA
The zona-free hamster egg penetration test (HEPT) has been used in many laboratories to predict male fertility (Blasco, 1984; Prasad, 1984; Yanagimachi, 1984; Barros et al, 1988; Margalioth et al., 1989; Gwatkin et al., 1990), to measure human sperm function after fertility or contraception therapy (Mygatt et al., 1982; Rogers, 1985; Plymate et al., 1987; Wu and Aitken, 1989) and to assess human sperm function for assisted reproductive technology (Rogers, 1986; Aitken, 1988; Kremer and Jager, 1990; Vazquez-Levin et al. (1990). The procedure of HEPT originally involved multiple washing of the semen sample in culture medium by centrifugation to remove the seminal plasma, before sperm capacitation and co-incubation with zona-free hamster eggs (Yanagimachi et al., 1976). Later, this procedure was modified to include a swim-up step after the initial washing to recover the motile spermatozoa (Wolf and Sokoloski, 1982). Recently, Mortimer (1991) reviewed the literature on human sperm separation techniques and in-vitro fertilization (TVF) of human eggs or zona-free hamster eggs; the evidence was summarized to show diat human sperm performance in HEPT can be improved by using alternative methods of sperm preparation, such as discontinuous Percoll gradient centrifugation. According to Aitken and Clarkson (1988), washing and centrifugal pelleting of unselected sperm populations from human ejaculates can cause production of reactive oxygen species (superoxide and hydroxyl radicals) within the washed sperm pellet, so inducing irreversible damage to die spermatozoa and impairment of their in-vitro fertilizing capacity.
'To whom correspondence should be addressed
A prospective study on 61 unselected semen samples from infertile patients was conducted to evaluate the effects of sperm preparation techniques on the outcomes of the zonafree hamster egg penetration test (HEFT) to assess the in-vitro fertilizing capacity of spermatozoa. Each semen sample was divided into two equal portions before separation of the spermatozoa from seminal plasma either by the single-tube swim-up method, or using a two-layer discontinuous Percoll gradient. Spermatozoa were incubated overnight for initiation of capacitation after which HEPT was performed. The swim-up spermatozoa were further divided into two subgroups before HEPT as follows: with or without (control) treatment for 20 min with 50% (v/v) pooled, human follicular fluid (hFF) which had not been heat-inactivated. It was demonstrated (P < 0.05) that the Percoll-separated spermatozoa exhibited higher penetration scores (percentage penetration rate and penetration index) than the control or the hFF-treated swim-up spermatozoa. A short exposure (20 min) to hFF significantly increased the penetration scores in HEPT for swim-up spermatozoa (P < 0.05) but the average results were still significantly lower (P < 0.05) than those of the Percoll separated spermatozoa, which had received no hFF treatment. Based on these findings, we conclude that the Percoll separation technique is better than the centrifugal pelleting and single-tube swim-up technique for reducing the false-negative results in HEPT. In addition, the use of hFF can significantly improve the performance of the swim-up sperm samples in HEPT. The centrifugal pelleting and swim-up technique might have caused some damage to the swim-up spermatozoa in comparison to the Percoll separated spermatozoa, thus resulting in a poorer performance in the HEPT. This last point might also explain the inability of hFF-treated swim-up spermatozoa to exhibit a comparable performance in HEPT to the Percoll separated spermatozoa, which had not received any hFF treatment.
Key words: human follicularfluid/PercoUgradient/sperm preparation/swim-up/zona-free hamster eggs
Recently, human follicular fluid (hFF) collected from female subjects undergoing induction of superovulation for FVF programmes has been demonstrated significantly to enhance the penetration of zona-free hamster eggs by spermatozoa separated from seminal plasma using conventional washing and swim-up procedures (Yee and Cummings, 1988; Blumenfeld and Nahhas, 1989; Fukuda etal., 1989; Siegel etal., 1990). Apparently, the hFF treatment could be used to reduce the impairment of sperm in-vitro fertilizing capacity caused by the reactive oxygen species generated during the centrifugation and swim-up process (Aitken and Clarkson, 1988; Mortimer, 1991), even though die nature and action mechanisms of the specific stimulatory factors in hFF are still not clearly understood. Modification of the original HEPT procedure to incorporate the hFF treatment step should theoretically increase the sensitivity of the test and minimize falsenegative results obtained using spermatozoa separated by the swim-up method. In a small study (Morales et al., 1991), 12 samples prepared
© Oxford University Press
255
S.Y.W.Chan and MJ.Tucker
either by swim-up or Percoll, showed no significant difference in KEPT results between the two methods. However, there have been no published studies specifically comparing hFF-treated swim-up spermatozoa and spermatozoa separated by other preparation methods, such as the discontinuous Percoll gradient centrifugation, for their performance in HEPT. Thus, we undertook a prospective study to compare HEPT results using Percoll separated spermatozoa and swim-up spermatozoa, which were either treated or not treated with hFF after capacitation.
pellet were washed once in 3 ml Earle's medium containing 0.35% BSA-Earle's medium by centrifugation at 150 g for 10 min to remove the Percoll. After centrifugation, the supernatant was discarded and the sperm pellet resuspended in 0.35% BSA-Earle's medium to give a final motile sperm concentration of 15-20 x 106/ml before overnight (24 h) capacitation in a 5% CO2 humidified incubator, ready for the HEPT procedure on the following day. After capacitation, the motile sperm concentration was adjusted to 3.5 x lO^ml before HEPT.
Materials and methods
Computerized analysis of sperm movement characteristics The movement characteristics of spermatozoa in the original semen samples and post-capacitation Percoll or swim-up (control or hFF-treated) sperm preparations were analysed by the automated CellSoft semen analyser (Cryo Resources Ltd, New York, NY, USA), according to the method previously described (Chan et al., 1989a,b, 1990, 1991). The movement characteristics evaluated by automated CellSoft analysis included the curvilinear velocity (/im/s), linearity (the straightxurvilinear distance ratio x 10), mean amplitude of lateral head displacement (mean ALH; and head beat frequency (Hz; 1/s).
Semen samples A total of 61 semen samples, of which 54 were normozoospermic (sperm concentration >20 x 106/ml) and seven were oligozoospermic (sperm concentration
