Human Reproduction vol.5 no.3 pp.298-303, 1990
Relationships between human sperm acrosin, acrosomes, morphology and fertilization in vitro
D.Y.Liu1 and H.W.G.Baker Department of Obstetrics and Gynaecology, University of Melbourne and Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia 'To whom correspondence should be addressed
Acrosin was measured in the semen used for sperm preparation for in-vitro fertilization (1VF) in 118 patients. Acrosin levels correlated with the proportion of spermatozoa with normal intact acrosomes determined with Pisum sativum agglutinin labelled with fluorescein. However, acrosin levels and the proportion of spermatozoa with normal intact acrosomes in semen were not significantly related to the fertilization rate in vitro. Only the percentage normal morphology and sperm concentration in the insemination medium were independently significantly related to the fertilization rate by logistic regression analysis. In patients with fewer than 30% of spermatozoa with normal morphology, although acrosin levels were not correlated with the fertilization rate, the proportion of spermatozoa with normal intact acrosomes in the insemination medium was the only significant factor in the logistic regression analysis. In conclusion, acrosin levels have no prognostic value for fertilization in vitro but the proportion of spermatozoa with normal intact acrosomes may be a useful clinical marker of fertilizing ability in men with poor sperm morphology. Key words: acrosin/acrosomes/IVF/morphology
Introduction Although semen quality is related to the success of human invitro fertilization (IVF) (Mahadevan and Trounson, 1984; Jeulin et al., 1986), the standard semen characteristics are of limited value in predicting IVF rates (Belaisch-Allart et al, 1984; Edwards et al., 1984; Garcia et al., 1984; Trounson and Wood, 1984). Recent studies have shown that sperm morphology is one of the most significant factors related to fertilization rate (Kruger et al, 1986, 1988a,b; Liu etal, 1987, 1988; Liu and Baker, 1988). However, some patients with poor sperm morphology (abnormal >80%) still fertilize most or all of the oocytes inseminated (Liu et al, 1987, 1988). Thus additional tests of sperm function are necessary to improve the prediction of sperm fertilizing ability in vitro. It is known that the acrosome is important for fertilization both in vivo and in vitro. The morphological and functional integrity of the acrosome and the ability of sperm to undergo the acrosome reaction are essential for species-specific zona binding and 298 Downloaded from https://academic.oup.com/humrep/article-abstract/5/3/298/737907 by University of Durham user on 13 January 2018
penetration and fusion with the oolemma (Soupart and Strong, 1974; Yanagimachi, 1981; Sathananthan etal, 1982; Singer et al, 1985; Chen and Sathananthan, 1986; Moore and Bedford, 1983). Spermatozoa with small round heads or normal shaped spermatozoa with abnormally small acrosomes are associated with male infertility (Jeyendran etal, 1985; Schill etal, 1988). Round-headed spermatozoa without acrosomes do not have the ability to bind or penetrate zona-free hamster ova (Weissenberg et al., 1983). However, the condition of completely acrosome free spermatozoa is rare. Acrosin, a neutral proteinase, is one of the most important acrosomal enzymes and is believed to play a role in sperm-zona penetration (Rogers and Bentwood, 1982). It has been reported that fertilization does not oocur when the activity of this enzyme is inhibited (Zaneveld, 1976). It has been suggested also that low acrosin activity is associated with male infertility (Goodpasture et al., 1980; Mohsenian et al., 1982) and low fertilization rates in vitro (Acosta etal, 1986; Kennedy et al, 1989). In order to evaluate further whether measurement of acrosin is of clinical value for predicting sperm fertilizing ability in vitro, the present study was carried out to determine the relationship between acrosin activity, the proportion of spermatozoa with normal intact acrosomes and fertilization rates in a clinical IVF programme.
Materials and methods Patients Investigations were performed on 118 couples who underwent IVF at the Royal Women's Hospital between June and December 1987, where the wife's oocytes were inseminated with the husband's spermatozoa. Diagnoses were unexplained infertility in 11, male factor infertility in nine, tubal occlusion in 56 and endometriosis in 21. The other 21 patients had more than one problem in the following combinations: tubal occlusion with endometriosis in five, tubal occlusion with male factor in eight, endometriosis with male factor in six and tubal occlusion, endometriosis and male factor in two. The diagnoses were determined before IVF was performed and patients with antisperm antibodies in either partner were excluded. In-vitro fertilization Multiple follicular development was induced with clomiphene citrate (CC) and human menopausal gonadotrophin (HMG): CC 100 mg daily for 5 days starting 10 days before the calculated midpoint of the cycle and HMG 150 units intramuscularly daily starting the day after 9 days before the calculated midpoint. Doses of HMG were based on urinary oestrone measurements and © Oxford University Press
Acrosin, acrosomes, morphology and IVF
increased if necessary to a maximum of 300 units per day. During the time of this study, oocyte collection was performed laparoscopically or by vaginal ultrasound-guided aspiration, 34—36 h after injection of human chorionic gonadotrophin (HCG) or the estimated time of commencement of the spontaneous luteinizing hormone (LH) surge. Laboratory procedures were the same for all patients. Semen was collected by masturbation 2 h prior to the expected time of insemination and the spermatozoa for insemination of oocytes were prepared by a swim-up technique as described previously (Asch et ai, 1985). Approximately 100 000 motile spermatozoa were added to each oocyte in 1 ml of culture medium (human tubal fluid medium supplemented with 10% human cord serum) in a plastic multiwell tray. However, the actual concentration used in the insemination medium varied between individuals (Table I) and mainly depended on the quality of semen produced by the husband and the number of motile spermatozoa obtained by the swim-up procedure. Fertilization was assessed at 18-20 h of incubation at 37°C in 5% CO2 in air and embryos were transferred to the uterus 40-45 h after insemination. Oocytes which had apparently failed to fertilize were re-examined up to 66 h after insemination before concluding that fertilization had not occurred. Semen analysis Analysis were performed within 2 —3 h of collection on semen remaining after preparation of spermatozoa for IVF and also on a sample of the sperm suspension for insemination after selection by the swim-up procedure. The sperm concentration in semen and in the insemination medium were determined using a haemocytometer. Sperm motility and motility index were measured subjectively in semen (Liu etai., 1988). Sperm viability (% live sperm) was determined by exclusion of eosin Y (World Health Organization, 1987). Sperm morphology was assessed on stained smears prepared from semen after washing with 0.9% w/v sodium chloride and adjustment of the sperm concentration to ~80 x 106/ml. The smears were stained using the Shorr method (Jeulin et al., 1986) and 200 spermatozoa were assessed under oil immersion with a magnification of 1000 and bright field illumination. Normal sperm morphology was considered according to the criteria of the World Health Organization (1987). Some slides were assessed by two observers with good agreement (Spearman r = 0.462, n = 75, P < 0.001). When there was more than one result for morphology, the average was used for subsequent analysis. Acrosome assessment Sperm acrosomes were assessed according to the method of Cross etai (1986) as modified by Liu and Baker (1988). The modification of the method of Cross et al. (1986), namely fixing the spermatozoa on glass slides, was shown not to affect the proportion of spermatozoa with intact acrosomes when compared with fixing the spermatozoa in suspension (Liu and Baker, 1988). This slight modification was made for more convenience in processing a large number of samples. In brief, spermatozoa in semen or in the insemination medium were washed twice with 10 ml of 0.9% w/v sodium chloride with centrifugation at 600 g for 10 min and the sperm pellet was smeared on a glass slide. The smear was fixed in 95% ethanol for 1 h after air drying,
washed in distilled water for 10 min with three to four changes, and stained for 2 h with 30 /ig/ml fluorescein isothiocyanateconjugated Pisum sativum agglutinin (FITC-PSA, Sigma Co., St Louis, MO) in Dulbecco's phosphate buffered saline (pH 7.4, Commonwealth Serum Laboratory, Melbourne, Victoria, Australia). Finally, the slide was washed and mounted with distilled water and 200 spermatozoa were counted using a fluorescence microscope (Dialux 200, Leitz, Wetzlar, West Germany) at a magnification of 400X (oil lens). When more than half the sperm head was brightly and uniformly fluorescing, the acrosome was considered to be normal and intact (Figure 1; Liu and Baker, 1988). Some samples for acrosome assessment were examined by two different observers with good agreement (semen, Spearman r = 0.681, n = 55, P < 0.001; insemination medium, Spearman r = 0.720, n = 56, P < 0.001). When there was more than one result for acrosomes, the average was used for subsequent analysis. Acrosin measurement Acrosin was extracted according to the method of Mohsenian et al. (1982) as follows: ~50 X 106 spermatozoa (five oligozoospermic semen samples had 50%) had low acrosin levels (Figure 3). In contrast, the proportions of spermatozoa with normal intact acrosomes in semen and in the insemination medium were correlated with all other sperm tests except for normal intact acrosomes in semen and sperm concentration in the insemination medium. There was a highly significant correlation between the proportion of spermatozoa with normal intact acrosomes in semen and in the insemination medium (Spearman r = 0.502, n = 83, P < 0.001). Correlation between sperm tests, diagnoses and IVF rates Acrosin levels were not significantly correlated with the IVF rate. However, the proportion of spermatozoa with normal morphology (Spearman r = 0.346, P < 0.001) and diagnosis of male infertility (Spearman r = -0.244, P < 0.01) were most strongly correlated with IVF rates. Sperm concentrations in semen (Spearman r = 0.236, P < 0.05) and in the insemination medium (Spearman r = 0.196, P < 0.05) were also significant. The proportion of spermatozoa with normal intact acrosomes and other sperm tests were not significantly related to IVF rates.
Statistical analysis Correlations between normal intact acrosomes, acrosin levels and other sperm tests, and between sperm tests and fertilization rate were examined by a nonparametric (Spearman) test. To determine which combinations of tests were independently related to the fertilization rate, all variables were analysed by logistic regression.
25
Results CD
Outcome of IVF For the 118 couples studied, the average number of oocytes recovered and inseminated was 7.7 (range 1—21) and the average fertilization rate was 64% (range 0-100%).
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Acrosin, acrosomes and other sperm tests (Table I)
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Relationships between human sperm acrosin, acrosomes, morphology and fertilization in vitro
D.Y.Liu1 and H.W.G.Baker Department of Obstetrics and Gynaecology, University of Melbourne and Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia 'To whom correspondence should be addressed
Acrosin was measured in the semen used for sperm preparation for in-vitro fertilization (1VF) in 118 patients. Acrosin levels correlated with the proportion of spermatozoa with normal intact acrosomes determined with Pisum sativum agglutinin labelled with fluorescein. However, acrosin levels and the proportion of spermatozoa with normal intact acrosomes in semen were not significantly related to the fertilization rate in vitro. Only the percentage normal morphology and sperm concentration in the insemination medium were independently significantly related to the fertilization rate by logistic regression analysis. In patients with fewer than 30% of spermatozoa with normal morphology, although acrosin levels were not correlated with the fertilization rate, the proportion of spermatozoa with normal intact acrosomes in the insemination medium was the only significant factor in the logistic regression analysis. In conclusion, acrosin levels have no prognostic value for fertilization in vitro but the proportion of spermatozoa with normal intact acrosomes may be a useful clinical marker of fertilizing ability in men with poor sperm morphology. Key words: acrosin/acrosomes/IVF/morphology
Introduction Although semen quality is related to the success of human invitro fertilization (IVF) (Mahadevan and Trounson, 1984; Jeulin et al., 1986), the standard semen characteristics are of limited value in predicting IVF rates (Belaisch-Allart et al, 1984; Edwards et al., 1984; Garcia et al., 1984; Trounson and Wood, 1984). Recent studies have shown that sperm morphology is one of the most significant factors related to fertilization rate (Kruger et al, 1986, 1988a,b; Liu etal, 1987, 1988; Liu and Baker, 1988). However, some patients with poor sperm morphology (abnormal >80%) still fertilize most or all of the oocytes inseminated (Liu et al, 1987, 1988). Thus additional tests of sperm function are necessary to improve the prediction of sperm fertilizing ability in vitro. It is known that the acrosome is important for fertilization both in vivo and in vitro. The morphological and functional integrity of the acrosome and the ability of sperm to undergo the acrosome reaction are essential for species-specific zona binding and 298 Downloaded from https://academic.oup.com/humrep/article-abstract/5/3/298/737907 by University of Durham user on 13 January 2018
penetration and fusion with the oolemma (Soupart and Strong, 1974; Yanagimachi, 1981; Sathananthan etal, 1982; Singer et al, 1985; Chen and Sathananthan, 1986; Moore and Bedford, 1983). Spermatozoa with small round heads or normal shaped spermatozoa with abnormally small acrosomes are associated with male infertility (Jeyendran etal, 1985; Schill etal, 1988). Round-headed spermatozoa without acrosomes do not have the ability to bind or penetrate zona-free hamster ova (Weissenberg et al., 1983). However, the condition of completely acrosome free spermatozoa is rare. Acrosin, a neutral proteinase, is one of the most important acrosomal enzymes and is believed to play a role in sperm-zona penetration (Rogers and Bentwood, 1982). It has been reported that fertilization does not oocur when the activity of this enzyme is inhibited (Zaneveld, 1976). It has been suggested also that low acrosin activity is associated with male infertility (Goodpasture et al., 1980; Mohsenian et al., 1982) and low fertilization rates in vitro (Acosta etal, 1986; Kennedy et al, 1989). In order to evaluate further whether measurement of acrosin is of clinical value for predicting sperm fertilizing ability in vitro, the present study was carried out to determine the relationship between acrosin activity, the proportion of spermatozoa with normal intact acrosomes and fertilization rates in a clinical IVF programme.
Materials and methods Patients Investigations were performed on 118 couples who underwent IVF at the Royal Women's Hospital between June and December 1987, where the wife's oocytes were inseminated with the husband's spermatozoa. Diagnoses were unexplained infertility in 11, male factor infertility in nine, tubal occlusion in 56 and endometriosis in 21. The other 21 patients had more than one problem in the following combinations: tubal occlusion with endometriosis in five, tubal occlusion with male factor in eight, endometriosis with male factor in six and tubal occlusion, endometriosis and male factor in two. The diagnoses were determined before IVF was performed and patients with antisperm antibodies in either partner were excluded. In-vitro fertilization Multiple follicular development was induced with clomiphene citrate (CC) and human menopausal gonadotrophin (HMG): CC 100 mg daily for 5 days starting 10 days before the calculated midpoint of the cycle and HMG 150 units intramuscularly daily starting the day after 9 days before the calculated midpoint. Doses of HMG were based on urinary oestrone measurements and © Oxford University Press
Acrosin, acrosomes, morphology and IVF
increased if necessary to a maximum of 300 units per day. During the time of this study, oocyte collection was performed laparoscopically or by vaginal ultrasound-guided aspiration, 34—36 h after injection of human chorionic gonadotrophin (HCG) or the estimated time of commencement of the spontaneous luteinizing hormone (LH) surge. Laboratory procedures were the same for all patients. Semen was collected by masturbation 2 h prior to the expected time of insemination and the spermatozoa for insemination of oocytes were prepared by a swim-up technique as described previously (Asch et ai, 1985). Approximately 100 000 motile spermatozoa were added to each oocyte in 1 ml of culture medium (human tubal fluid medium supplemented with 10% human cord serum) in a plastic multiwell tray. However, the actual concentration used in the insemination medium varied between individuals (Table I) and mainly depended on the quality of semen produced by the husband and the number of motile spermatozoa obtained by the swim-up procedure. Fertilization was assessed at 18-20 h of incubation at 37°C in 5% CO2 in air and embryos were transferred to the uterus 40-45 h after insemination. Oocytes which had apparently failed to fertilize were re-examined up to 66 h after insemination before concluding that fertilization had not occurred. Semen analysis Analysis were performed within 2 —3 h of collection on semen remaining after preparation of spermatozoa for IVF and also on a sample of the sperm suspension for insemination after selection by the swim-up procedure. The sperm concentration in semen and in the insemination medium were determined using a haemocytometer. Sperm motility and motility index were measured subjectively in semen (Liu etai., 1988). Sperm viability (% live sperm) was determined by exclusion of eosin Y (World Health Organization, 1987). Sperm morphology was assessed on stained smears prepared from semen after washing with 0.9% w/v sodium chloride and adjustment of the sperm concentration to ~80 x 106/ml. The smears were stained using the Shorr method (Jeulin et al., 1986) and 200 spermatozoa were assessed under oil immersion with a magnification of 1000 and bright field illumination. Normal sperm morphology was considered according to the criteria of the World Health Organization (1987). Some slides were assessed by two observers with good agreement (Spearman r = 0.462, n = 75, P < 0.001). When there was more than one result for morphology, the average was used for subsequent analysis. Acrosome assessment Sperm acrosomes were assessed according to the method of Cross etai (1986) as modified by Liu and Baker (1988). The modification of the method of Cross et al. (1986), namely fixing the spermatozoa on glass slides, was shown not to affect the proportion of spermatozoa with intact acrosomes when compared with fixing the spermatozoa in suspension (Liu and Baker, 1988). This slight modification was made for more convenience in processing a large number of samples. In brief, spermatozoa in semen or in the insemination medium were washed twice with 10 ml of 0.9% w/v sodium chloride with centrifugation at 600 g for 10 min and the sperm pellet was smeared on a glass slide. The smear was fixed in 95% ethanol for 1 h after air drying,
washed in distilled water for 10 min with three to four changes, and stained for 2 h with 30 /ig/ml fluorescein isothiocyanateconjugated Pisum sativum agglutinin (FITC-PSA, Sigma Co., St Louis, MO) in Dulbecco's phosphate buffered saline (pH 7.4, Commonwealth Serum Laboratory, Melbourne, Victoria, Australia). Finally, the slide was washed and mounted with distilled water and 200 spermatozoa were counted using a fluorescence microscope (Dialux 200, Leitz, Wetzlar, West Germany) at a magnification of 400X (oil lens). When more than half the sperm head was brightly and uniformly fluorescing, the acrosome was considered to be normal and intact (Figure 1; Liu and Baker, 1988). Some samples for acrosome assessment were examined by two different observers with good agreement (semen, Spearman r = 0.681, n = 55, P < 0.001; insemination medium, Spearman r = 0.720, n = 56, P < 0.001). When there was more than one result for acrosomes, the average was used for subsequent analysis. Acrosin measurement Acrosin was extracted according to the method of Mohsenian et al. (1982) as follows: ~50 X 106 spermatozoa (five oligozoospermic semen samples had 50%) had low acrosin levels (Figure 3). In contrast, the proportions of spermatozoa with normal intact acrosomes in semen and in the insemination medium were correlated with all other sperm tests except for normal intact acrosomes in semen and sperm concentration in the insemination medium. There was a highly significant correlation between the proportion of spermatozoa with normal intact acrosomes in semen and in the insemination medium (Spearman r = 0.502, n = 83, P < 0.001). Correlation between sperm tests, diagnoses and IVF rates Acrosin levels were not significantly correlated with the IVF rate. However, the proportion of spermatozoa with normal morphology (Spearman r = 0.346, P < 0.001) and diagnosis of male infertility (Spearman r = -0.244, P < 0.01) were most strongly correlated with IVF rates. Sperm concentrations in semen (Spearman r = 0.236, P < 0.05) and in the insemination medium (Spearman r = 0.196, P < 0.05) were also significant. The proportion of spermatozoa with normal intact acrosomes and other sperm tests were not significantly related to IVF rates.
Statistical analysis Correlations between normal intact acrosomes, acrosin levels and other sperm tests, and between sperm tests and fertilization rate were examined by a nonparametric (Spearman) test. To determine which combinations of tests were independently related to the fertilization rate, all variables were analysed by logistic regression.
25
Results CD
Outcome of IVF For the 118 couples studied, the average number of oocytes recovered and inseminated was 7.7 (range 1—21) and the average fertilization rate was 64% (range 0-100%).
a
20
CO o
15 3
E c
Acrosin, acrosomes and other sperm tests (Table I)
10
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