Department of Obstetrics and Gynaecology, University of Melbourne, Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia

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1 FERTILITY AND STERILITY Copyright c 1988 The American Fertility Society Vol. 50, No. 2, August 1988 Printed in U.S.A. The proportion of human sperm with poor morphology but normal intact acrosomes detected with pisum sativum agglutinin correlates with fertilization in vitro De Yi Liu, B.Sc. * H. W. Gordon Baker, M.D., Ph.D. Department of Obstetrics and Gynaecology, University of Melbourne, Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia Sperm acrosomes were examined with fluorescein-labeled pisum sativum agglutinin in 137 in vitro fertilization treatments. The percentage of sperm with normal intact aerosomes in semen was correlated with sperm concentration, morphology, viability, and motility. The proportion of sperm with normal intact acrosomes was significantly increased by the "swim up" selection from semen. Fertilization rates in the whole group studied were significantly related to percentage normal morphology, concentration of sperm inseminated and cause of infertility. However, with normal morphology <30%, the proportion of sperm with normal intact acrosomes in the insemination medium was the only significant factor related to fertilization rate in the logistic regression analysis. Thus, assessment of acrosomes may be of clinical value for predicting the fertility of men with poor sperm morphology. Fertil Steril 50:288, 1988 Recently, a number of studies on sperm factors affecting human in vitro fertilization (IVF) have shown that spermatozoal morphology is the most significant correlate of fertilization rate. 1-5 However, we also found that some semen samples with poor sperm morphology (abnormal morphology >70%) still fertilized most or all of the oocytes inseminated.4 5 Therefore, we wished to develop a new test which would help to improve our ability to predict success of IVF, particularly for patients with poor sperm morphology. It is known that the acrosome reaction is essential for mammalian fertilization because it is required for sperm penetration through zona pellucida and for fusion with the oolemma. 6-9 Roundheaded sperm without acrosomes will not fertilize Received February 8, 1988; revised and accepted April 26, *Reprint requests: D. Y. Liu, B.Sc., Department of Obstetrics and Gynaecology, University of Melbourne, Parkville 3052, Melbourne, Australia. 288 Liu and Baker Sperm morphology, acrosomes and IVF human oocytes either in vivo or in vitro Absence of the acrosome reaction in vitro has been reported in patients with polyzoospermia. 13 Topfer-Petersen et ap4 suggested that the kinetics of the acrosome reaction may be important functional variables affecting fertility of human spermatozoa. However, there has been no simple method available for investigating acrosome status in human spermatozoa. The human acrosome is too small to examine by phase-contrast light microscopy, and transmission electron microscopy is costly and unsuitable for examining large numbers of samples. Other techniques such as the triple stain, indirect immunofiuorescence with monoclonal antibodies, and chlortetracycline fiuorescence have not been widely used because they are difficult to apply Cross et ap 8 have reported that the lectin-pisum sativum agglutinin (PSA) labeled with ftuorescein isothiocyanate (FITC) binds to components in the acrosome and can be used to differentiate acrosome intact from acrosome reacted human sperm. This method is simple and Fertility and Sterility

2 has been used in the present study to determine if the proportion of sperm with normal intact aerosomes is related to fertilization rates in vitro. Patients MATERIALS AND METHODS Investigations were performed on the semen of all couples (n = 137) who underwent IVF at the Royal Women's Hospital between May and October 1987, where the wife's oocytes were inseminated with the husband's sperm except for couples who had severe oligospermia and where insufficient semen was left over after sperm preparation. Diagnoses were unexplained infertility in 36, male factor infertility in 26, tubal occlusion in 45, and endometriosis in 30. The diagnoses were determined before IVF was performed, and a number of couples with previously normal semen tests had poorer semen at the time of IVF. A few couples with previously poor tests had normal semen analysis results at the time of treatment. Patients with sperm antibodies in either partner were excluded. In Vitro Fertilization Multiple follicular development was induced with sequential clomiphene and human menopausal gonadotropin (hmg) treatment: clomiphene citrate (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 CC. Doses of hmg were based on serum estradiol (E2 ) measurements, increased if necessary and continued until the serum E 2 reached 4 nmol/1. During the time of this study, human chorionic gonadotropin (hcg) was given and oocyte collection performed laparoscopically or by vaginal ultrasound-guided aspiration 34 to 36 hours after hcg injection or the estimated time of commencement of spontaneous luteinizing hormone (LH) surges. Semen was collected by masturbation 2 hours before the expected time of insemination and the sperm prepared by a swim-up technique described previously. 19 Approximately 100,000 motile spermatozoa were added to each oocyte in 1 ml of culture medium in a plastic multiwell tray. Fertilization was assessed 18 to 20 hours later and embryos transferred to the uterus 2 days after insemination. Oocytes that had apparently failed to fertilize were re-examined up to 60 hours after insemination before concluding that fertilization had not occurred. Vol. 50, No.2, August 1988 Semen Analysis Semen analysis was performed on semen remaining after preparation of sperm for IVF within 2 to 3 hours of collection and on a sample of the sperm suspension for insemination obtained after selection by the swim-up procedure. Sperm concentrations in semen and insemination medium were determined with a hemocytometer. Sperm motility and motility index were measured in semen with three grades of forward progression: 1 = zero progressive motility, 2 = fair progressive motility, and 3 = good progressive motility. The index is the sum of the percentage of sperm in each grade multiplied by the grade number. 4 Sperm viability (% live sperm) was determined by exclusion of eosin Y. 20 Sperm morphology was assessed on stained smears prepared from semen after washing with 0.9% sodium chloride and adjustment of sperm concentration to approximately 80 X 10 6 /ml. The smears were stained with the Shorr method, 21 and 200 sperm were assessed under oil immersion with magnification of 1000X, and bright field illumination. Sperm normal morphology was considered according to the criteria of the World Health Organization. 20 All samples for morphology assessment were examined by two observers (Spearman r = 0.60, n = 137, P < 0.001), and the average result was used for subsequent analysis. Human Acrosome Assessment Sperm acrosomes were examined by staining with PSA labeled with FITC described by Cross et ap 8 with slight modification as follows: sperm in semen, or insemination medium, were washed with 10 ml of 0.9% sodium chloride twice by centrifugation at 600 X g for 10 minutes, and the sperm pellet was smeared on a glass microscope slide. The smear was fixed in 95% ethanol for 1 hour after air drying. Slides were washed in distilled water for 10 minutes with three to four changes, exposed to 30 }tg/ml PSA labeled with FITC (Sigma Co. St. Louis, MO) in Dulbecco phosphate buffered saline (Commonwealth Serum Laboratory, Melbourne, Victoria, Australia) for 2 hours, and finally, the slide washed and mounted with distilled water. At least 200 sperm were counted under a fluorescence microscope (Dialux 20, Leitz Wetzlar, Germany) with 400X (oil lens) magnification. When more than half a sperm head was brightly and uniformly fluorescing the acrosome was considered to be normal (Fig. 1). The present modification is more convenient for examining large numbers of sperm Liu and Baker Sperm morphology, acrosomes and IVF 289

3 Figure 1 (A, and B) Human acrosomes stained with PSA labeled with FITC: a, normal intact acrosome; b, abnormal acrosome; c, acrosome reacted sperm (magnification l,ooox). samples. Preliminary studies showed there was good agreement between the results of fixing the sperm before or after smearing on the slides (n = 10, b = 1.04, r = 0.92). Most samples were assessed by two observers {Spearman r = 0.580, n = 98, P < 0.001), and average results were used for subsequent analysis. Statistical Analysis Correlations between percentages of sperm with normal intact acrosomes and other sperm tests and between sperm tests and fertilization rate were examined by Spearman tests. Differences between mean percentages of sperm characteristics in semen and in insemination medium were assessed by Student's paired t-test. Differences between proportions were examined by X 2 tests. To determine which combinations of tests were independently related to fertilization rate, all variables were analyzed by logistic regression. 4 semination medium after selection by swim-up compared with the original semen. The acrosome results in insemination medium and semen were strongly correlated (n = 121, Spearman r = 0.533, P < 0.001). Despite this, there were 25 patients who had a lower percentage of sperm with normal intact acrosomes in the insemination medium following the swim up selection than in the original semen. This decrease occurred more commonly with poor sperm morphology; 12 of 33 patients with <30% sperm normal morphology (38%) compared with 13 of 89 patients with >30% sperm normal morphology (14%, X 2 = 6.19, P < 0.05). Similarly, 16 of 121 patients were found to have <30% of sperm with normal intact acrosomes in the insemination medium, and 10 were in the group of 33 patients with <30% normal morphology (31%) compared with 6 in the group of 89 patients with >30% normal morphology (7%, x 2 = 10.28, P < 0.01). The proportion of sperm with normal intact RESULTS Table 1 Results of Sperm Tests Outcome of IVF For the 137 couples studied, the average number of oocytes recovered and inseminated was 7.9 (range 1 to 31), the average fertilization rate was 60% and the pregnancy rate was 19%. Sperm Tests Results of the sperm tests are shown in Table 1. There was a wide range for all results including the percentage of sperm with normal acrosomes. The mean percentage of sperm with normal intact acrosomes was significantly increased in the in- n Mean Range Sperm concentration (semen) 10 6 /ml Sperm concentration (insemination medium) 10 3 /ml Motility (semen) % Motility index (semen) Viability (semen) % Normal morphology (semen) % Normal intact acrosomes (semen)% " 3-85 Normal intact acrosomes (insemination medium) % " 4-95 Comparison of 44 and 51, P < 0.05 (t-test). 290 Liu and Baker Sperm morphology, acrosomes and IVF Fertility and Sterility

4 acrosomes in semen or in insemination medium was correlated with all other sperm tests except for sperm concentration in the insemination medium. It was particularly strongly correlated with normal morphology and viability (Table 2). Correlation Between Sperm Tests and IVF Rate The percentage of sperm with normal morphology in semen was most strongly correlated (n = 132, Spearman r = 0.368, P < 0.001) with IVF rate. Sperm concentration in semen (n = 132, Spearman r = 0.243, P < 0.01) and in insemination medium (n = 133, Spearman r = 0.218, P < 0.05) was also significantly correlated with IVF rate. The percentage normal acrosomes in semen was just significant (n = 137, Spearman r = 0.210, P < 0.05). All other tests were not significantly correlated with IVF rate. When data from patients with <30% sperm normal morphology were examined, only percentage normal intact acrosomes in the insemination medium was significantly correlated with fertilization rate (Fig. 2). All other tests were not significantly correlated. In the poor morphology group (Fig. 2), one of nine couples with <30% normal intact aerosomes had a good fertilization rate (two of three oocytes fertilized). In this couple, IVF was being performed for male infertility; the sperm concentration was 7 X 10 6 /ml, motility 25%, motility index 29, viability 40%, normal morphology 20% and normal intact acrosomes 11% in semen at the time of this treatment and a higher than usual concentration of sperm was inseminated (500,000 per oocyte). In contrast, there were four other oligospermic men with similar sperm characteristics who Table 2 Spearman Correlation Coefficients Between Percentage Normal Intact Acrosomes in Semen and Insemination Medium (Insem), and Other Sperm Characteristics Sperm concentration (semen) 10 6 /ml Sperm concentration (insem) 10 3 /ml Motility (semen) % Motility index (semen) Viability (semen) % Normal morphology (semen) % a P < b p < 'p < Vol. 50, No.2, August 1988 Normal intact acrosomes (%) Semen Insem ' 0.341' ' 0.216" 0.331' 0.260b 0.451' 0.288b 0.472' 0.376' m % N. Acrosome (insem) Figure 2 Correlation between percentage of sperm with normal intact acrosome(% N. acrosome) in the insemination medium (insem) for patients with <30% sperm normal morphology and IVF rate (n = 33, Spearman r = 0.357, P < 0.05). had approximately 100,000 sperm per oocyte inseminated and they had lower fertilization rates averaging 10% (range 6% to 17%). Only two patients had high proportions of sperm with normal intact acrosomes (>60%) and poor fertilization (<50%, Fig. 2). Logistic Regression Analysis Data for 121 subjects with results for all variables examined (those for 16 subjects with any missing values were excluded) were analyzed by logistic regression. The proportion of sperm with normal intact acrosomes in semen or in the insemination medium was not significantly related to fertilization rates. The most significant factor related to IVF rate was normal morphology in semen (regression coefficient = 0.023, standard error (SE) = 0.005, z = 4.6, P < 0.001), the second most significant factor was sperm concentration in insemination medium (regression coefficient = 0.007, SE = 0.002, z = 3.5, P < 0.001), and the third was diagnosis of male infertility (regression coefficient = , SE = 0.174, z = -2.3, P < 0.01). Other sperm tests were not significant. However, when data from the 33 subjects with <30% sperm normal morphology in semen were examined by logistic regression analysis, percentage normal intact aerosomes in insemination medium was the strongly and only significant factor related to fertilization rate (regression coefficient = 0.030, SE = 0.008, z = 3.75, p < 0.001). DISCUSSION The acrosome reaction is important for human fertilization. However, the process of the acrosome reaction during human fertilization is not fully un- Liu and Baker Sperm morphology, acrosomes and IVF 291

5 derstood. In some mammals, sperm attachment to the zona pellucida must precede completion of the acrosome reaction since sperm without acrosomes are unable to bind to zonae.6 7 Whether this is so in the human is unclear since both acrosome reacted and intact spermatozoa are found on the surface of the zona pellucida.8 An acrosome reaction does seem to be required for zona penetration.8 9 The acrosome stabilizing factor, a glycoprotein in rabbit seminal plasma, was found to block the induction of the rabbit sperm acrosome reaction in vitro. 22 Whether there is a similar factor in human seminal plasma is unknown. It is known that there are a number of enzymes associated with the sperm acrosome, such as hyaluronidase and acrosin, which appear to play an essential role in the fertilization process by enabling the sperm to either undergo the acrosome reaction or penetrate into the vestments surrounding the egg.23 Jeulin et al.21 reported that good sperm acrosome morphology was more common in patients who had successful IVF than in these with poor fertilization rates. However, it has been difficult to show a correlation between sperm acrosin content and their fertilizing ability.3 It has also been difficult to obtain evidence to show a relationship between the ability of human sperm to undergo capacitation and acrosome react, and to fertilize oocytes in vitro.24 The present study shows that the proportion of sperm with normal intact acrosomes in the insemination medium was significantly related to the fertilization rate in vitro only for patients with poor sperm morphology (<30% normal). The percentage of sperm with normal intact acrosomes in semen was not significant probably because the population of sperm with normal intact acrosomes was changed by selection of the motile sperm by the swim-up technique. In general, the number of sperm with normal intact acrosomes was increased by swim-up selection, and there was significant correlation between the proportion of sperm with normal intact acrosomes in semen and in insemination medium. However, the proportion of sperm with normal intact acrosome in semen and insemination medium was variable between individuals. There was a decrease in the proportion of sperm with normal intact acrosomes in the insemination medium after selection in some patients, particularly in those with poor sperm morphology. This result possibly indicates that'in these patients the acrosomes were less stable or reacted faster in the culture medium because of defects of development of sperm or subsequent changes during epididymal passage or ejaculation that alter morphology of the sperm head.14'25 Cross et al.18 reported that the use of a supravital stain in conjunction with the PSA acrosome assessment to differentiate living from dead sperm because acrosome-reacted living sperm cannot be distinguished from dead sperm by the use of PSA alone. We did not use this approach in this study because we have found that only a few dead sperm ( <5% determined by eosin Y exclusion) were present in the insemination medium after selection by swim up.26 Also, sperm motility was usually being 80% to 95% in the insemination medium. The present study further confirms previous reports that morphology is the most significant sperm factor related to fertilization rate in vitro.1-5 The percentage normal intact acrosomes was not. significant for the whole group studied possibly because it was strongly correlated with sperm morphology and most other sperm tests. Many sperm test results are correlated with both because similar functions are measured and defects of sperm number, viability, motility, and morphology usually occur in combination. 4 6 The low fertilization rate with sperm with low percentage normal morphology does not appear to be explained by any specific morphologic abnormality. Some sperm samples with poor morphology (abnormal morphology > 70%) still fertilized most or all of the oocytes inseminated.4 5 Therefore, the value of using only sperm morphology for predicting success in IVF is limited. However, this study shows that the samples with high proportions of sperm with poor morphology that produced good fertilization rates also usually had a high proportion of sperm in the insemination medium showing normal intact aerosomes by PSA staining. It may be that some sperm have a defect of sperm morphology without a severe associated abnormality of the acrosome.25 For example, one semen sample with a very poor sperm morphology (only 6% normal morphology) fertilized 9 of 11 oocytes inseminated, but 70% of the sperm in the insemination medium had normal acrosomes. The second most significant factor related to fertilization rate was number of sperm used for insemination, which was similar to our previous reports.4 6 One sperm sample from an oligospermic man that was very poor (see results) fertilized two of three oocytes, possibly because a high concentration of sperm was used for insemination, about five times higher than the usual for in our IVF program. 292 Liu and Baker Sperm morphology, acrosomes and IVF Fertility and Sterility

6 Sperm motility was not significantly related to fertilization in this and our previous studies. 4 5 This may result from the effectiveness of the swim-up technique for collection of sperm with good motility and because sperm motility was correlated with other tests, such as sperm morphology, which were so significant in the logistic regression. In conclusion, fluorescein-labeled PSA is simple to use for assessing the human acrosome. Measurement of the proportion of sperm with normal intact acrosomes obtained by the swim-up technique may be of clinical value for predicting fertility of men with poor sperm morphology. Acknowledgments. We thank Miss Carolyn J. Wiltshire for technical assistance and Miss Yvonne P. Duplessis, B.Sc., and her staff in the IVF laboratory for preparing sperm samples. REFERENCES 1. Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, van Zyl JA, Smith K: Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril46:1118, Kruger TF, Acosta AA, Simmon KF, Swanson RJ, Matta JF, Oehninger S: Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 49:112, Kruger TF, Oehninger S, Haggue D, Acosta AA, Pleban P, Swanson RJ, Simmons KF, Matta JF, Morshedi M: Correlation between sperm morphology, acrosin and fertilization in in vitro fertilization. (Abstr. 217) Presented at the Annual Meeting of the American Fertility Society, Reno, Nevada, September 28 to , Published by The American Fertility Society, p Liu DY, DuPlessis YP, Nayudu PL, Johnston WIH, Baker HWG: The use of in vitro fertilization to evaluate putative tests of human sperm function. Fertil Steril 49:272, Liu DY, Elton RA, Johnston WIH, Baker HWG: Spermatozoal nuclear chromatin decondensation in vitro, a test for sperm immaturity, correlation with results of human in vitro fertilization. Clin Reprod Fertil 5:191, Y anagimachi R: Mechanisms of fertilization in mammals. 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Fertil Steril 27:677, World Health Organization Laboratory Manual for Examination of Human Semen and Semen-Cervical Mucus Interaction, Edited by MA Betsey, R Eliasson, AJ Gellegos, KS Moghissi, CA Paulsen, MRN Prasad. Singapore, Press Concern, 1980, p Jeulin C, Feneux D, Serres C, Jouannet P, Guillet-Rosso F, Belaisch-Allart J, Frydman R, Testart J: Sperm factors related to failure of human in vitro fertilization. J Reprod Fertil 76:1, Eng LE, Oliphant G: Rabbit sperm reversible decapacitation by membrane stabilization with a light purified glycoprotein from seminal plasma. Bioi Reprod 19:1083, Rogers BJ, Bentwood BJ: Capacitation, acrosome reaction and fertilization. In Biochemistry of Mammalian Reproduction, Edited by LJD Zaneveld, RJ Chatterton. New York, John Wiley & Sons, 1982, p Plachot M, Mandelbaum J, Junca MA: Acrosome reaction of human sperm used for in in vitro fertilization. Fertil Steril 42:418, Zamboni L: The ultrastructural pathology of the spermatozoon as a cause of infertility: the role of electron microscopy in the evaluation of semen quality. Fertil Steril48:711, Liu DY, Baker HWG: Unpublished data Vol. 50, No.2, August 1988 Liu and Baker Sperm morphology, acrosomes and IVF 293