Vol. 58, No.6, December 1992

FERTILITY AND STERILITY

Printed on acid-free paper in U.S.A.

Copyright Q 1992 The American Fertility Society

Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding, and fertilization rates in vitro*

De Yi Liu, Ph.D.t H. W. Gordon Baker, M.D., Ph.D. University of Melbourne Department of Obstetrics and Gynaecology and Reproductive Biology Unit, Royal Women's Hospital, Melbourne, Victoria, Australia

Objective: To study whether the results of tests of sperm chromatin and deoxyribonucleic acid (DNA) normality are related to fertilization rates in vitro. Design: Normal morphology, nuclear maturity determined by acidic aniline blue stain, and DNA normality determined by acridine orange fluorescence of sperm in insemination medium and the number of sperm bound to the zona pellucida (ZP) of the oocytes that had failed to fertilize in vitro were determined. The relationship between sperm test results and fertilization rates were analyzed by logistic regression. Setting: Samples were obtained from patients undergoing in vitro fertilization (IVF) treatment. Results: The number of sperm bound to the ZP, the percentage of sperm with normal morphology, and the percentage of sperm with normal DNA were the most significant factors related to fertilization rates in vitro. In patients with normal morphology z 15% or with >10 sperm bound per ZP, the percentage of sperm with normal DNA, the number of sperm bound to the ZP, and motility grade were significantly related to IVF rates. Conclusion: In patients with normal morphology z 15%, failure of fertilization may be because of defects of sperm-ZP binding or abnormal DNA. Assessment of DNA normality of motile sperm in the insemination medium may aid prediction of fertilization rates in addition to normal morphology and sperm-ZP binding. Fertil Steril1992;58:1178-84 Key Words: Sperm morphology, nuclear chromatin, zona binding, in vitro fertilization

Use of the results of in vitro fertilization (IVF) to evaluate clinical tests of human sperm function is a powerful approach because sperm used for insemination of oocytes can be tested and the results of sperm tests directly related to IVF rates. Logistic regression analysis is useful to determine which groups of sperm characteristics are most independently significantly related to fertilization rates (1).

Received May 15, 1992; revised and accepted August 24, 1992. * Presented at the 10th Annual Scientific Meeting of Fertility

Society of Australia, Lome, Victoria, Australia, November 18 to 22, 1991. t Reprint requests: De Yi Liu, Ph.D., Department of Obstetrics and Gynaecology, University of Melbourne, Royal Women's Hospital, Carlton, Victoria 3053, Australia. 1178

Liu and Baker

A number of such studies, including our reports, have shown that the percentage of normal morphology assessed with an improved method is one of the most significant sperm factors related to fertilization rates (1-3). However, the accuracy of prediction of IVF rates using sperm morphology alone is currently still poor. Therefore, other new sperm function tests such as sperm-zona pellucida (ZP) binding and assessment of sperm with normal intact acrosomes have been developed and used in combination with normal morphology to improve prediction of IVF rates (4-6). Using these sperm function tests, the majority of patients with failure of fertilization are found to have defects of sperm morphology, acrosomes, and ZP binding. However, the causes of failure of IVF for the other 25% of patients is unclear (Liu DY,

Sperm nuclear chromatin normality and IVF

Fertility and Sterility

Baker HWG, unpublished observations). Therefore, it is necessary to study other defects of sperm or oocytes as causes of poor IVF results, particularly in those patients without severe defects of sperm morphology and sperm-ZP binding. Jeulin et al. (7) reported that assessment of sperm nuclear maturity with acidic aniline blue stain could provide additional information for predicting fertility in vitro. However, our previous study in 106 IVF patients showed the percentage of sperm with immature sperm stained with acidic aniline blue was not significantly related to IVF rates (1). Others reported that assessment of sperm deoxyribonucleic acid (DNA) normality by staining with acridine orange (AO) may help in the prediction of fertility (8, 9). Although these tests are useful for studying some aspects of human sperm physiology, the relationship between the results and fertility has not been studied in detail. In the present study, we have determined if the percentages of sperm with immaturity of nuclear chromatin determined with acidic aniline blue and abnormality of DNA determined with AO fluorescence of sperm in the insemination medium are related to failure of IVF, particularly in those patients without severe defects of morphology and sperm -ZP binding. MATERIALS AND METHODS Patients

Investigations were performed in 91 IVF patients. In these couples, the wife's oocytes (~3) were inseminated with husband's sperm, and some or all of the oocytes failed to fertilize. All the patients (n = 42) with zero fertilization from January to December 1991 were included in the present study. Other patients (n = 49) were selected because they had IVF on the same day as those with zero fertilization, and a proportion of their oocytes failed to fertilize. The clinical and laboratory aspects of the IVF procedures are described elsewhere (1). The swim-up technique was used for selection of motile sperm for insemination of the oocytes. Diagnoses were male infertility in 22, unexplained infertility in 28, tubal occlusion in 16, endometriosis in 6, and mixed male and female (tubal occlusion or/and endometriosis) factors in 19. Sperm Samples

Sperm in the insemination medium were obtained after embryos or unfertilized oocytes had been reVol. 58, No.6, December 1992

moved at 48 to 60 hours after insemination. The insemination medium from all the culture dishes was pooled for the same patient. The sperm were washed with 10 mL of 0.9% NaCI (sterile intravenous saline), and the sperm pellet was resuspended in 0.1 mL of 0.9% NaCl. Washed sperm were smeared on a glass slide, and three separate slides were prepared for staining of morphology, nuclear maturity, and DNA normality. Sperm in semen and swim-up preparation were not examined in this study because many of the patients had poor semen, and all the sample was used for sperm preparation for in semi nation of oocytes. Assessment of Sperm Concentration, Motility, and Morphology

Sperm concentration in the insemination suspension was determined using a hemocytometer. Sperm motility and motility grade in the insemination suspension were determined using phasecontrast microscope according to the methods of World Health Organization (WHO) (10). The percentage motility was calculated by counting 100 sperm. Four grades were used for classifying sperm motility as follows: 1 = immotile; 2 = nonprogressive motility; 3 = progressive but not rapid linear movement; 4 = rapid and linear progression. Morphology smears were prepared after adjusting the sperm concentration to approximately 80 X 106 /mL. The slides were stained with the Shorr method after the smear was fixed in 90% ethanol for 30 minutes (1, 7). Normal sperm morphology was assessed following WHO's criteria for the silhouette plus internal staining characteristics with the acrosomal region being clearly seen, regular in shape, and occupying at least half of the sperm head (1, 10, 11). The percentage of sperm with normal morphology was determined by assessing 200 sperm from more than 10 individual fields under oil immersion with magnification of Xl,OOO and brightfield illumination. Assessment of Sperm Nuclear Maturity and DNA Normality

Maturity of nuclear chromatin was examined with acidic aniline blue (BDH Chemical, Poole, England) stain method described elsewhere (12). Briefly, after air drying, the sperm smear was fixed for 30 minutes in 3% glutaraldehyde in phosphate-buffered saline. The smear was stained for 15 minutes in 5% aqueous aniline blue in 4% acetic acid (pH 3.5). Sperm heads with immature nuclear chromatin stain dark blue

Liu and Baker

Sperm nuclear chromatin normality and IVF

1179

with acidic aniline blue and those with a mature nucleus do not stain (10). The percentage of sperm stained with aniline blue was determined by counting 200 sperm under oil immersion with X1,000 magnification and bright-field illumination. The normality of DNA was assessed by the AO fluorescence method described by Tejada et al. (8). After air drying, the sperm smear was fixed in Carnoy's solution (3 parts of methanol and 1 part glacial acetic acid) for at least 3 hours or overnight. The Carnoy's solution was prepared daily. The slides were then washed in distilled water and allowed to air dry for a few minutes before staining. The AO (Sigma Chemical Co., St Louis, MO) staining solution was prepared as follows: 10 mL of 1 % AO in distilled water was added to a mixture of 40 mL of 0.1 M citric acid and 2.5 mL of 0.3 M Na2HP047H20, pH 2.5. The AO staining solution was prepared daily. The 1 % AO stock solution was stored in the dark at 4°C for 4 weeks. Sperm smear was stained for 5 minutes. Then the slide was gently rinsed and mounted with distilled water. The percentage of sperm with normal DNA was determined by counting 200 sperm under a fluorescence microscope (Dialux 20; Leitz, Wetzlar, Germany) with X400 (oil lens) magnification with excitation of 450 to 490 nm. Sperm with normal (double-stranded) DNA fluoresce green, and those with denatured or singlestranded DNA fluoresce red or yellow. Assessment of Number of Sperm Bound to the ZP

The number of sperm bound to the ZP of all the unfertilized oocytes were counted with an inverted phase-contrast microscope with magnification of X250 after removing the cumulus or corona cells if present (13). The oocytes were pipetted gently several times to dislodge any sperm not tightly bound to the zona. When there were >100 sperm bound to one ZP, it is difficult to count the number of sperm accurately. In this situation the number was recorded as 100. To determine whether the ZP selectively bound sperm with a mature nucleus and normal DNA, 00cytes with >20 sperm bound to the ZP from the same patient were pooled together and smeared on a glass slide. These slides were stained to assess nuclear maturity and DNA normality of the sperm bound to the ZP. Statistical Analysis

The significance of differences between mean results of sperm tests for patients with zero (IVF rate 1180

Liu and Baker

= 0) or some fertilization (IVF rates> 0) was performed by t-test. Paired t-tests were used for differences between the percentages of sperm with normal DNA and mature nuclei bound to the ZP and in the insemination medium. Weighted mean binding rates were also calculated (14). Correlations between sperm test results and between sperm test results and IVF rates were performed by nonparametric (Spearman) test. Relationships between sperm test results and IVF rates were examined by logistic regression analysis using the statistical package SPIDA (Macquarie University, Sydney, Australia). The proportion of the deviance explained by each model was calculated from the difference between the deviance of the fitted model and the deviance of a null model containing only the constant. The resulting r values with logistic regression are lower than with linear regression because of the binary response.

RESULTS Sperm Test and IVF Results

The simple comparison of mean sperm test results between patients with no oocytes and some oocytes fertilized is shown in Table 1. There was a wide range for all the sperm test results and also the number of oocytes inseminated. There were significant differences in the percentages of sperm with mature nuclei, normal DNA, normal morphology, and the number of sperm bound to the ZP between patients

Table 1 In Vitro Fertilization and Sperm Test Results for Patients With 0 and >0 Fertilization Rates Fertilization rates

0 No. of subjects No. of oocytes inseminated No. of oocytes fertilized Fertilization rates (%) No. of sperm inseminated (103/mL) Motility (insemination medium, %) No. of sperm bound/ZP No. of patients with ~1O sperm bound/ZP Normal morphology (insemination medium, %) Normal DNA (insemination medium, %) Mature nucleus (insemination medium, %)

'P < t P
0

42 10 (3 to 22) 0 0

49

(3 to 27) 6 (1 to 19) 50 (4 to 89) 11

220 (30 to 900)

144 (60 to 500)'

68 00 to 100) 9 (0 to 81)

78 (5 to 100) 30 (1 to 100)t

11

31

10 (0 to 39)

25 (3 to 53)t

40 (1 to 91)

56 (6 to 95)'

74 0

82 (15 to 97)'

to

98)

0.05. 0.001. Fertility and Sterility

with IVF rates = 0 and IVF rates > O. There was an average of 10% of sperm with normal morphology in patients with zero fertilization compared with 25% in those with some fertilization. Similarly, patients with zero fertilization had on average 9 sperm bound per ZP compared with 30 in those with some oocytes fertilized. There was no significant difference in the number of oocytes inseminated between the two groups. Because it is current practice to increase the number of sperm in the insemination medium in patients with high proportions of sperm with abnormal morphology or previous failure of fertilization, there was a significantly higher number of sperm inseminated in patients with IVF rates = 0 than in those with IVF rates> O. Patients with diagnoses of male infertility, including those with mixed male and female factors infertility (23 of 41,56%), had a significantly higher frequency of zero fertilization (x 2 = 4.8, P < 0.05) than did patients with only female infertility (6 of 22, 27%). Of patients with unexplained infertility, 47% (13 of 28) had zero fertilization: not significantly different from male factor patients. Correlations Between Sperm Test Results

There were significant correlations between most of the sperm test results, such as between motility, motility grade, and morphology; between morphology, motility, and motility grade and number of sperm bound to the ZP; and between morphology, normal DNA, and mature nuclei (Table 2). Overall, sperm morphology was most strongly correlated with the number of sperm bound to the ZP and other characteristics such as motility and mature nuclei and less significantly correlated with normal DNA. Correlation Between Sperm Tests Results and IVF Rates

When all the data from 91 patients were analyzed by Spearman's correlation test, there were highly

Table 2

significant correlations between percentage of sperm with normal morphology (r = 0.607, P < 0.001), the number of sperm bound to the ZP (r = 0.530, P < 0.001), and the percentage of sperm with normal DNA (r = 0.302, P < 0.001), and IVF rates. Sperm motility (r = 0.296, P < 0.05), motility grade (r = 0.274, P < 0.05), and mature nuclei (r = 0.252, P < 0.05) were less significant. Logistic Regression Analysis

To determine which groups of sperm characteristics were independently related to IVF rates, all of the data were examined by logistic regression analysis. Only number of sperm bound to the ZP, the percentages of sperm with normal morphology and normal DNA, and motility grade were strongly significantly related to IVF rates. All the other variables were not significant (Table 3). Because there was a significantly lower IVF rate (mean ± SD, 10 ± 22 versus 42 ± 31; P < 0.001) for patients with normal morphology < 15% (n = 39) than for patients with normal morphology ~ 15% (n = 52), the data from patients with normal morphology ~ 15% were selected and analyzed by logistic regression. In these, only number of sperm bound to the ZP, the percentages of sperm with normal DNA and mature nucleus, and motility grade were significantly related to IVF rates. Normal morphology and other test results were not significant (Table 3). Because patients with no oocytes fertilized usually have on average

Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding, and fertilization rates in vitro.

To study whether the results of tests of sperm chromatin and deoxyribonucleic acid (DNA) normality are related to fertilization rates in vitro...
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