Vol. 28, No.2, February 1977 Printed in U.s.A.
FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society
ON THE MORPHOLOGY OF LIVE SPERMATOZOA OF HUMAN SEMEN
BENGT FREDRICSSON, M.D., PH.D. GOREL WAXEGARD, M.D. SUSANNA BREGE INGER LUNDBERG
Department of Obstetrics and Gynecology, Sabbatsberg Hospital, S-113 82 Stockholm, Sweden
Live and dead spermatozoa ofhuman semen were distinguished and evaluated separately with respect to their morphology after supravital staining with buffered eosin. This evaluation was shown to be almost as reliable as that ofthe commonly stained smear. Different samples from the same individual were found to be similar with respect to both the total population and the live population. Different types of abnormalities were not equally common in the live and dead populations. Tapered heads were more common in the live population and abnormal midpieces more common in the dead, whereas abnormal tails and cytoplasmic droplets occurred with the same frequency in both populations. However, neither the frequency of all spermatozoal abnormalities nor that of abnormal heads or midpieces studied separately showed strong correlations in the two populations, in contrast to the frequencies of tapered heads, cytoplasmic droplets, and abnormal tails. No constant relationship was found between the live population and the entire sperm population with respect to numerical differences or quotients between the figures for abnormal cell frequency. Therefore, study of the morphology of the live sperm population gives supplementary information about the qualities ofsemen not attained by previous methods of study.
evaluation than the study of the whole semen sample. This study presents an evaluation of the possibilities of studying the morphology of the living sperm population separately and its relationship to the whole population as studied by conventional methods. This was achieved by supravital staining of the semen with eosin and morphologic evaluation of the stained dead spermatozoa and the unstained living spermatozoa separately under a phase-contrast microscope.
In contrast to the semen of many other species, human semen contains a very heterogeneous population of spermatozoa. Even in normal, fertile men, the frequency of immotile and morphologically defective spermatozoa is unusually high. Accordingly, it is not astonishing that the fertility of an individual cannot be predicted with reasonable accuracy from the data of the conventional semen analysis. Motility and morphology are better correlated to fertility than are other parameters of the semen analysis. 1 Although it is known from studies on the semen of many animal species that morphologic aberrations appear with the death of spermatozoa,2.3 it is not known to what degree human living spermatozoa also are morphologically normal. We may suppose that the study of the morphology of the living sperm population would give information more pertinent to the fertility
MATERIALS AND METHODS
The following calculations are based on data obtained from semen samples delivered at the laboratory during investigations for infertility. Samples with low sperm counts were initially omitted from the study; otherwise there was no selection. Smears were made and stained with hematoxylin-eosin for conventional morphologic
Accepted September 16, 1976.
168
Vol. 28, No. 2
MORPHOLOGY OF SPERMATOZOA
analysis. Smears were also mounted unstained for morphologic evaluation under a phase-contrast microscope. Supravital staining with eosin was achieved by mixing a small portion of the semen sample with an equal volume of 0.5% eosin Y in 0.15 M phosphate buffer (pH 7.4).4 Smears of this mixture were then made, air-dried, and mounted in Eukitt. Since semen specimens with low sperm counts are laborious to examine, the following method was developed to overcome this difficulty. After determination of the sperm concentration, the specimen is centrifuged at 400 x g for 20 minutes. The sediment is then resuspended in a portion of the supernatant, using a shaking apparatus (Super-Mixer; Lab-Line Instruments, Melrose Park, Ill). This suspension is then used for supravital staining. The treatment was shown not to interfere with either vitality or morphology and to have a reasonable safety margin, as centrifugation at 600 x g did not have any demonstrable effect on these parameters whereas centrifugation at 800 x g resulted in some reduction of vitality but did not impair morphology. The preparations were studied microscopically under negative phase-contrast with a x 100 oil-immersion lens (Anoptral phase-contrast; Reichert); dead cells are stained yellow, whereas live cells are whitish blue. The evaluation of the smears was more convenient with this equipment but was also possible with ordinary phase-contrast. Unstained preparations were similarly examined. The supravitally stained preparations were studied with respect to the morphology of both the unstained live and the stained dead populations. Morphologic evaluation of spermatozoa is practiced in the following way in our laboratory. The figures are based on examination of 200 spermatozoa appearing consecutively under the microscope. First, the frequency of spermatozoa with abnormal heads is determined. Tapered forms are included in this figure, but the frequency of tapered heads is also recorded separately. The frequency of spermatozoa with normal heads but abnormal mid pieces is then recorded. The category abnormal midpieces also includes abaxial insertion at the head. The frequency of spermatozoa with normal heads and midpieces but abnormal tails is then presented. Last, the frequency of spermatozoa normal in all respects except for the presence of cytoplasmic droplets exceeding one-half the size of the head is determined. The morphologic evaluation is based on the principles of Eliasson. 1 Their application in our
169
laboratory can be expressed by the mean and standard deviation calculated from the analyses of the first sample of all new patients referred in 1975 because of infertility. The distribution of the morphologic evaluations of these samples is shown in Figure 1. The mean frequency and standard deviation (within parentheses) of abnormal forms are as follows: total frequency of abnormal spermatozoa, 57.72% (14.66); of abnormal heads, 46.89% (15.45); of tapered spermatozoa, 3.37% (3.53); of abnormal midpieces, 6.75% (3.93); of abnormal tails, 2.64% (3.13); of cytoplasmic droplets, 1.40% (0.95). The protocols were not available during the examination of the different slides, to avoid bias. The method was evaluated by submitting the results to different correlations as follows, and the reliability was also tested.
Calculations How Accurate Are the Morphologic Evaluations? Two investigators were engaged in the study. However, all of the morphologic evaluations for each semen sample were performed by the same investigator. After 3 months the investigators were given a number of slides which they had evaluated previously and, in addition, some slides which had been evaluated by the other investigator in the first turn. The intra- and interindividual differences in the two evaluations of the same slides were calculated and are presented in Table 1. The differences are in no case significant. Is the Evaluation of Spermatozoal Morphology under Negative Phase-Contrast Equally as Reliable as Conventional Analysis of the Stained Preparation? This and the following regression analyses 1975
n
X
100
=
5 -
57.7 14.7
n = 530 80
60
40
20
16- 21- 26- 3120 25 30 35
36- 4,- 46- 51- 56- 6'- 66- 71- 76- 81- 86- 91- 9640 45 50 55 60 65 70 7S 80 85 90 95 100
% abnormal sperm
FIG. 1. Numerical distribution of the occurrence of abnonnal spennatozoa in all new infertility cases referred for analysis in 1975.
FREDRICSSON ET AL.
170
February 1977
TABLE 1. Differences between First and Second Evaluation of Seminal Smears: Intra- and Interindividual Differences for Investigators A and B Morphology
Frequency of live spermatozoa Dead spermatozoa
All spermatozoa
Live spermatozoa
-0.8 ± 1.6 4.6 (8)
-4.5 ± 3.3 8.8 (7)
2.1 ± 3.2 8.4 (7)
-3.9 ± 2.1 5.7 (7)
-1.6 ± 1.7 5.1 (10)
-0.7 ± 2.5 7.5 (10)
2.6 ± 1.3 4.0 (10)
2.7 ± 1.8 4.5 (10)
1.6 ± 2.2 5.4 (7)
-2.1 ± 3.3 8.1 (7)
8.0 ± 5.7 14.1 (7)
-2.4 ± 2.0 4.8 (7)
Intraindividual difference Investigator A Mean ± SE SD (no.) Investigator B Mean ± SE SD (no.) Interindividual difference A - B, mean ± SE SD (no.)
are based on the mean values obtained from the two evaluations of the same slide, as obtained in the preceding paragraph. Figure 2 shows that there was a good correlation between the morphology evaluated in the conventionally stained preparation and that evaluated in an unstained preparation examined under phase-contrast (r = 0.88; P < 0.001). The line does not point to the origin, which indicates the presence of an error. The deviation from the expected correlation is shown in the graph. This phenomenon is discussed later. How Reliable Is the Morphologic Evaluation of the Supravitally Stained Preparation under PhaseContrast? In this preparation the live spermatozoa are not stained at all and can be evaluated only by virtue of the optical principles of the phasecontrast microscope. In the preceding paragraph
this evaluation was shown to be equal to that obtained with the ordinary microscope, employing a conventionally stained specimen. In contrast, dead spermatozoa are stained by eosin. Therefore, they show up under phase-contrast with a much more pronounced halo phenomenon than do living spermatozoa, and thus are somewhat more difficult to evaluate. That this difficulty does not invalidate the evaluation is evident from the following calculation. VVhen both the percentage of live and dead spermatozoa and the morphology of the live and dead populations are known, a figure may be calculated which should correspond to the total number of abnormal spermatozoa. The correlation between the calculated values and those obtained %abn ,conventional stain
80 % 80
/'"
abn. conventional stain
'" '" '"
'"
'" '"
",e
60
'"'"
","'e
60
e",
40
e
~'"
.'"
'"
'"'"
'"
'"
'" '"'"
'"'"
40
e
20
-'" '"
'"
'" '" '"
'"
'" '" '"
'"'"
'" '"'"
'"
2
20
40
60
80 % abn calculated
20
40
60
80 %
abn.
unstained phase contrast
FIG. 2. Correlation of sperm morphology evaluated under phase-contrast with that evaluated in the stained preparation. The regression line, y = 0.79x + 15.9, deviates slightly from the expected line, y = x (---). The curved lines represent the 95% confidence interval.
3. Correlation of the calculated frequency of abnormal sperm with that obtained by evaluation of the stained preparation. The calculated values were obtained from the frequency of dead spermatozoa and the figures obtained by evaluation of the dead and live spermatozoa separately. The regression line, y = 0.94x + 9.86, deviates slightly from the expected line (---). The curved lines represent the 95% confidence interval: r = 0.92, t = 10.734, P < 0.001. FIG.
MORPHOLOGY OF SPERMATOZOA
Vol. 28, No.2 Yo
abn. live
80
60
4
20
20
40
60
80 % abn. dead
FIG. 4. Correlation of the morphology of the dead and live sperm populations,y = 0.7lx - 2.4. The curved lines represent the 95% confidence interval.
by conventional analysis of the stained smear is very good (r = 0.92; P < 0.001) (Fig. 3). This proves that the supra vitally stained preparation may be used for morphologic evaluation and that comparisons of the live and dead populations with the total population are justifiable.
Correlations We may now wonder which relationships may exist particularly between the live sperm population and the whole population. The following comparisons and calculations are intended to give the live sperm population a better-defined profile. The following aspects are considered: 1. Are there any correlations between the morphology of live spermatozoa and that of dead spermatozoa? 2. Is the difference between the morphology of live and dead spermatozoal populations related to the over-all morphology of the semen sample? 3. Is the morphology oflive spermatozoa correlated to their frequency? 4. In those cases in which correlations exist, are they more connected to one or another of the different morphologic subgroups? 5. Are the morphologic characteristics of the live population related to the individual patient and thus reproducible in different semen samples? The correlation between the live and dead sperm populations with respect to their morphology is shown in Figure 4. The correlation is statistically highly significant (r = 0.65; t = 3.870; P < 0.001). Although the correlation is highly significant,
171
the coefficient of correlation (r) is low, as evidenced by the wide scattering of the dots in Figure 4. Considering in the same way the different types of abnormalities, quite different regressions become evident (Table 2A). The most significant correlations are present when tails, tapering forms, and cytoplasmic droplets are considered. Less significant correlations are found when abnormalities in mid pieces and heads are considered separately. (Note that the coefficients of regression vary from 0.59 to 1.50, indicating that these correlations have different meanings.) The relationship between the living population and the total population was tested similarly. These relationships were stronger except for cytoplasmic droplets (Table 2B). The correlation is strong both for heads and tapering forms, but the coefficients are different. Therefore, the relationship for the heads, excluding tapering forms, was determined. A much weaker correlation was then evidenced (Table 2B). Next we tested whether the difference between the live and dead sperm populations was related to the over-all morphology of the semen sample. Regression analysis showed that there was no TABLE 2. Comparison of the Morphology of Live, Dead, and Total Sperm Populations" Correlation
A. Dead cells (,x) vs. live cells (y) Abnormal heads Tapering forms Abnormal midpieces Abnormal tails Cytoplasmic droplets All abnormalities B. All cells (,x) vs. live cells (y) Abnormal heads Tapering forms Abnormal heads except tapering forms Abnormal midpieces Abnormal tails Cytoplasmic droplets All abnormalities
Equation
P
y
= 0.59x - 0.70 0.59
3.232
FERTILITY AND STERILITY Copyright © 1977 The American Fertility Society
ON THE MORPHOLOGY OF LIVE SPERMATOZOA OF HUMAN SEMEN
BENGT FREDRICSSON, M.D., PH.D. GOREL WAXEGARD, M.D. SUSANNA BREGE INGER LUNDBERG
Department of Obstetrics and Gynecology, Sabbatsberg Hospital, S-113 82 Stockholm, Sweden
Live and dead spermatozoa ofhuman semen were distinguished and evaluated separately with respect to their morphology after supravital staining with buffered eosin. This evaluation was shown to be almost as reliable as that ofthe commonly stained smear. Different samples from the same individual were found to be similar with respect to both the total population and the live population. Different types of abnormalities were not equally common in the live and dead populations. Tapered heads were more common in the live population and abnormal midpieces more common in the dead, whereas abnormal tails and cytoplasmic droplets occurred with the same frequency in both populations. However, neither the frequency of all spermatozoal abnormalities nor that of abnormal heads or midpieces studied separately showed strong correlations in the two populations, in contrast to the frequencies of tapered heads, cytoplasmic droplets, and abnormal tails. No constant relationship was found between the live population and the entire sperm population with respect to numerical differences or quotients between the figures for abnormal cell frequency. Therefore, study of the morphology of the live sperm population gives supplementary information about the qualities ofsemen not attained by previous methods of study.
evaluation than the study of the whole semen sample. This study presents an evaluation of the possibilities of studying the morphology of the living sperm population separately and its relationship to the whole population as studied by conventional methods. This was achieved by supravital staining of the semen with eosin and morphologic evaluation of the stained dead spermatozoa and the unstained living spermatozoa separately under a phase-contrast microscope.
In contrast to the semen of many other species, human semen contains a very heterogeneous population of spermatozoa. Even in normal, fertile men, the frequency of immotile and morphologically defective spermatozoa is unusually high. Accordingly, it is not astonishing that the fertility of an individual cannot be predicted with reasonable accuracy from the data of the conventional semen analysis. Motility and morphology are better correlated to fertility than are other parameters of the semen analysis. 1 Although it is known from studies on the semen of many animal species that morphologic aberrations appear with the death of spermatozoa,2.3 it is not known to what degree human living spermatozoa also are morphologically normal. We may suppose that the study of the morphology of the living sperm population would give information more pertinent to the fertility
MATERIALS AND METHODS
The following calculations are based on data obtained from semen samples delivered at the laboratory during investigations for infertility. Samples with low sperm counts were initially omitted from the study; otherwise there was no selection. Smears were made and stained with hematoxylin-eosin for conventional morphologic
Accepted September 16, 1976.
168
Vol. 28, No. 2
MORPHOLOGY OF SPERMATOZOA
analysis. Smears were also mounted unstained for morphologic evaluation under a phase-contrast microscope. Supravital staining with eosin was achieved by mixing a small portion of the semen sample with an equal volume of 0.5% eosin Y in 0.15 M phosphate buffer (pH 7.4).4 Smears of this mixture were then made, air-dried, and mounted in Eukitt. Since semen specimens with low sperm counts are laborious to examine, the following method was developed to overcome this difficulty. After determination of the sperm concentration, the specimen is centrifuged at 400 x g for 20 minutes. The sediment is then resuspended in a portion of the supernatant, using a shaking apparatus (Super-Mixer; Lab-Line Instruments, Melrose Park, Ill). This suspension is then used for supravital staining. The treatment was shown not to interfere with either vitality or morphology and to have a reasonable safety margin, as centrifugation at 600 x g did not have any demonstrable effect on these parameters whereas centrifugation at 800 x g resulted in some reduction of vitality but did not impair morphology. The preparations were studied microscopically under negative phase-contrast with a x 100 oil-immersion lens (Anoptral phase-contrast; Reichert); dead cells are stained yellow, whereas live cells are whitish blue. The evaluation of the smears was more convenient with this equipment but was also possible with ordinary phase-contrast. Unstained preparations were similarly examined. The supravitally stained preparations were studied with respect to the morphology of both the unstained live and the stained dead populations. Morphologic evaluation of spermatozoa is practiced in the following way in our laboratory. The figures are based on examination of 200 spermatozoa appearing consecutively under the microscope. First, the frequency of spermatozoa with abnormal heads is determined. Tapered forms are included in this figure, but the frequency of tapered heads is also recorded separately. The frequency of spermatozoa with normal heads but abnormal mid pieces is then recorded. The category abnormal midpieces also includes abaxial insertion at the head. The frequency of spermatozoa with normal heads and midpieces but abnormal tails is then presented. Last, the frequency of spermatozoa normal in all respects except for the presence of cytoplasmic droplets exceeding one-half the size of the head is determined. The morphologic evaluation is based on the principles of Eliasson. 1 Their application in our
169
laboratory can be expressed by the mean and standard deviation calculated from the analyses of the first sample of all new patients referred in 1975 because of infertility. The distribution of the morphologic evaluations of these samples is shown in Figure 1. The mean frequency and standard deviation (within parentheses) of abnormal forms are as follows: total frequency of abnormal spermatozoa, 57.72% (14.66); of abnormal heads, 46.89% (15.45); of tapered spermatozoa, 3.37% (3.53); of abnormal midpieces, 6.75% (3.93); of abnormal tails, 2.64% (3.13); of cytoplasmic droplets, 1.40% (0.95). The protocols were not available during the examination of the different slides, to avoid bias. The method was evaluated by submitting the results to different correlations as follows, and the reliability was also tested.
Calculations How Accurate Are the Morphologic Evaluations? Two investigators were engaged in the study. However, all of the morphologic evaluations for each semen sample were performed by the same investigator. After 3 months the investigators were given a number of slides which they had evaluated previously and, in addition, some slides which had been evaluated by the other investigator in the first turn. The intra- and interindividual differences in the two evaluations of the same slides were calculated and are presented in Table 1. The differences are in no case significant. Is the Evaluation of Spermatozoal Morphology under Negative Phase-Contrast Equally as Reliable as Conventional Analysis of the Stained Preparation? This and the following regression analyses 1975
n
X
100
=
5 -
57.7 14.7
n = 530 80
60
40
20
16- 21- 26- 3120 25 30 35
36- 4,- 46- 51- 56- 6'- 66- 71- 76- 81- 86- 91- 9640 45 50 55 60 65 70 7S 80 85 90 95 100
% abnormal sperm
FIG. 1. Numerical distribution of the occurrence of abnonnal spennatozoa in all new infertility cases referred for analysis in 1975.
FREDRICSSON ET AL.
170
February 1977
TABLE 1. Differences between First and Second Evaluation of Seminal Smears: Intra- and Interindividual Differences for Investigators A and B Morphology
Frequency of live spermatozoa Dead spermatozoa
All spermatozoa
Live spermatozoa
-0.8 ± 1.6 4.6 (8)
-4.5 ± 3.3 8.8 (7)
2.1 ± 3.2 8.4 (7)
-3.9 ± 2.1 5.7 (7)
-1.6 ± 1.7 5.1 (10)
-0.7 ± 2.5 7.5 (10)
2.6 ± 1.3 4.0 (10)
2.7 ± 1.8 4.5 (10)
1.6 ± 2.2 5.4 (7)
-2.1 ± 3.3 8.1 (7)
8.0 ± 5.7 14.1 (7)
-2.4 ± 2.0 4.8 (7)
Intraindividual difference Investigator A Mean ± SE SD (no.) Investigator B Mean ± SE SD (no.) Interindividual difference A - B, mean ± SE SD (no.)
are based on the mean values obtained from the two evaluations of the same slide, as obtained in the preceding paragraph. Figure 2 shows that there was a good correlation between the morphology evaluated in the conventionally stained preparation and that evaluated in an unstained preparation examined under phase-contrast (r = 0.88; P < 0.001). The line does not point to the origin, which indicates the presence of an error. The deviation from the expected correlation is shown in the graph. This phenomenon is discussed later. How Reliable Is the Morphologic Evaluation of the Supravitally Stained Preparation under PhaseContrast? In this preparation the live spermatozoa are not stained at all and can be evaluated only by virtue of the optical principles of the phasecontrast microscope. In the preceding paragraph
this evaluation was shown to be equal to that obtained with the ordinary microscope, employing a conventionally stained specimen. In contrast, dead spermatozoa are stained by eosin. Therefore, they show up under phase-contrast with a much more pronounced halo phenomenon than do living spermatozoa, and thus are somewhat more difficult to evaluate. That this difficulty does not invalidate the evaluation is evident from the following calculation. VVhen both the percentage of live and dead spermatozoa and the morphology of the live and dead populations are known, a figure may be calculated which should correspond to the total number of abnormal spermatozoa. The correlation between the calculated values and those obtained %abn ,conventional stain
80 % 80
/'"
abn. conventional stain
'" '" '"
'"
'" '"
",e
60
'"'"
","'e
60
e",
40
e
~'"
.'"
'"
'"'"
'"
'"
'" '"'"
'"'"
40
e
20
-'" '"
'"
'" '" '"
'"
'" '" '"
'"'"
'" '"'"
'"
2
20
40
60
80 % abn calculated
20
40
60
80 %
abn.
unstained phase contrast
FIG. 2. Correlation of sperm morphology evaluated under phase-contrast with that evaluated in the stained preparation. The regression line, y = 0.79x + 15.9, deviates slightly from the expected line, y = x (---). The curved lines represent the 95% confidence interval.
3. Correlation of the calculated frequency of abnormal sperm with that obtained by evaluation of the stained preparation. The calculated values were obtained from the frequency of dead spermatozoa and the figures obtained by evaluation of the dead and live spermatozoa separately. The regression line, y = 0.94x + 9.86, deviates slightly from the expected line (---). The curved lines represent the 95% confidence interval: r = 0.92, t = 10.734, P < 0.001. FIG.
MORPHOLOGY OF SPERMATOZOA
Vol. 28, No.2 Yo
abn. live
80
60
4
20
20
40
60
80 % abn. dead
FIG. 4. Correlation of the morphology of the dead and live sperm populations,y = 0.7lx - 2.4. The curved lines represent the 95% confidence interval.
by conventional analysis of the stained smear is very good (r = 0.92; P < 0.001) (Fig. 3). This proves that the supra vitally stained preparation may be used for morphologic evaluation and that comparisons of the live and dead populations with the total population are justifiable.
Correlations We may now wonder which relationships may exist particularly between the live sperm population and the whole population. The following comparisons and calculations are intended to give the live sperm population a better-defined profile. The following aspects are considered: 1. Are there any correlations between the morphology of live spermatozoa and that of dead spermatozoa? 2. Is the difference between the morphology of live and dead spermatozoal populations related to the over-all morphology of the semen sample? 3. Is the morphology oflive spermatozoa correlated to their frequency? 4. In those cases in which correlations exist, are they more connected to one or another of the different morphologic subgroups? 5. Are the morphologic characteristics of the live population related to the individual patient and thus reproducible in different semen samples? The correlation between the live and dead sperm populations with respect to their morphology is shown in Figure 4. The correlation is statistically highly significant (r = 0.65; t = 3.870; P < 0.001). Although the correlation is highly significant,
171
the coefficient of correlation (r) is low, as evidenced by the wide scattering of the dots in Figure 4. Considering in the same way the different types of abnormalities, quite different regressions become evident (Table 2A). The most significant correlations are present when tails, tapering forms, and cytoplasmic droplets are considered. Less significant correlations are found when abnormalities in mid pieces and heads are considered separately. (Note that the coefficients of regression vary from 0.59 to 1.50, indicating that these correlations have different meanings.) The relationship between the living population and the total population was tested similarly. These relationships were stronger except for cytoplasmic droplets (Table 2B). The correlation is strong both for heads and tapering forms, but the coefficients are different. Therefore, the relationship for the heads, excluding tapering forms, was determined. A much weaker correlation was then evidenced (Table 2B). Next we tested whether the difference between the live and dead sperm populations was related to the over-all morphology of the semen sample. Regression analysis showed that there was no TABLE 2. Comparison of the Morphology of Live, Dead, and Total Sperm Populations" Correlation
A. Dead cells (,x) vs. live cells (y) Abnormal heads Tapering forms Abnormal midpieces Abnormal tails Cytoplasmic droplets All abnormalities B. All cells (,x) vs. live cells (y) Abnormal heads Tapering forms Abnormal heads except tapering forms Abnormal midpieces Abnormal tails Cytoplasmic droplets All abnormalities
Equation
P
y
= 0.59x - 0.70 0.59
3.232
