Bourne GH (ed): Aspects of Some Vitamins, Minerals and Enzymes in Health and Disease. World Rev Nutr Diet. Basel, Karger, 1990, vol 62, pp 1-26
Relationship between Ascorbic Acid and Male Fertility Earl B. Dawson, William A. Harris, Leslie C. Powell Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Tex., USA
Contents Introduction A Comparison of Semen AA and Agglutination Levels Materials and Methods Results Effect of AA Dietary Supplementation on Sperm Qualities Materials and Methods Results Sperm Quality and Fertility Changes with Age Materials and Methods Results Discussion References
1 5 5 6 10 10 11 16 16 17 20 25
Ascorbic acid (AA, C6H608) is a ketolactone with a molecular weight of 176.1 g. Most mammals can synthesize AA from glucuronic acid derived from glucose. However, primates (including man) and the guinea pig lack the intracellular enzyme gulonolactone oxidase and require the dietary supplement of this vitamin for normal growth, development, and maintenance.
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Introduction
2
In 1926, Lindsay and ledes [1] studied the histological damage caused by feeding male guinea pigs a diet deficient in AA for one month, prior to sacrifice and microscopic study of various organ tissues. Second only to the adrenomedulla, the testes were the most severely affected of all the tissues damaged by ÁA deficiency. They were surprised to observe massive degeneration of the germinal epithelium with total desquamation of the lamina into the epithelial tubules. In 1941, Berg et al. [2] reported that the mean concentration of AA in the seminal plasma of man was 12.8 ± 1.6 mg/dl and is actively secreted by the seminal vesicles during ejaculation. This concentration of AA is extremely high when compared to the average serum values between 0.4 and 1.0 mg/dl and implied an important biological role for AA in the seminal plasma. In 1947, Jungek et al. [3] reported that a study comparing male blood hormone levels with corresponding infertility showed no correlation; however, a study of seminal plasma levels of vitamins A, E, and C showed that the majority of infertile males exhibited lowered seminal plasma AA levels. Lindahl and Kilstrom [4], in a series of studies between 1952 and 1954, reported the presence of a sperm agglutinating substance in the seminal plasma of man, horse, swine, and rabbit. Further, this substance was equally capable of causing the agglutination of sperm between these different species, and the substance was subsequently called `nonspecific sperm agglutinin' (NSA). Their studies showed that the NSA existed in one of two forms, either oxidized or reduced. The reduced form attaches to and coats the surface of the spermatozoa but loses this ability of spermatozoa fixation when oxidized. The reduced NSA forms a molecular layer over the surface of the heads of the sperm, the active group sticking to the cell surface and an extruding protein residue increasing the actual sperm surface. The NSA appeared to constitute a component of the seminal plasma of great importance for functioning mammalian sperm, to the extent that the absence of this substance or its occurrence in the inactive oxidized form impaired normal fertility by resulting in clumping of the sperm and preventing normal forward movement (motility). Further studies in vivo demonstrated the prostate gland was the source of NSA, and analysis revealed the NSA molecule was a medium-sized protein with a saccharide segment and containing active sulfhydryl groups, and apparently vitamin E [4, 5]. In 1954, Wilson [6] identified sperm agglutination as the cause of infertility in several of his patients. In 1958, Kupperman and Epstein [7]
Downloaded by: Université de Paris 193.51.85.197 - 1/24/2020 9:53:14 AM
Dawson/Harris/Powell
Relationship between Ascorbic Acid and Male Fertility
3
Table 1. Changes in the visible physical properties of semen from 20 infertile men (> 25 % Agglutination) after Dietary ascorbic acid supplementation (1.0 g/day for 60 days) Test Volume, ml Sperm count, million/ml Motility, % Abnormal, % Precursors, million/ml
Beforea 2.4±0.2 46 ± 6 26±3 56±1 4.95 ± 0.46
Afters 2.9±0.2 72 ± 10 34±3 51 ± 1 3.75 ± 0.35
t 2.207b 2.270b 2.342b 2.580° 2.125b
also reported the occurrence of infertility in their male patients due to excessive sperm agglutination. This was diagnosed by microscopic examination showing the inhibition of sperm motility associated with sperm clumping. They reported the use of AA in effectively overcoming agglutination within a few days with dietary supplementation of 100 mg of AA three times a day. They recommended the use of AA supplementation when there was agglutination of the sperm accompanied by a normal sperm count. These studies were confirmed by in vitro studies of Lindahl and Kihlstrom [8] in 1960 that showed NSA can be readily oxidized with dilute concentrations of hydrogen peroxide and reversibly reduced by such substances as AA, glutathione, ergatothionine, or sulfite, all of which occur in the seminal plasma of man and other mammals. Of these, ÁA is normally present in the greatest concentration. In 1979, Harris et al. [9] reported that dietary AA supplementation in men with sperm agglutination in excess of 25 % resulted in improvement in semen volume, sperm count, sperm motility, a reduction in the number of abnormal sperm, and sperm quality, in addition to a marked reduction in sperm agglutination associated with increased fertility (table 1). These effects were observed after a dietary supplementation of 1 g of AA per day for 60 days. In contrast, no changes were noted in any of these measurements in a control group who received no AA supplementation during the same period of time (table 2). At the end of 60 days, all of the group who had received AA had impregnated their wives, and none of the control
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a Values are means ± standard error. b Confidence level < 0.05. Confidence level < 0.02.
4
Dawson/Hams/Powell
Table 2. Changes in the sperm qualities of 7 infertile men (> 25% agglutination) during 60-day interval Test
Beforea
Afters
t
Volume, ml Sperm count, million/ml Motility, % Abnormal, % Precursors, million/ml
1.8 ±0.3 84±26 22±3 57 ± 2 6.6 ± 0.6
2.2 ± 0.2 72± 18 24±3 52±1 6.6± 0.6
1.686 0.820 0.810 2.040 0.000
Values are means ± standard error. Confidence levels: no significant differences.
subjects reported pregnancies. Comparison of the sperm qualities before and after the 60-day period showed marked improvement in all of the measured properties in the group receiving Ø supplementation and no change in the control group. Most notably, there was observed a significant increase in sperm count and motility, and a decrease in abnormal sperm, sperm precursors (p < 0.05), and agglutination (p < 0.001). The group mean ± SEM of the seminal plasma Ø levels of the control group before the study was 1.41 ± 0.40 and 1.25 ± 0.32 mg/dl at the end. The group mean ± SEM seminal plasma levels of Ø of the supplemented group was 2.29 ± 0.96 mg/dl prior to supplementation and 6.75 ± 0.43 mg/dl after 60 days of ΑΑ supplementation. These results indicated a beneficial effect of Ø on both the mature sperm present in the seminal plasma and upon the germinal epithelium of the testes and were in agreement with previous studies. Harris et al. [9] noted that the sperm of fertile men may show some level of sperm agglutination up to 25 %, but when the percentage of agglutination of normal sperm exceeds 25 %, the subject is considered infertile. In 1986, the studies of Lindsay and Medes [ 1 ] were confirmed by Chinoy et al. [10] when male guinea pigs with proven fertility were placed on a diet deficient in ΑΑ for 3 weeks and sacrificed. The most severe histological damage was observed in the testes. Further, the most pronounced effect was in the cauda epididymis, wherein sperm undergo maturation, with thickened peritubular muscle layer and connective tissue, decreased tubular diameter and epithelial cell height, and nuclear pycnosis. Similarly affected were the vas deferens and accessory sex glands, although to a lesser degree. Sperm analysis of semen samples collected
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a
Relationship between Ascorbic Acid and Male Fertility
5
from testis, cauda epididymis, and vas deferens showed decline in total sperm count and sperm motility, as well as an increase in the number of abnormal sperm. The purposes of the present studies were: (1) to confirm the relationship between ÁA and sperm agglutination and AA and the germinal epithelium; (2) to obtain a more precise level of AA dietary supplementation and length of supplementation necessary to obtain the desired effects, and (3) to determine the effect of age on the occurrence of sperm agglutination and other sperm qualities.
Materials and Methods Semen samples were obtained from 28 apparently healthy young men between the ages of 23 and 37 years with a demonstrated normal sperm count (at least 20 million/ml). The study subjects were recruited on a volunteer basis, and each signed a consent form as required by the Institutional Review Board for Human Use before samples were collected. Four of the subjects were routine semen donors with proven fertility, six were housekeepers, seven were maintenance workers, two were gardeners, two were graduate students, and seven were laboratory personnel. The study population was a broad spectrum of male employment within the age range of probable maximum productivity. The only reward for participation was a copy of their semen analysis for their own knowledge. Routinely, all samples were obtained within the laboratory. The sperm analysis was with an American Optical Binocular Microscope (AO Scientific Instruments, Buffalo, N.Y.) and was completed within 10 min [9]. The sperm qualities determined were: total counts as millions of sperm per milliliter; viability as percent of sperm count showing movement; motility as percentage of sperm count showing progressive forward movement; sperm precursors as millions per milliliter; abnormal sperm as percentage of sperm count; and sperm agglutination as percentage of sperm count clumped together with no forward movement, but obviously viable (fig. 1, 2). All of these counts were in triplicate of separated fields with a highpowered lens (fig. 1, 2). Sperm precursors are easily discernable: (1) they are larger in size than white blood cells, which are usually present, (2) the nucleus appears as a small tip on the outer perimeter of the precursor, and (3) vacuoles are present.
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A Comparison of Semen AA and Agglutination Levels
Dawson/Harris/Powell
6
Attenuated Head
Pi·ecursore
Fig. 1. Abnormal mature sperm and sperm precursors.
Results The average seminal plasma AA level of the study population was 9.1 mg/dl, with a range between 3.7 and 14.7 mg/dl. Sperm agglutination aver-
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After microscopic examination, the semen specimens were centrifuged (300 g, 27 °C) to obtain clear seminal plasma for the preparation of trichloroacetic acid (TCA) extracts, which were stored frozen (— 60 °C) until assayed for AA. All AA measurements were made by the 2,4- dinítrophenylhydrazine method of Lowry et al. [ 11 ], with anhydrous L-ascorbic acid used as the standard for comparison (A-7506, Sigma Chemical Company, St. Louis, Mo.). The results were calculated as mg/dl. The measured data were grouped into tertiles of increasing percent agglutination: 0%, 5-25%, and > 25%, and the group means ± SEM of percent agglutination and seminal plasma AA levels (mg/dl) were calculated. The Student's t-test was used to determine the significant differences among the group means of the three tertiles, and linear regression was used to correlate the semen ascorbic acid levels with the percent agglutination, determined simultaneously.
Relationship between Ascorbic Acid and Male Fertility
7
aged 11.4 %, with a range from 0 to 40%. The viability averaged 52%, with a range from 20 to 75 %. The semen volume averaged 2.6 ml, ranging from 1.2 to 2.75 ml. The sperm count averaged 65 million/ml, ranging from 25 to 88 million/ml. The percent motility averaged 62%, ranging from 38 to 80%. The percent of abnormal sperm averaged 55%, ranging from 42 to 60 %. The sperm precursor count averaged 6 million/ml, ranging from 4.5 to 7.2 million/ml. The percent agglutination of the individual sperm counts was the basis for grouping the study tertiles of table 3 and the group mean percent agglutination of the three tertiles would be significantly different by design. There were 13 semen samples with no sperm agglutination, 8 semen samples with sperm agglutination between 5 and 24 %, and 7 samples with 25 % sperm agglutination or higher. The group mean percent agglutination of the 0% tertile was 0%, the group mean percent agglutination of the 5-25% tertile was 9 ± 1%, and 32 ± 1% for the > 25% tertile. The differences between the group means of both the 0% and 5-25% tertiles and the 0% and > 25% tertiles were statistically significant (p 0.001) (table 3).
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Fig. 2. Agglutinated pair of normal mature sperm and two sperm precursors. Highpower magnification.
Dawson/Harris/Powell
8
Table 3. Semen physical properties of 28 random samples grouped according to tertiles of percent nonspecific sperm agglutination: 0, 5-25, and > 25 Physical properties
Agglutination, % 0
Semen volume, ml Sperm count, 10/ml Sperm motility, % Abnormal sperm,% Precursors, 10/ml Sperm agglutination, % Ascorbic acid, mg/dl N
2.5±03 76±20 50±4 58±5 5±0.7 0 9.06±0.96 13
5-25 2.9±0.4 60±16 38±4b 49±6 6±0.6 9± l a
7.56±1.13 8
> 25 1.8±0.4 58±26 16±6a 58±1 5±0.6 32 ± l 6.00±0.91b 7
Nο significant difference was observed between the 0 and 5-25 % tertiles in group mean Ø levels, but the mean of the > 25 % tertile was significantly lower than the 0% tertile (6.0 ± 0.9 vs 9.1 ± 0.9, p < 0.05) (table 3). There were no significant differences in the tertile group means: 7.5 ± 0.3 ml for the 0% tertile, 2.9 ± 0.4 for the 5-25 % tertile, and 0.8 ± 0.4% for the > 25% tertile. There were no significant differences between the three tertiles in the sperm count. The group mean count for the 0% tertile was 79 ± 20 million/ml, 60 ± 16 millionml for the 5-25 % tertile, and 58 ± 20 million/mi for the > 25% tertile. However, there was an average 30 % difference in sperm count between the 0 and the > 25 % tertiles. There were significant differences between all three tertiles in group mean sperm viability (table 3). The group mean viability was significantly lower than the 0% tertile in both the 5-25 and the >25% tertile. The group mean sperm viability of the 5-25 % tertile was 38 ± 4 % compared to the group mean οf 50 ± 4% for the 0% tertile (p < 0.05). The group mean sperm viability of the > 25% tertile was 15 ± 6% compared to 50 ± 4% for the 0% tertile (p < 0.001). The group mean sperm motility was 50 ± 4% for the 0% tertile, 38 ± 4% for the 5-25% tertile and 16 ± 6% for the > 25 % tertile. The group mean of the 5-25 % tertile was significantly higher than the 0% tertile (p < 0.05) as was the group mean of the > 25% tertile (p < 0.001).
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ap < 0.001; bp < 0.05.
Relationship between Ascorbic Acid and Male Fertility
4-0 30 c o
._c
9
Υ intercept: 31.2 Slope: -2.64 `r': -0.6140 p < 0.001 n: 28
20
rn
m 10 Ε
ι
0
2 0 4 6 Semen ascorbic acid, mg/dl
10
12
There was no significant difference between the group mean percent of abnormal sperm between the three tertiles (table 3). The group means of the three tertiles were: 49 ± 5% for the 0% tertile, 53 ± 11 % for the 5-25% tertile, and 58 ± 5% for the -25% tertile. Although the group mean differences were small between the three tertiles, there was an 8% increase in abnormal sperm between the 0 and the 5-25 % tertiles and a 9% increase between the 5-25 and the >25% tertiles. The total increase in abnormal sperm between the 0 and the >25% tertile was 18%. There was virtually no measurable difference between the three study tertiles in the group mean sperm precursor counts (table 3). The group mean for the 0% tertile was 5 ± 0.7 million/ml, 6 ± 0.6 million/ml for the 5-25 % tertile, and 5 ± 0.6 million/ml for the > 25 % tertile. The group mean precursor count for all three tertiles may be considered identical. The viability was significantly lower in both the 5-25 and > 25 % tertiles than in the 0% tertile (p < 0.05, 0.001). The group mean agglutination of both the 5-25 % and > 25 % tertile was significantly higher than the 0% tertile (p < 0.001). The sperm agglutination measurements and seminal plasma AA levels were compared by linear regression (fig. 3). This analysis showed a negative correlation (r) of 0.6140, which was statistically significant (p < 0.001). This correlation shows that every 1.0 mg/ml increase in seminal plasma AA level was associated with a 2.64% decrease in sperm agglutina-
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Fig. 3. A linear regression analysis comparing semen ascorbic acid levels with percentage of nonspecific sperm agglutination in 28 random study patients.
Dawson/Hams/Powell
10
tun. Predictably, when the seminal plasma AA level is zero in this study population, approximately 31 % of the sperm are agglutinated. Also, there is no agglutination when the seminal plasma AA is 11.0 mg/dl or higher.
Materials and Methods The study subjects were 30 men between the ages of 25 and 45 with sperm agglutination over 25% when their sperm was initially studied by microscopic analysis [9]. Sperm antibody studies, according to the method of Franklin and Dukes [12, 13], were negative in each subject. All were considered physically normal and showed no clinical signs of inflammatory disease. The research protocol was described to the subjects, human consent forms signed, and 10 ml of blood was obtained for the measurement of serum AA. An aliquot of seminal plasma was prepared for AA measurement. By random selection, three groups of 10 subjects each were provided one month's supply of a daily supplement of 1,000 or 200 mg of AA, or a placebo. The pure L-ascorbic acid used for supplement and placebo were provided by Hoffmann-La Roche, Inc., Nutley, N.J. Each subject was told he was receiving AA and expected improvement in sperm quality. The subjects returned weekly for 3 weeks and were requested to refrain from sexual activity during the length of the study. After microscope examination of the sperm, blood samples were obtained by venipuncture. Semen and blood samples were analyzed as in the previous study. The measurement of AA was in a separate laboratory after samples had been collected and stored, and the subject identity of the numbered test tubes containing the TCA extracts was unknown at the time of analysis. After completion of laboratory analysis, the sample data were grouped according to the dietary supplementation provided each subject and the week of sampling. The group mean and the standard error of the mean ( ± SEM) was calculated for the placebo, 200 mg AA, and the 1,000mg AA-supplemented groups for each of the 4 weeks during the study: 0, 1, 2, and 3. The samples obtained prior to subject selection and supplementation were at 0 weeks, and the groups of samples obtained during weeks 1, 2, and 3 were during supplementation. The Student's t-test was used to determine the statistical significance of the weekly group mean change from the presupplementation levels in the serum and seminal plasma AA and the sperm qualities during supplementation.
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Effect of ΑΑ Dietary Supplementation on Sperm Qualities
Relationship between Ascorbic Acid and Male Fertility
11
Table 4. Changes in mean ± SEM serum and seminal plasma vitamin C levels (mg/dl) during study for the three study groups (10 in each group) Study weeks
Serum Placebo Supplemented Seminal plasma Placebo Supplemented
1
2
3
200 mg 1,000 mg
0.57±0.16 0.40±0.12 0.20±0.01
0.61±0.11 0.90±0.20a 1.30±0.30c
0.65±0.08 1.80±0.144 2.50±0.154
0.65±0.09 2.20±0.104 2.40±0.15d
200 mg 1,000 mg
3.7 ± 1.9 2.0±0.5 4.2±0.7
2.9±2.0 3.8±0.4b 12.7±1.1d
3.0 ± 1.1 6.3±0.3d 11.2±1.3d
2.0 ± 1.1 8.1±0.54 13.1±1.3d
< 0.05;bp < 0.02; Cp
Relationship between Ascorbic Acid and Male Fertility Earl B. Dawson, William A. Harris, Leslie C. Powell Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Tex., USA
Contents Introduction A Comparison of Semen AA and Agglutination Levels Materials and Methods Results Effect of AA Dietary Supplementation on Sperm Qualities Materials and Methods Results Sperm Quality and Fertility Changes with Age Materials and Methods Results Discussion References
1 5 5 6 10 10 11 16 16 17 20 25
Ascorbic acid (AA, C6H608) is a ketolactone with a molecular weight of 176.1 g. Most mammals can synthesize AA from glucuronic acid derived from glucose. However, primates (including man) and the guinea pig lack the intracellular enzyme gulonolactone oxidase and require the dietary supplement of this vitamin for normal growth, development, and maintenance.
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Introduction
2
In 1926, Lindsay and ledes [1] studied the histological damage caused by feeding male guinea pigs a diet deficient in AA for one month, prior to sacrifice and microscopic study of various organ tissues. Second only to the adrenomedulla, the testes were the most severely affected of all the tissues damaged by ÁA deficiency. They were surprised to observe massive degeneration of the germinal epithelium with total desquamation of the lamina into the epithelial tubules. In 1941, Berg et al. [2] reported that the mean concentration of AA in the seminal plasma of man was 12.8 ± 1.6 mg/dl and is actively secreted by the seminal vesicles during ejaculation. This concentration of AA is extremely high when compared to the average serum values between 0.4 and 1.0 mg/dl and implied an important biological role for AA in the seminal plasma. In 1947, Jungek et al. [3] reported that a study comparing male blood hormone levels with corresponding infertility showed no correlation; however, a study of seminal plasma levels of vitamins A, E, and C showed that the majority of infertile males exhibited lowered seminal plasma AA levels. Lindahl and Kilstrom [4], in a series of studies between 1952 and 1954, reported the presence of a sperm agglutinating substance in the seminal plasma of man, horse, swine, and rabbit. Further, this substance was equally capable of causing the agglutination of sperm between these different species, and the substance was subsequently called `nonspecific sperm agglutinin' (NSA). Their studies showed that the NSA existed in one of two forms, either oxidized or reduced. The reduced form attaches to and coats the surface of the spermatozoa but loses this ability of spermatozoa fixation when oxidized. The reduced NSA forms a molecular layer over the surface of the heads of the sperm, the active group sticking to the cell surface and an extruding protein residue increasing the actual sperm surface. The NSA appeared to constitute a component of the seminal plasma of great importance for functioning mammalian sperm, to the extent that the absence of this substance or its occurrence in the inactive oxidized form impaired normal fertility by resulting in clumping of the sperm and preventing normal forward movement (motility). Further studies in vivo demonstrated the prostate gland was the source of NSA, and analysis revealed the NSA molecule was a medium-sized protein with a saccharide segment and containing active sulfhydryl groups, and apparently vitamin E [4, 5]. In 1954, Wilson [6] identified sperm agglutination as the cause of infertility in several of his patients. In 1958, Kupperman and Epstein [7]
Downloaded by: Université de Paris 193.51.85.197 - 1/24/2020 9:53:14 AM
Dawson/Harris/Powell
Relationship between Ascorbic Acid and Male Fertility
3
Table 1. Changes in the visible physical properties of semen from 20 infertile men (> 25 % Agglutination) after Dietary ascorbic acid supplementation (1.0 g/day for 60 days) Test Volume, ml Sperm count, million/ml Motility, % Abnormal, % Precursors, million/ml
Beforea 2.4±0.2 46 ± 6 26±3 56±1 4.95 ± 0.46
Afters 2.9±0.2 72 ± 10 34±3 51 ± 1 3.75 ± 0.35
t 2.207b 2.270b 2.342b 2.580° 2.125b
also reported the occurrence of infertility in their male patients due to excessive sperm agglutination. This was diagnosed by microscopic examination showing the inhibition of sperm motility associated with sperm clumping. They reported the use of AA in effectively overcoming agglutination within a few days with dietary supplementation of 100 mg of AA three times a day. They recommended the use of AA supplementation when there was agglutination of the sperm accompanied by a normal sperm count. These studies were confirmed by in vitro studies of Lindahl and Kihlstrom [8] in 1960 that showed NSA can be readily oxidized with dilute concentrations of hydrogen peroxide and reversibly reduced by such substances as AA, glutathione, ergatothionine, or sulfite, all of which occur in the seminal plasma of man and other mammals. Of these, ÁA is normally present in the greatest concentration. In 1979, Harris et al. [9] reported that dietary AA supplementation in men with sperm agglutination in excess of 25 % resulted in improvement in semen volume, sperm count, sperm motility, a reduction in the number of abnormal sperm, and sperm quality, in addition to a marked reduction in sperm agglutination associated with increased fertility (table 1). These effects were observed after a dietary supplementation of 1 g of AA per day for 60 days. In contrast, no changes were noted in any of these measurements in a control group who received no AA supplementation during the same period of time (table 2). At the end of 60 days, all of the group who had received AA had impregnated their wives, and none of the control
Downloaded by: Université de Paris 193.51.85.197 - 1/24/2020 9:53:14 AM
a Values are means ± standard error. b Confidence level < 0.05. Confidence level < 0.02.
4
Dawson/Hams/Powell
Table 2. Changes in the sperm qualities of 7 infertile men (> 25% agglutination) during 60-day interval Test
Beforea
Afters
t
Volume, ml Sperm count, million/ml Motility, % Abnormal, % Precursors, million/ml
1.8 ±0.3 84±26 22±3 57 ± 2 6.6 ± 0.6
2.2 ± 0.2 72± 18 24±3 52±1 6.6± 0.6
1.686 0.820 0.810 2.040 0.000
Values are means ± standard error. Confidence levels: no significant differences.
subjects reported pregnancies. Comparison of the sperm qualities before and after the 60-day period showed marked improvement in all of the measured properties in the group receiving Ø supplementation and no change in the control group. Most notably, there was observed a significant increase in sperm count and motility, and a decrease in abnormal sperm, sperm precursors (p < 0.05), and agglutination (p < 0.001). The group mean ± SEM of the seminal plasma Ø levels of the control group before the study was 1.41 ± 0.40 and 1.25 ± 0.32 mg/dl at the end. The group mean ± SEM seminal plasma levels of Ø of the supplemented group was 2.29 ± 0.96 mg/dl prior to supplementation and 6.75 ± 0.43 mg/dl after 60 days of ΑΑ supplementation. These results indicated a beneficial effect of Ø on both the mature sperm present in the seminal plasma and upon the germinal epithelium of the testes and were in agreement with previous studies. Harris et al. [9] noted that the sperm of fertile men may show some level of sperm agglutination up to 25 %, but when the percentage of agglutination of normal sperm exceeds 25 %, the subject is considered infertile. In 1986, the studies of Lindsay and Medes [ 1 ] were confirmed by Chinoy et al. [10] when male guinea pigs with proven fertility were placed on a diet deficient in ΑΑ for 3 weeks and sacrificed. The most severe histological damage was observed in the testes. Further, the most pronounced effect was in the cauda epididymis, wherein sperm undergo maturation, with thickened peritubular muscle layer and connective tissue, decreased tubular diameter and epithelial cell height, and nuclear pycnosis. Similarly affected were the vas deferens and accessory sex glands, although to a lesser degree. Sperm analysis of semen samples collected
Downloaded by: Université de Paris 193.51.85.197 - 1/24/2020 9:53:14 AM
a
Relationship between Ascorbic Acid and Male Fertility
5
from testis, cauda epididymis, and vas deferens showed decline in total sperm count and sperm motility, as well as an increase in the number of abnormal sperm. The purposes of the present studies were: (1) to confirm the relationship between ÁA and sperm agglutination and AA and the germinal epithelium; (2) to obtain a more precise level of AA dietary supplementation and length of supplementation necessary to obtain the desired effects, and (3) to determine the effect of age on the occurrence of sperm agglutination and other sperm qualities.
Materials and Methods Semen samples were obtained from 28 apparently healthy young men between the ages of 23 and 37 years with a demonstrated normal sperm count (at least 20 million/ml). The study subjects were recruited on a volunteer basis, and each signed a consent form as required by the Institutional Review Board for Human Use before samples were collected. Four of the subjects were routine semen donors with proven fertility, six were housekeepers, seven were maintenance workers, two were gardeners, two were graduate students, and seven were laboratory personnel. The study population was a broad spectrum of male employment within the age range of probable maximum productivity. The only reward for participation was a copy of their semen analysis for their own knowledge. Routinely, all samples were obtained within the laboratory. The sperm analysis was with an American Optical Binocular Microscope (AO Scientific Instruments, Buffalo, N.Y.) and was completed within 10 min [9]. The sperm qualities determined were: total counts as millions of sperm per milliliter; viability as percent of sperm count showing movement; motility as percentage of sperm count showing progressive forward movement; sperm precursors as millions per milliliter; abnormal sperm as percentage of sperm count; and sperm agglutination as percentage of sperm count clumped together with no forward movement, but obviously viable (fig. 1, 2). All of these counts were in triplicate of separated fields with a highpowered lens (fig. 1, 2). Sperm precursors are easily discernable: (1) they are larger in size than white blood cells, which are usually present, (2) the nucleus appears as a small tip on the outer perimeter of the precursor, and (3) vacuoles are present.
Downloaded by: Université de Paris 193.51.85.197 - 1/24/2020 9:53:14 AM
A Comparison of Semen AA and Agglutination Levels
Dawson/Harris/Powell
6
Attenuated Head
Pi·ecursore
Fig. 1. Abnormal mature sperm and sperm precursors.
Results The average seminal plasma AA level of the study population was 9.1 mg/dl, with a range between 3.7 and 14.7 mg/dl. Sperm agglutination aver-
Downloaded by: Université de Paris 193.51.85.197 - 1/24/2020 9:53:14 AM
After microscopic examination, the semen specimens were centrifuged (300 g, 27 °C) to obtain clear seminal plasma for the preparation of trichloroacetic acid (TCA) extracts, which were stored frozen (— 60 °C) until assayed for AA. All AA measurements were made by the 2,4- dinítrophenylhydrazine method of Lowry et al. [ 11 ], with anhydrous L-ascorbic acid used as the standard for comparison (A-7506, Sigma Chemical Company, St. Louis, Mo.). The results were calculated as mg/dl. The measured data were grouped into tertiles of increasing percent agglutination: 0%, 5-25%, and > 25%, and the group means ± SEM of percent agglutination and seminal plasma AA levels (mg/dl) were calculated. The Student's t-test was used to determine the significant differences among the group means of the three tertiles, and linear regression was used to correlate the semen ascorbic acid levels with the percent agglutination, determined simultaneously.
Relationship between Ascorbic Acid and Male Fertility
7
aged 11.4 %, with a range from 0 to 40%. The viability averaged 52%, with a range from 20 to 75 %. The semen volume averaged 2.6 ml, ranging from 1.2 to 2.75 ml. The sperm count averaged 65 million/ml, ranging from 25 to 88 million/ml. The percent motility averaged 62%, ranging from 38 to 80%. The percent of abnormal sperm averaged 55%, ranging from 42 to 60 %. The sperm precursor count averaged 6 million/ml, ranging from 4.5 to 7.2 million/ml. The percent agglutination of the individual sperm counts was the basis for grouping the study tertiles of table 3 and the group mean percent agglutination of the three tertiles would be significantly different by design. There were 13 semen samples with no sperm agglutination, 8 semen samples with sperm agglutination between 5 and 24 %, and 7 samples with 25 % sperm agglutination or higher. The group mean percent agglutination of the 0% tertile was 0%, the group mean percent agglutination of the 5-25% tertile was 9 ± 1%, and 32 ± 1% for the > 25% tertile. The differences between the group means of both the 0% and 5-25% tertiles and the 0% and > 25% tertiles were statistically significant (p 0.001) (table 3).
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Fig. 2. Agglutinated pair of normal mature sperm and two sperm precursors. Highpower magnification.
Dawson/Harris/Powell
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Table 3. Semen physical properties of 28 random samples grouped according to tertiles of percent nonspecific sperm agglutination: 0, 5-25, and > 25 Physical properties
Agglutination, % 0
Semen volume, ml Sperm count, 10/ml Sperm motility, % Abnormal sperm,% Precursors, 10/ml Sperm agglutination, % Ascorbic acid, mg/dl N
2.5±03 76±20 50±4 58±5 5±0.7 0 9.06±0.96 13
5-25 2.9±0.4 60±16 38±4b 49±6 6±0.6 9± l a
7.56±1.13 8
> 25 1.8±0.4 58±26 16±6a 58±1 5±0.6 32 ± l 6.00±0.91b 7
Nο significant difference was observed between the 0 and 5-25 % tertiles in group mean Ø levels, but the mean of the > 25 % tertile was significantly lower than the 0% tertile (6.0 ± 0.9 vs 9.1 ± 0.9, p < 0.05) (table 3). There were no significant differences in the tertile group means: 7.5 ± 0.3 ml for the 0% tertile, 2.9 ± 0.4 for the 5-25 % tertile, and 0.8 ± 0.4% for the > 25% tertile. There were no significant differences between the three tertiles in the sperm count. The group mean count for the 0% tertile was 79 ± 20 million/ml, 60 ± 16 millionml for the 5-25 % tertile, and 58 ± 20 million/mi for the > 25% tertile. However, there was an average 30 % difference in sperm count between the 0 and the > 25 % tertiles. There were significant differences between all three tertiles in group mean sperm viability (table 3). The group mean viability was significantly lower than the 0% tertile in both the 5-25 and the >25% tertile. The group mean sperm viability of the 5-25 % tertile was 38 ± 4 % compared to the group mean οf 50 ± 4% for the 0% tertile (p < 0.05). The group mean sperm viability of the > 25% tertile was 15 ± 6% compared to 50 ± 4% for the 0% tertile (p < 0.001). The group mean sperm motility was 50 ± 4% for the 0% tertile, 38 ± 4% for the 5-25% tertile and 16 ± 6% for the > 25 % tertile. The group mean of the 5-25 % tertile was significantly higher than the 0% tertile (p < 0.05) as was the group mean of the > 25% tertile (p < 0.001).
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ap < 0.001; bp < 0.05.
Relationship between Ascorbic Acid and Male Fertility
4-0 30 c o
._c
9
Υ intercept: 31.2 Slope: -2.64 `r': -0.6140 p < 0.001 n: 28
20
rn
m 10 Ε
ι
0
2 0 4 6 Semen ascorbic acid, mg/dl
10
12
There was no significant difference between the group mean percent of abnormal sperm between the three tertiles (table 3). The group means of the three tertiles were: 49 ± 5% for the 0% tertile, 53 ± 11 % for the 5-25% tertile, and 58 ± 5% for the -25% tertile. Although the group mean differences were small between the three tertiles, there was an 8% increase in abnormal sperm between the 0 and the 5-25 % tertiles and a 9% increase between the 5-25 and the >25% tertiles. The total increase in abnormal sperm between the 0 and the >25% tertile was 18%. There was virtually no measurable difference between the three study tertiles in the group mean sperm precursor counts (table 3). The group mean for the 0% tertile was 5 ± 0.7 million/ml, 6 ± 0.6 million/ml for the 5-25 % tertile, and 5 ± 0.6 million/ml for the > 25 % tertile. The group mean precursor count for all three tertiles may be considered identical. The viability was significantly lower in both the 5-25 and > 25 % tertiles than in the 0% tertile (p < 0.05, 0.001). The group mean agglutination of both the 5-25 % and > 25 % tertile was significantly higher than the 0% tertile (p < 0.001). The sperm agglutination measurements and seminal plasma AA levels were compared by linear regression (fig. 3). This analysis showed a negative correlation (r) of 0.6140, which was statistically significant (p < 0.001). This correlation shows that every 1.0 mg/ml increase in seminal plasma AA level was associated with a 2.64% decrease in sperm agglutina-
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Fig. 3. A linear regression analysis comparing semen ascorbic acid levels with percentage of nonspecific sperm agglutination in 28 random study patients.
Dawson/Hams/Powell
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tun. Predictably, when the seminal plasma AA level is zero in this study population, approximately 31 % of the sperm are agglutinated. Also, there is no agglutination when the seminal plasma AA is 11.0 mg/dl or higher.
Materials and Methods The study subjects were 30 men between the ages of 25 and 45 with sperm agglutination over 25% when their sperm was initially studied by microscopic analysis [9]. Sperm antibody studies, according to the method of Franklin and Dukes [12, 13], were negative in each subject. All were considered physically normal and showed no clinical signs of inflammatory disease. The research protocol was described to the subjects, human consent forms signed, and 10 ml of blood was obtained for the measurement of serum AA. An aliquot of seminal plasma was prepared for AA measurement. By random selection, three groups of 10 subjects each were provided one month's supply of a daily supplement of 1,000 or 200 mg of AA, or a placebo. The pure L-ascorbic acid used for supplement and placebo were provided by Hoffmann-La Roche, Inc., Nutley, N.J. Each subject was told he was receiving AA and expected improvement in sperm quality. The subjects returned weekly for 3 weeks and were requested to refrain from sexual activity during the length of the study. After microscope examination of the sperm, blood samples were obtained by venipuncture. Semen and blood samples were analyzed as in the previous study. The measurement of AA was in a separate laboratory after samples had been collected and stored, and the subject identity of the numbered test tubes containing the TCA extracts was unknown at the time of analysis. After completion of laboratory analysis, the sample data were grouped according to the dietary supplementation provided each subject and the week of sampling. The group mean and the standard error of the mean ( ± SEM) was calculated for the placebo, 200 mg AA, and the 1,000mg AA-supplemented groups for each of the 4 weeks during the study: 0, 1, 2, and 3. The samples obtained prior to subject selection and supplementation were at 0 weeks, and the groups of samples obtained during weeks 1, 2, and 3 were during supplementation. The Student's t-test was used to determine the statistical significance of the weekly group mean change from the presupplementation levels in the serum and seminal plasma AA and the sperm qualities during supplementation.
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Effect of ΑΑ Dietary Supplementation on Sperm Qualities
Relationship between Ascorbic Acid and Male Fertility
11
Table 4. Changes in mean ± SEM serum and seminal plasma vitamin C levels (mg/dl) during study for the three study groups (10 in each group) Study weeks
Serum Placebo Supplemented Seminal plasma Placebo Supplemented
1
2
3
200 mg 1,000 mg
0.57±0.16 0.40±0.12 0.20±0.01
0.61±0.11 0.90±0.20a 1.30±0.30c
0.65±0.08 1.80±0.144 2.50±0.154
0.65±0.09 2.20±0.104 2.40±0.15d
200 mg 1,000 mg
3.7 ± 1.9 2.0±0.5 4.2±0.7
2.9±2.0 3.8±0.4b 12.7±1.1d
3.0 ± 1.1 6.3±0.3d 11.2±1.3d
2.0 ± 1.1 8.1±0.54 13.1±1.3d
< 0.05;bp < 0.02; Cp
