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Acridine Orange Male Fertility Test
I I
Acridine Orange Male Fertility Test To the Editor: In a recent article, 1 the authors present a method purported to be a practical test for evaluation of male fertility. The authors stained fixed, but unheated, sperm with 750 ~-tM acridine orange (AO) and then scored cells as having entirely green heads or as having a red component. Cells were scored "red" when the head was yellow or when discrete red granules were seen. Confirmed fertile donors showed a broad distribution in the range of 40% to 90% "green" sperm with a mode at 65% to 70%, while infertility patients were broadly distributed as having from 10% to 90% green sperm with no discernible mode. When combined with the sperm count into an "effective sperm count," the authors showed a substantial separation of their infertility patients from confirmed fertile donors. The reported basis for this work was the findings of Evenson et al., 2 • 3 which showed a correlation between resistance of sperm chromatin to heat denaturation, sperm count, and fertility. The authors used an extremely high concentration of AO (750 ~-tM), which is several orders of magnitude greater than the 27 ~-tM employed by Evenson et al. 2 • 3 or the range of 2 to 20 ~-tM used by us in a variety of earlier studies, including some with slide photometry. 4 - 7 While AO has a high degree of selectivity for cellular biopolymers, it is not absolute, 5 and the number of substances in the cell to which it is capable of binding rises sharply with the concentration of AO used to stain the cell. In our experience with even 100 ~-tM, the complexity of cellular staining is so great as to defy interpretation. Moreover, deoxyribonucleic acid (DNA) normally develops a yellow color at high AO concentrations due to weak dye/dye interactions. 4 • 6 • 7 Thus, the claim by Tejada et al. 1 that red fluorescence measured DNA denaturation under the conditions they employed is questionable at best. While the authors achieved a separation of fertility patients from confirmed fertile donors, such separations in no way prove a proposed test has any merit. In the absence of epidemiologic data, there is no way of knowing whether the observed difference is due to fertility differences or other factors. Such factors could include age, socioeconomic class, frequency of ejaculation, diet, and similar factors which could clearly affect either or 154
Letters to the editor
both the sperm count or composition. These factors clearly must be considered in view of the very high concentrations of AO used by the authors. In summary, we believe that the authors' conclusion that this test can be used for the "practical evaluation of male fertility" is, at best, highly premature.
Robert E. Hurst, Ph.D. Johnny B. Roy, M.D. Department of Urology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma 73190 September 27, 1984 REFERENCES 1. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S: A test for the practical evaluation of male fertility by acridine orange (AO) .fluorescence. Fertil Steril 42:87, 1984 2. Evenson DP, Darzynkiewicz Z, Melamed MR: Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131, 1980 3. Evenson DP, Melamed MR: Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J Histochem Cytochem 31:248, 1983 4. West SS: Fluorescence microspectrophotometry of supravitally stained cells. In Physical Techniques in Biological Research, Edited by AW Pollister. New York, Academic Press, 1969, p 253 5. Nakamura NN, Hurst RE, West SS, Menter JM, Golden JF, Corliss DA, Jones DD: Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells. J Histochem Cytochem 28:223, 1980 6. Golden JF, West SS, Echols CK, Shingleton HM: Quantitative fluorescence spectrophotometry of acridine orangestained unfixed cells. J Histochem Cytochem 24:315, 1976 7. Hemstreet GP III, West SS, Weems WL, Echols CK, McFarland S, Lewin J, Lindseth G: Quantitative fluorescence measurements of AO-stained normal and malignant bladder cells. Int J Cancer 31:577, 1983
Reply of the Authors: With regard to our recent article, 1 we would like to address the thoughtful criticisms outlined in a letter from Drs. Hurst and Roy. Our work was indeed based upon that of Evenson et al., 2 who reported differences in the response of sperm to deoxyribonucleic acid (DNA) denaturation by heat dependent upon the fertility of the donor individual. That is, boiled sperm from infertile individuals shifted more into the Fertility and Sterility
l
red (relative to the unheated control) when stained with acridine orange (AO) than did sperm from infertile individuals. Our inference from these results is that resistance of the sperm to denaturation stress is a parameter of fertility, and the aim of our study was the development of a simple office procedure based on this principle. Instead of a separate denaturation step involving heat or chemical treatment, our procedure employs a high concentration of AO to denature the DNA. The theory that AO is an active agent, capable of denaturing DNA, was advanced by Darzynkiewicz et al. 3 to explain the shift from green to red fluorescence observed with high dye concentrations. We are therefore using AO both as an active agent and as a probe of DNA condition. Recent work4 has provided evidence that the red/green ratio is determined quite early in spermatogenesis, at the preleptotene spermatocyte stage or sooner. Furthermore, in vivo irradiation of murine spermatogenic cells causes the red/ green ratio to be altered in a manner that suggests the red sperm have poorer survival characteristics than the green. This phenomenon suggests a possible explanation for the relative fertility of the two subpopulations. Of course, no new test can be accepted without widespread evaluation in a number of laboratories. It is our hope that the results of this study, which identified 96% of the fertile individuals tested and 98% of the infertile, will prompt other clinics to assess the value of this procedure. POSTSCRIPT
Regarding our article, 1 we would like to correct a printed error on page 89, second line. "0.3 M" should read "0.2M Na2HP04·7H20."
Rafael I. Tejada, M.D. J. Cameron Mitchell, M.S. Laboratory of Biomedical and Environmental Sciences University of California, Los Angeles Los Angeles, California 90024 November 7, 1984
2. Evenson DP, Darzynkiewicz Z, Melamed MR: Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131, 1980 3. Darzynkiewicz Z, Evenson DP, Kapuscinski J, Melamed MR: Denaturation of RNA and DNA in situ induced by acridine orange. Exp Cell Res 148:31, 1983 4. Mitchell JC: Analysis of DNA structure by acridine orange denaturation in situ. Master's Thesis, University of California, Los Angeles, 1984
Sex Ratio and Clomiphene Treatment To the Editor: After reading the letter of Shettles1 in the May issue of Fertility and Sterility where he reported that 66% of the offspring of 39 patients treated with clomiphene citrate (CC) were females, we want to give our results of 84 CC pregnancies. The conceptions during 109 CC cycles (50 mg CC daily, cycles days 5 to 9) resulted in 84 deliveries (19 abortions and 6 ectopic pregnancies), and 89 children (3 sets of twins and 1 set of triplets) were born. Forty-eight of the children were girls (53.9%), and 41 were boys (46.1 %), which is exactly the same sex distribution as reported by Sampson et al. 2 . Shettles did not mention the CC dosage used in his study. The fact that we did not use more than 50 mg CC daily could be one explanation for the less prominent female dominance in our patients. Another possible reason for the female predominance, other than the "disturbed" cervix factor, during CC cycles could be the increased abortion rate (22.6% in our material) of CC pregnancies, which also may have an influence on the sex ratio.
Lars Ronnberg, M.D., Ph.D. Susanna Huuskonen, M.D. Departments of Obstetrics and Gynecology University ofOulu Oulu, Finland November 8, 1984 REFERENCES
REFERENCES 1. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman
S: A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 42:87, 1984 Vol. 43, No. 1, January 1985
1. Shettles LB: Letter to the Editor. Fertil Steril 41:786,
1984 2. Sampson JH, Alexander NJ, Fulgham DL, Burry KA: Gender after artificial induction of ovulation and artificial insemination. Fertil Steril 40:481, 1983 Letters to the editor
155
Acridine Orange Male Fertility Test To the Editor: In a recent article, 1 the authors present a method purported to be a practical test for evaluation of male fertility. The authors stained fixed, but unheated, sperm with 750 ~-tM acridine orange (AO) and then scored cells as having entirely green heads or as having a red component. Cells were scored "red" when the head was yellow or when discrete red granules were seen. Confirmed fertile donors showed a broad distribution in the range of 40% to 90% "green" sperm with a mode at 65% to 70%, while infertility patients were broadly distributed as having from 10% to 90% green sperm with no discernible mode. When combined with the sperm count into an "effective sperm count," the authors showed a substantial separation of their infertility patients from confirmed fertile donors. The reported basis for this work was the findings of Evenson et al., 2 • 3 which showed a correlation between resistance of sperm chromatin to heat denaturation, sperm count, and fertility. The authors used an extremely high concentration of AO (750 ~-tM), which is several orders of magnitude greater than the 27 ~-tM employed by Evenson et al. 2 • 3 or the range of 2 to 20 ~-tM used by us in a variety of earlier studies, including some with slide photometry. 4 - 7 While AO has a high degree of selectivity for cellular biopolymers, it is not absolute, 5 and the number of substances in the cell to which it is capable of binding rises sharply with the concentration of AO used to stain the cell. In our experience with even 100 ~-tM, the complexity of cellular staining is so great as to defy interpretation. Moreover, deoxyribonucleic acid (DNA) normally develops a yellow color at high AO concentrations due to weak dye/dye interactions. 4 • 6 • 7 Thus, the claim by Tejada et al. 1 that red fluorescence measured DNA denaturation under the conditions they employed is questionable at best. While the authors achieved a separation of fertility patients from confirmed fertile donors, such separations in no way prove a proposed test has any merit. In the absence of epidemiologic data, there is no way of knowing whether the observed difference is due to fertility differences or other factors. Such factors could include age, socioeconomic class, frequency of ejaculation, diet, and similar factors which could clearly affect either or 154
Letters to the editor
both the sperm count or composition. These factors clearly must be considered in view of the very high concentrations of AO used by the authors. In summary, we believe that the authors' conclusion that this test can be used for the "practical evaluation of male fertility" is, at best, highly premature.
Robert E. Hurst, Ph.D. Johnny B. Roy, M.D. Department of Urology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma 73190 September 27, 1984 REFERENCES 1. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S: A test for the practical evaluation of male fertility by acridine orange (AO) .fluorescence. Fertil Steril 42:87, 1984 2. Evenson DP, Darzynkiewicz Z, Melamed MR: Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131, 1980 3. Evenson DP, Melamed MR: Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J Histochem Cytochem 31:248, 1983 4. West SS: Fluorescence microspectrophotometry of supravitally stained cells. In Physical Techniques in Biological Research, Edited by AW Pollister. New York, Academic Press, 1969, p 253 5. Nakamura NN, Hurst RE, West SS, Menter JM, Golden JF, Corliss DA, Jones DD: Biophysical cytochemical investigations of intracellular heparin in neoplastic mast cells. J Histochem Cytochem 28:223, 1980 6. Golden JF, West SS, Echols CK, Shingleton HM: Quantitative fluorescence spectrophotometry of acridine orangestained unfixed cells. J Histochem Cytochem 24:315, 1976 7. Hemstreet GP III, West SS, Weems WL, Echols CK, McFarland S, Lewin J, Lindseth G: Quantitative fluorescence measurements of AO-stained normal and malignant bladder cells. Int J Cancer 31:577, 1983
Reply of the Authors: With regard to our recent article, 1 we would like to address the thoughtful criticisms outlined in a letter from Drs. Hurst and Roy. Our work was indeed based upon that of Evenson et al., 2 who reported differences in the response of sperm to deoxyribonucleic acid (DNA) denaturation by heat dependent upon the fertility of the donor individual. That is, boiled sperm from infertile individuals shifted more into the Fertility and Sterility
l
red (relative to the unheated control) when stained with acridine orange (AO) than did sperm from infertile individuals. Our inference from these results is that resistance of the sperm to denaturation stress is a parameter of fertility, and the aim of our study was the development of a simple office procedure based on this principle. Instead of a separate denaturation step involving heat or chemical treatment, our procedure employs a high concentration of AO to denature the DNA. The theory that AO is an active agent, capable of denaturing DNA, was advanced by Darzynkiewicz et al. 3 to explain the shift from green to red fluorescence observed with high dye concentrations. We are therefore using AO both as an active agent and as a probe of DNA condition. Recent work4 has provided evidence that the red/green ratio is determined quite early in spermatogenesis, at the preleptotene spermatocyte stage or sooner. Furthermore, in vivo irradiation of murine spermatogenic cells causes the red/ green ratio to be altered in a manner that suggests the red sperm have poorer survival characteristics than the green. This phenomenon suggests a possible explanation for the relative fertility of the two subpopulations. Of course, no new test can be accepted without widespread evaluation in a number of laboratories. It is our hope that the results of this study, which identified 96% of the fertile individuals tested and 98% of the infertile, will prompt other clinics to assess the value of this procedure. POSTSCRIPT
Regarding our article, 1 we would like to correct a printed error on page 89, second line. "0.3 M" should read "0.2M Na2HP04·7H20."
Rafael I. Tejada, M.D. J. Cameron Mitchell, M.S. Laboratory of Biomedical and Environmental Sciences University of California, Los Angeles Los Angeles, California 90024 November 7, 1984
2. Evenson DP, Darzynkiewicz Z, Melamed MR: Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210:1131, 1980 3. Darzynkiewicz Z, Evenson DP, Kapuscinski J, Melamed MR: Denaturation of RNA and DNA in situ induced by acridine orange. Exp Cell Res 148:31, 1983 4. Mitchell JC: Analysis of DNA structure by acridine orange denaturation in situ. Master's Thesis, University of California, Los Angeles, 1984
Sex Ratio and Clomiphene Treatment To the Editor: After reading the letter of Shettles1 in the May issue of Fertility and Sterility where he reported that 66% of the offspring of 39 patients treated with clomiphene citrate (CC) were females, we want to give our results of 84 CC pregnancies. The conceptions during 109 CC cycles (50 mg CC daily, cycles days 5 to 9) resulted in 84 deliveries (19 abortions and 6 ectopic pregnancies), and 89 children (3 sets of twins and 1 set of triplets) were born. Forty-eight of the children were girls (53.9%), and 41 were boys (46.1 %), which is exactly the same sex distribution as reported by Sampson et al. 2 . Shettles did not mention the CC dosage used in his study. The fact that we did not use more than 50 mg CC daily could be one explanation for the less prominent female dominance in our patients. Another possible reason for the female predominance, other than the "disturbed" cervix factor, during CC cycles could be the increased abortion rate (22.6% in our material) of CC pregnancies, which also may have an influence on the sex ratio.
Lars Ronnberg, M.D., Ph.D. Susanna Huuskonen, M.D. Departments of Obstetrics and Gynecology University ofOulu Oulu, Finland November 8, 1984 REFERENCES
REFERENCES 1. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman
S: A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 42:87, 1984 Vol. 43, No. 1, January 1985
1. Shettles LB: Letter to the Editor. Fertil Steril 41:786,
1984 2. Sampson JH, Alexander NJ, Fulgham DL, Burry KA: Gender after artificial induction of ovulation and artificial insemination. Fertil Steril 40:481, 1983 Letters to the editor
155