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Heat in male contraception (hot water 60 °C, infrared, microwave, and ultrasound)
HEAT IN MALE CONTRACEPTION (HOT WATER 6o"c,
INFRARED, MICROWAVE,AND ULTRASOUND) M.S. Fahim, Z. Fahim, R. Der, D.G. Hall, and J. Harman Department of Obstetrics and Gynecology University of Missouri Columbia, Missouri 65201 ABSTRACT Spermatogenesis in the mammalian male is disturbed by high ambient temperature. 300 male rats weighing 250-300 gm were divided into 5 were exposed for 15 minutes groups. Group I, control; Group II, testes to water at 60°C; Group III, testicular temperature was raised to 600C for 15 minutes by infrared spot heater concentrating radiant energy from a 750 watt iodine cycle lamp focused on the testes; Group IV, testes were treated with a microwave diathermy unit radiating energy at 2450 megacycles/second for 1, 5, or 15 minutes; Group V, ultrasound 1.0 w/sq cm was applied once or twice, or 2.0 w/sq cm was applied once. The animals were mated with proestrus females 24 hours after treatment, then followed each 5 days with another female until pregnancy was documented to have occurred in females. This was continued for 10 months, the duration of this experiment. Group II males impregnated females after 30-35 days, while those in Group III impregnated females after 60-75 days. Groups IV and V impregnated females after 65-80 days, after 150-210 days, or did not impregnate females during the lo-month stuay--depending on the power and duration in the case of microwave, and the dosage, duration, and frequency of application in the case of ultrasound. No significant difference in blood testosterone levels occurred in treated animals. Seminiferous tubules were either normal in histological appearance or showed partial degranulation, especially in Groups IV and V. Long-term effects of recurrent heat application are under investigation. This pilot study offers the possibility of reversible and irreversible sterilization for human males as well as a new method of animal population control in the U.S.A. Presented at the FASEB meeting in Atlantic City, April 1974.
Accepted
MAY
1975
for
publication
VOL.
11 NO. 5
March
10,
1975
549
CONTRACEPTION
INTRODUCTION The scrotal testis of mammals is normally at a temperature several degrees (3-4OC) below that of the body core. Crew in 1922 (1) was probably the first to suggest that the damage to the seminiferous epithelium of cryptorchid testes is the result of exposure of such testes to intra-abdominal temperature, which is higher than the intrascrotal temperature. This suggestion has been supported by observations that intrascrotal testes exposed experimentally to elevated temperatures respond with rapid degeneration of the seminiferous epithelium, noted by Fukui in 1923 (2), Moore in 1924 (3), and Young in 1927 (4). In man there are a number of everyday possibilities for heat effect on testes, such as: hot bath (2), (5); tight clothing (2), (5); fever (6), (7); vocation or situation in which man is required to labor under hot conditions (8), (9); and posture (10). Thus, man probably more than any other species is likely to be subjected to conditions which are detrimental to sperm production. Vasectomy has become an important and frequently employed method of permanent contraception. In the United States, approximately 2.5 million men have undergone vasectomy. Recognized complications of vasectomy include hematomata, edema, pain on ejaculation, sperm granulomata, and vas recanalization. Certain long-term effects of vasectomy in laboratory animals are potential causes for concern (11). Pharmaceutical inhibition of spermatogenesis in the male to date has been productive of undesirable side effects. In considering an approach to male contraception which would be neither surgical nor pharmacological, the authors elected to reinvestigate, utilizing modern technology, the effect of heat in suppressing spermatogenesis without alteration of Leydig cell secretion. MATERIALS AND METHODS 300 male Sesco rats of the Haltzman strain, weighing 250-300 grams, were divided into control and 5 treated groups in which 9 different treatments were performed. The control group and treatment groups each contained 30 animals. Following treatment and progeny tests, 10 males from each treatment group were terminated at 2 weeks and 60 days,respectiveFive of these 10 animals sacrificed at 2 weeks and 5 of the 10 1Y. sacrificed at 60 days were also used to measure intratesticular temperature. The 10 remaining males in each treatment group were progeny tested on a regular basis for 10 months and sacrificed. To measure the temperature inside the testis, the selected animals had their testes cooled to 280C by cold water application before heat applications were started. Because of the possible damage to the testes resulting from thermocouple needle puncture, one testis was employed for temperature measurements , and the contralateral testis was used to study the histologic effects of heat treatment.
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In order to expose rat testes only, a plexiglass restraining table with multiple adjustments for animal size and testicular position was built. An adjustable testicle cup had neoprene membrane at the uppermost end to receive the testicles. Water inside the cup circulated by a pumpsiphon system. A lubricant was applied to the testis before heat treatment. Rectal temperatures and scrotal temperature were recorded by rectal telethermometer and digital thermometer. Animals were anesthetized with an interperitoneal injection of Nembutal 15 mg/kg, before heat application. Group I was the control; Group II testes were exposed to 60°C water for 15 minutes. Group III testicular temperature was raised to 60°C for 15 minutes by infrared spot heater concentrating radiant energy from a 750 watt iodine cycle source. Group IV testes were exposed to a microwave diathermy unit radiating energy at 2450 megacycles/second. This machine (Burdick MW-208) is used medically as a therapeutic heat source in physical medicine and rehabilitation. The wave length is 12.2 cm with a maximum output of 100 watts. The microwave exposure to testes of different subgroups varied in the percent of power, duration of exposure, and distance from the skin. We standardized the distance in this trial at 3". Subgroup 1 2 3 4
Period
Power 100% 20% 20% 20%
5 1 5 15
minutes minute minutes minutes
In this experiment6 testicular temperature was raised to 65'C in subgroup 1, while it was 39 C in subgroups 2 and 3, and 45OC in subgroup 4. Group V testes were exposed to ultrasound (Burdick UT 1420 Pulsed Unit) with 2 doses, 1.0 w/sq cm or 2.0 w/sq cm for five minutes. The 1.0 w/sq cm treatment group was divided into 2 subgroups. Subgroup 1 was exposed to one treatment. Subgroup 2 received 2 treatments; the second was performed 48 ho rs after the first treatment. Temperature of ti testes was raised to 38-40 C for all ultrasound-treated animals. All animals were mated with proestrus females 24 hours after treatment, then followed each 5 days with another female until pregnancy was documented to have occurred in females. The endpoint for fertility was the amount of time required for every surviving male in the treatment group to impregnate a female. Data was collected for: a) blood testosterone, determined by protein binding assay (12) b) sex organ weights; histology of testes, seminal vesicles, and prostate were prepared with routine H&E stain.
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RESULTS 60'C*Water. Females were found impregnated 30-35 days after male treatment. Testes were quartered and 50 slides from each quarter were fixed for histological examination. Histology was obtained 2 weeks after animal treatment and (in all groups) revealed a significant reduction of sperm and spermatid as compared to the control group (Fii. 1 and 2). The intratesticular temperature of this group was 59 f 0.5 C. Infrared. Treated animals impregnated their females 60-75 days after infrared application; notice this figure is doubled from the hot water-treated group. The testicular temperature was raised to 59 + 0.5'C in this group also. 20-25% of seminiferous tubules indicated absence of spermatogenesis stages and no spermatozoa were present (Fig. 3). Microwave. When 100% power appligd to the testes was used, the inside temperature was raised to 63-65 C during the 5-minute treatment. Ten months later the males had still not impregnated the females. Testes histology indicated 100% absence of spermatogenesis (Fig. 4). For subgroup 2, the treatment was not effectixe (one minute at 20% power; the testes temperature was raised to 39-40 C). 70% of the males in subgroup 2 impregnated their females at the first mating. The other 30% impregnated their females within lo-14 days. There were sperm present in histological sections of the testes (Fig. 5).
In subgroup 3 when the period of treating animals was raised to 5 minutes, elevating testicular temperature to 39 f 0.4oC, there were more effects on spermatid and spermatogenesis than from the one-minute exposure, as noted on the histologic section of testes (Fig. 6). In this subgroup, the treated males impregnated the females after 65-80 days. In subgroup 4, with the testes treated for 15 minutes, the testes temperature was raised to 45 ?r0.6 C; absence of spermatogenesis stages and of spermatozoa was noted (Fig. 7). Ten months after male treatment, no female had been impregnated. Ultrasound. When 1 w/sq cm was applied for 5 minutes, the testicular temperature was raised to 38'C and the testes indicated reduction in sperm, spermatid, and secondary spermatid (Fig. 8). The females were impregnated 150-210 days after male treatment. When the dose of ultrasound was doubled (2 w/sq cm), the testes temperature was raised to 40 f 0.5Oc. 65% of seminiferous tubules showed absence of spermatogenesis stages. The females had not conceived after 10 months (Fig. 9). On the other hand, when 1 w/sq cm was applied twice, the second application after 48 hours, 100% of seminiferous tubules showed an absence of spermatogenesis stages (Fig. lo), and the tested females did not become pregnant throughout the period of the experiment (10 months). Sex Organ Weights. There were no significant changes of sex organ weights (testis, epididymis, prostate, and seminal vesicle) in treated animals after 2 weeks, 60 days, or 10 months.
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Fig. 1. Histobgk section of testes from contml animal. stages. o( 200)
Ndke presence of spermatogenesis
~~I~QIS, IW weeps aner ~_ .._____ ter 6@ for 15 minutes. Notice significant reduction of spe rmdirl I I t.nAcrrnn&,-.tmsrlr4,.-*--
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1975 VOL. 11 NO. 5
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Histologic section of testes in animals, two weeks after treatment with infrared at 64 for 15 minutes. Notice the absence of spermatcqenesis stages and no spermatozoa present. W3l)
Hlstobgic section of testes In animals. two weeks after treatment with microwave radfating energy at 245fJ megacycles/second for fhre minutes with 100% paver and three inches distance from the testes. Notice the absence of spermatqenesis stages. WOO)
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Histologic section of testes in animals, two weeks after treatment wfth mkrowava. power was 20% for one minute and three inches distance from testes. Notice spermatozoa, spermatids, and secondary spermatccytes have not been affected. (x200)
H&ologic section of testes in animals, two weeks alter treatment with microwave power 2C% for five minutes and three inches from testes. Notice there are more effects on spermatid and secondary spermatocytes than one-minute exposure. WOO)
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Histologic section of testes in animals, two weeks atier treatment wfth micrmvave at 2096 power for 15 minutes and three inches from testes. Notice the absence of spermatogenesis stages and no spermatozoa present. (x200)
spermatdcytes.
556
(x2001
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Histologic section of testes in animals, two weeks after treatment with ultrasound 2 wattslsq cm for five minutes. Notice the absence of spermatogenesis stages. (x200)
Histologic section of testes in animals, two weeks after two treatments with ultrasound 1 wattlsq cm for five minutes. Second treatment 48 hours after first. Notice complete absence of spermatcgenesis stages. (x200)
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Testosterone Levels. There were no significant changes of blood testosterone levels due to heat treatment after 2 weeks and after 60 days (Fig. 11); this observation was also confirmed after 10 months of heat treatment. Control animals had 7.4 ng/ml (S.E. r 0.16) testosterone in blood, while the average for all heat treated animals was 7.2 ng/ml (S.E. + 0.21). Histology of Testes After 60 Days and After 10 Months The testes from Group II, treated with 60°C water, showed normal histological sections and presence of spermatogenesis stages at 60 days and at 10 months after treatment. However, in Group III (infrared treatment), 8% of the seminiferous tubules indicated absence of sperm and spermatids at 60 days, but testes were normal after 10 months. In Group IV (microwave treatment), subgroup 1 animals showed absence of spermatogenesis at 60 days and at 10 months after treatment. In subgroup 2, normal spermatogenesis was shown after 60 days and after 10 months. In subgroup 3, lo-12% of the seminiferous tubules indicated absence of sperm and spermatids after 60 days. However, at 10 months the tubules were normal. In subgroup 4, absence of spermatogenesis stages was noted at 60 days and at 10 months after treatment. In Group V, when animals were treated with ultrasound 1 w/sq cm for five minutes, a reduction in spermatids and secondary spermatocytes was noted in the seminiferous tubules after 60 days; but sperm were observed after 10 months. However, when the dose of ultrasound was doubled or the treatment was repeated twice, the seminiferous tubules showed absence of spermatogenesis stages at both 60 days and 10 months (Fig. 12). DISCUSSION It has been noted that heat affects spermatogenesis, resulting in reduction of sperm production (l-10). In this investigation, we confirmed this finding and noted the following: 1) When the rat testes were exposed to 600C water for 15 minutes, the animals impregnated the females in half the time as the infraredtreated animals with the same exposure period and same temperature. However, in subgroup 1 of the microwave-treated group (100% power), when the testicular temperature was raised to 63 C for only 5 minutes, no spermatogenesis stages were present for 10 months. This indicates that intracellular penetration of heat is more effective by microwave than by water or infrared. 2) Duration of treatment was an important factor, as we noticed in subgroups 2, 3, and 4 of Group IV (microwave treatment). When the mechanical power and the distance from the testes were standardized, the intratesticular temperatures varied (39'C, 39oC, and 45OC) with the varying of exposure time (1, 5, and 15 minutes, respectively). The
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1 ?Standard
10
.
Error
n
Control
Water 600 lnfared 60’
,SUbgrOUP~ Microwaves
,l w/Cm’
2 w/Cm;:
Ultrasound
Fig. 11. Effect of heat treatment
after treatment.
on blood testosterone level, sixty days Ten animals were used in each treatment.
Histologic section of testes in animals, ten months after two treatments with ultrasound 1 wattlsq cm for five minutes. Second treatment I hours after first. Notice the This animal had ken mated wRh sixty female rats, absence of spermatoqenesis stages. none of them conceived. (x400)
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increase in temperature and time correlated with the effectiveness of contraception: the one-minute treatment was not effective; but the 5-minute treatment was effective for 65-80 days, and the 15-minute treatment was effective for the duration of the study. 3) In comparing the hot water-treated group, the infrared-treated group, and the microwave-treated subgroup 1, we found that the intratesticular temperatures of all 3 groups were nearly the same (59OC for water, 59'C for infrared, and 63OC for microwave subgroup 1). However, the hot water-treated group recovered spermatogenesis within 30 days; the infrared-treated group recovered spermatogenesis within 60 days; but the microwave-treated subgroup 1 never recovered spermatogenesis during the course of this study. In the microwave-treated subgroup 3 (20% power), the intratesticular temperature was raised to 39OC; animals impregnated their females at 65-80 days. When treated with ultrasound at 1 w/sq cm, animals impregnated females after 150-210 days and their intratesticular temperature was increased to 3 OC. When the dosage was increased to 2 w/sq cm, the 8 temperature was 40 C, and no female conceived after 10 months. When the 1 w/sq cm treatment of ultrasound was applied twice, temperature was raised to 38'C; again, no female became pregnant after 10 months. These data indicate that, although the intratesticular temperatures in these groups were similar, electronic means of heat induction was more effective than the other methods of contraception. In comparing microwave and ultrasound, we found that ultrasound was more effective at a lower temperature because of its combined effect of heat and mechanics. This combined effect could cause an ion exchange between the fluid in the seminiferous tubules and rete testis, creating an environment not suitable for spermatogenesis. This point is currently under intensive investigation. 4) No pain was observed in ultrasound-treated animals while the animals were awake. 5)
Heat treatment did not affect libido.
6) The heat treatment did not affect testosterone levels. Also, we did not note any pathological occurrence in male accessory glands. 7) In progeny tests, no abortions were noted among the tested females. The number and health of the pups were similar to control animals. 8) Long-term effects of recurrent heat application by modified electronic equipment, with consideration of semen analysis, are currently under investigation in rats, dogs, cats, and monkeys. This pilot study suggests a means of producing reversible or irreversible sterilization in the male, with neither surgical nor pharmacological procedures.
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1975
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ACKNOWLEDGEMENTS This investigation was partly supported from the University of Missouri Research Council and MSRP Medical School Research Fund. The authors are grateful to Dr. C. Peterson, Chairman of Physical Medicine and Rehabilitation, for his interest and help in the study. Our thanks also go to Dr. Louis Holroyd, Chairman of the Department of Physics, for his consultation; Dr. Sam Dwyer, Professor of Biological Engineering; Mr. J. Gauchan for photography; and Mr. J.C. Turner and Mr. J.C. Loyd from the Medical School Experimental Shop for building the restr*aining table and the circulation pump utilized in this investigation. University of Missouri patent disclosure no. 74-P-UMC-020 REFERENCES 1.
Crew, F.A.F. A suggestion as to the cause of'the aspermatic condition of the imperfectly descended testis. J. Anat. 56398 (1922).
2.
Fukui, W. Action of body temperature on the testicle. Med. World 3:160-163 (1923).
3.
Moore, C.R. Heat application and testicular degeneration: function of scrotum. Am. J. Anat. 34:373-380 (1924).
4.
Young, W.C. The influence of high temperature on the guinea pig testes: histological changes and effects on reproduction. J. Exp. Zool. 49:459-463 (1927).
5.
Rock, J. and Robinson, D. Effect of induced intrascrotal hyperthermia on testicular function in man. Amer. J. Obst. Gynec. 93:793-798 (1965).
6.
Cordes, H. Untersuchungen uber einfluss acuter und chronischer allgemeinerkrankungen auf die testikel, speziell auf die spermatogenese, sourie beobachtugen uber das auftreten von fett in der hoden. Arch. Path. Anat. Physiol. 151:402-404 (1898).
7.
Kar, J.K. Azoospermia: 7:42-48 (1953).
8.
Watanabe, A. The effect of heat on the human spermatogenesis. J. Med. Sci. lO:lOl-106 (1959).
9.
Lamar, N.J. and Rodger, R. Season and human fertility in Galveston. Texas Anat. Rec. 87:453 (1943).
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an analysis of 42 cases.
Japan the
Inter. J. Sexol.
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10.
Robinson, D., Rock, J., and Menkin, M.F. Control of human spermatogenesis by induced changes of intrascrotal temperature. JAMA
204:290-298
(1968).
11.
Nickell, M.D., Russell, R.L., Fahim, Z., and Fahim, M.S. Long-term effect of vasectomy on reproduction and liver function. Fed. Proc. Vol. 33, No. 3, March 1974, part 1.
12.
Anderson, D.C. A simple and clinically useful method for plasma testosterone-like substances by competitive protein binding. Clin. Chim. Acta 29:513-522 (1970).
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INFRARED, MICROWAVE,AND ULTRASOUND) M.S. Fahim, Z. Fahim, R. Der, D.G. Hall, and J. Harman Department of Obstetrics and Gynecology University of Missouri Columbia, Missouri 65201 ABSTRACT Spermatogenesis in the mammalian male is disturbed by high ambient temperature. 300 male rats weighing 250-300 gm were divided into 5 were exposed for 15 minutes groups. Group I, control; Group II, testes to water at 60°C; Group III, testicular temperature was raised to 600C for 15 minutes by infrared spot heater concentrating radiant energy from a 750 watt iodine cycle lamp focused on the testes; Group IV, testes were treated with a microwave diathermy unit radiating energy at 2450 megacycles/second for 1, 5, or 15 minutes; Group V, ultrasound 1.0 w/sq cm was applied once or twice, or 2.0 w/sq cm was applied once. The animals were mated with proestrus females 24 hours after treatment, then followed each 5 days with another female until pregnancy was documented to have occurred in females. This was continued for 10 months, the duration of this experiment. Group II males impregnated females after 30-35 days, while those in Group III impregnated females after 60-75 days. Groups IV and V impregnated females after 65-80 days, after 150-210 days, or did not impregnate females during the lo-month stuay--depending on the power and duration in the case of microwave, and the dosage, duration, and frequency of application in the case of ultrasound. No significant difference in blood testosterone levels occurred in treated animals. Seminiferous tubules were either normal in histological appearance or showed partial degranulation, especially in Groups IV and V. Long-term effects of recurrent heat application are under investigation. This pilot study offers the possibility of reversible and irreversible sterilization for human males as well as a new method of animal population control in the U.S.A. Presented at the FASEB meeting in Atlantic City, April 1974.
Accepted
MAY
1975
for
publication
VOL.
11 NO. 5
March
10,
1975
549
CONTRACEPTION
INTRODUCTION The scrotal testis of mammals is normally at a temperature several degrees (3-4OC) below that of the body core. Crew in 1922 (1) was probably the first to suggest that the damage to the seminiferous epithelium of cryptorchid testes is the result of exposure of such testes to intra-abdominal temperature, which is higher than the intrascrotal temperature. This suggestion has been supported by observations that intrascrotal testes exposed experimentally to elevated temperatures respond with rapid degeneration of the seminiferous epithelium, noted by Fukui in 1923 (2), Moore in 1924 (3), and Young in 1927 (4). In man there are a number of everyday possibilities for heat effect on testes, such as: hot bath (2), (5); tight clothing (2), (5); fever (6), (7); vocation or situation in which man is required to labor under hot conditions (8), (9); and posture (10). Thus, man probably more than any other species is likely to be subjected to conditions which are detrimental to sperm production. Vasectomy has become an important and frequently employed method of permanent contraception. In the United States, approximately 2.5 million men have undergone vasectomy. Recognized complications of vasectomy include hematomata, edema, pain on ejaculation, sperm granulomata, and vas recanalization. Certain long-term effects of vasectomy in laboratory animals are potential causes for concern (11). Pharmaceutical inhibition of spermatogenesis in the male to date has been productive of undesirable side effects. In considering an approach to male contraception which would be neither surgical nor pharmacological, the authors elected to reinvestigate, utilizing modern technology, the effect of heat in suppressing spermatogenesis without alteration of Leydig cell secretion. MATERIALS AND METHODS 300 male Sesco rats of the Haltzman strain, weighing 250-300 grams, were divided into control and 5 treated groups in which 9 different treatments were performed. The control group and treatment groups each contained 30 animals. Following treatment and progeny tests, 10 males from each treatment group were terminated at 2 weeks and 60 days,respectiveFive of these 10 animals sacrificed at 2 weeks and 5 of the 10 1Y. sacrificed at 60 days were also used to measure intratesticular temperature. The 10 remaining males in each treatment group were progeny tested on a regular basis for 10 months and sacrificed. To measure the temperature inside the testis, the selected animals had their testes cooled to 280C by cold water application before heat applications were started. Because of the possible damage to the testes resulting from thermocouple needle puncture, one testis was employed for temperature measurements , and the contralateral testis was used to study the histologic effects of heat treatment.
550
MAY
1975
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CONTRACEPTION
In order to expose rat testes only, a plexiglass restraining table with multiple adjustments for animal size and testicular position was built. An adjustable testicle cup had neoprene membrane at the uppermost end to receive the testicles. Water inside the cup circulated by a pumpsiphon system. A lubricant was applied to the testis before heat treatment. Rectal temperatures and scrotal temperature were recorded by rectal telethermometer and digital thermometer. Animals were anesthetized with an interperitoneal injection of Nembutal 15 mg/kg, before heat application. Group I was the control; Group II testes were exposed to 60°C water for 15 minutes. Group III testicular temperature was raised to 60°C for 15 minutes by infrared spot heater concentrating radiant energy from a 750 watt iodine cycle source. Group IV testes were exposed to a microwave diathermy unit radiating energy at 2450 megacycles/second. This machine (Burdick MW-208) is used medically as a therapeutic heat source in physical medicine and rehabilitation. The wave length is 12.2 cm with a maximum output of 100 watts. The microwave exposure to testes of different subgroups varied in the percent of power, duration of exposure, and distance from the skin. We standardized the distance in this trial at 3". Subgroup 1 2 3 4
Period
Power 100% 20% 20% 20%
5 1 5 15
minutes minute minutes minutes
In this experiment6 testicular temperature was raised to 65'C in subgroup 1, while it was 39 C in subgroups 2 and 3, and 45OC in subgroup 4. Group V testes were exposed to ultrasound (Burdick UT 1420 Pulsed Unit) with 2 doses, 1.0 w/sq cm or 2.0 w/sq cm for five minutes. The 1.0 w/sq cm treatment group was divided into 2 subgroups. Subgroup 1 was exposed to one treatment. Subgroup 2 received 2 treatments; the second was performed 48 ho rs after the first treatment. Temperature of ti testes was raised to 38-40 C for all ultrasound-treated animals. All animals were mated with proestrus females 24 hours after treatment, then followed each 5 days with another female until pregnancy was documented to have occurred in females. The endpoint for fertility was the amount of time required for every surviving male in the treatment group to impregnate a female. Data was collected for: a) blood testosterone, determined by protein binding assay (12) b) sex organ weights; histology of testes, seminal vesicles, and prostate were prepared with routine H&E stain.
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RESULTS 60'C*Water. Females were found impregnated 30-35 days after male treatment. Testes were quartered and 50 slides from each quarter were fixed for histological examination. Histology was obtained 2 weeks after animal treatment and (in all groups) revealed a significant reduction of sperm and spermatid as compared to the control group (Fii. 1 and 2). The intratesticular temperature of this group was 59 f 0.5 C. Infrared. Treated animals impregnated their females 60-75 days after infrared application; notice this figure is doubled from the hot water-treated group. The testicular temperature was raised to 59 + 0.5'C in this group also. 20-25% of seminiferous tubules indicated absence of spermatogenesis stages and no spermatozoa were present (Fig. 3). Microwave. When 100% power appligd to the testes was used, the inside temperature was raised to 63-65 C during the 5-minute treatment. Ten months later the males had still not impregnated the females. Testes histology indicated 100% absence of spermatogenesis (Fig. 4). For subgroup 2, the treatment was not effectixe (one minute at 20% power; the testes temperature was raised to 39-40 C). 70% of the males in subgroup 2 impregnated their females at the first mating. The other 30% impregnated their females within lo-14 days. There were sperm present in histological sections of the testes (Fig. 5).
In subgroup 3 when the period of treating animals was raised to 5 minutes, elevating testicular temperature to 39 f 0.4oC, there were more effects on spermatid and spermatogenesis than from the one-minute exposure, as noted on the histologic section of testes (Fig. 6). In this subgroup, the treated males impregnated the females after 65-80 days. In subgroup 4, with the testes treated for 15 minutes, the testes temperature was raised to 45 ?r0.6 C; absence of spermatogenesis stages and of spermatozoa was noted (Fig. 7). Ten months after male treatment, no female had been impregnated. Ultrasound. When 1 w/sq cm was applied for 5 minutes, the testicular temperature was raised to 38'C and the testes indicated reduction in sperm, spermatid, and secondary spermatid (Fig. 8). The females were impregnated 150-210 days after male treatment. When the dose of ultrasound was doubled (2 w/sq cm), the testes temperature was raised to 40 f 0.5Oc. 65% of seminiferous tubules showed absence of spermatogenesis stages. The females had not conceived after 10 months (Fig. 9). On the other hand, when 1 w/sq cm was applied twice, the second application after 48 hours, 100% of seminiferous tubules showed an absence of spermatogenesis stages (Fig. lo), and the tested females did not become pregnant throughout the period of the experiment (10 months). Sex Organ Weights. There were no significant changes of sex organ weights (testis, epididymis, prostate, and seminal vesicle) in treated animals after 2 weeks, 60 days, or 10 months.
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Fig. 1. Histobgk section of testes from contml animal. stages. o( 200)
Ndke presence of spermatogenesis
~~I~QIS, IW weeps aner ~_ .._____ ter 6@ for 15 minutes. Notice significant reduction of spe rmdirl I I t.nAcrrnn&,-.tmsrlr4,.-*--
MAY
1975 VOL. 11 NO. 5
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Histologic section of testes in animals, two weeks after treatment with infrared at 64 for 15 minutes. Notice the absence of spermatcqenesis stages and no spermatozoa present. W3l)
Hlstobgic section of testes In animals. two weeks after treatment with microwave radfating energy at 245fJ megacycles/second for fhre minutes with 100% paver and three inches distance from the testes. Notice the absence of spermatqenesis stages. WOO)
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Histologic section of testes in animals, two weeks after treatment wfth mkrowava. power was 20% for one minute and three inches distance from testes. Notice spermatozoa, spermatids, and secondary spermatccytes have not been affected. (x200)
H&ologic section of testes in animals, two weeks alter treatment with microwave power 2C% for five minutes and three inches from testes. Notice there are more effects on spermatid and secondary spermatocytes than one-minute exposure. WOO)
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Histologic section of testes in animals, two weeks atier treatment wfth micrmvave at 2096 power for 15 minutes and three inches from testes. Notice the absence of spermatogenesis stages and no spermatozoa present. (x200)
spermatdcytes.
556
(x2001
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Histologic section of testes in animals, two weeks after treatment with ultrasound 2 wattslsq cm for five minutes. Notice the absence of spermatogenesis stages. (x200)
Histologic section of testes in animals, two weeks after two treatments with ultrasound 1 wattlsq cm for five minutes. Second treatment 48 hours after first. Notice complete absence of spermatcgenesis stages. (x200)
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Testosterone Levels. There were no significant changes of blood testosterone levels due to heat treatment after 2 weeks and after 60 days (Fig. 11); this observation was also confirmed after 10 months of heat treatment. Control animals had 7.4 ng/ml (S.E. r 0.16) testosterone in blood, while the average for all heat treated animals was 7.2 ng/ml (S.E. + 0.21). Histology of Testes After 60 Days and After 10 Months The testes from Group II, treated with 60°C water, showed normal histological sections and presence of spermatogenesis stages at 60 days and at 10 months after treatment. However, in Group III (infrared treatment), 8% of the seminiferous tubules indicated absence of sperm and spermatids at 60 days, but testes were normal after 10 months. In Group IV (microwave treatment), subgroup 1 animals showed absence of spermatogenesis at 60 days and at 10 months after treatment. In subgroup 2, normal spermatogenesis was shown after 60 days and after 10 months. In subgroup 3, lo-12% of the seminiferous tubules indicated absence of sperm and spermatids after 60 days. However, at 10 months the tubules were normal. In subgroup 4, absence of spermatogenesis stages was noted at 60 days and at 10 months after treatment. In Group V, when animals were treated with ultrasound 1 w/sq cm for five minutes, a reduction in spermatids and secondary spermatocytes was noted in the seminiferous tubules after 60 days; but sperm were observed after 10 months. However, when the dose of ultrasound was doubled or the treatment was repeated twice, the seminiferous tubules showed absence of spermatogenesis stages at both 60 days and 10 months (Fig. 12). DISCUSSION It has been noted that heat affects spermatogenesis, resulting in reduction of sperm production (l-10). In this investigation, we confirmed this finding and noted the following: 1) When the rat testes were exposed to 600C water for 15 minutes, the animals impregnated the females in half the time as the infraredtreated animals with the same exposure period and same temperature. However, in subgroup 1 of the microwave-treated group (100% power), when the testicular temperature was raised to 63 C for only 5 minutes, no spermatogenesis stages were present for 10 months. This indicates that intracellular penetration of heat is more effective by microwave than by water or infrared. 2) Duration of treatment was an important factor, as we noticed in subgroups 2, 3, and 4 of Group IV (microwave treatment). When the mechanical power and the distance from the testes were standardized, the intratesticular temperatures varied (39'C, 39oC, and 45OC) with the varying of exposure time (1, 5, and 15 minutes, respectively). The
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1 ?Standard
10
.
Error
n
Control
Water 600 lnfared 60’
,SUbgrOUP~ Microwaves
,l w/Cm’
2 w/Cm;:
Ultrasound
Fig. 11. Effect of heat treatment
after treatment.
on blood testosterone level, sixty days Ten animals were used in each treatment.
Histologic section of testes in animals, ten months after two treatments with ultrasound 1 wattlsq cm for five minutes. Second treatment I hours after first. Notice the This animal had ken mated wRh sixty female rats, absence of spermatoqenesis stages. none of them conceived. (x400)
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increase in temperature and time correlated with the effectiveness of contraception: the one-minute treatment was not effective; but the 5-minute treatment was effective for 65-80 days, and the 15-minute treatment was effective for the duration of the study. 3) In comparing the hot water-treated group, the infrared-treated group, and the microwave-treated subgroup 1, we found that the intratesticular temperatures of all 3 groups were nearly the same (59OC for water, 59'C for infrared, and 63OC for microwave subgroup 1). However, the hot water-treated group recovered spermatogenesis within 30 days; the infrared-treated group recovered spermatogenesis within 60 days; but the microwave-treated subgroup 1 never recovered spermatogenesis during the course of this study. In the microwave-treated subgroup 3 (20% power), the intratesticular temperature was raised to 39OC; animals impregnated their females at 65-80 days. When treated with ultrasound at 1 w/sq cm, animals impregnated females after 150-210 days and their intratesticular temperature was increased to 3 OC. When the dosage was increased to 2 w/sq cm, the 8 temperature was 40 C, and no female conceived after 10 months. When the 1 w/sq cm treatment of ultrasound was applied twice, temperature was raised to 38'C; again, no female became pregnant after 10 months. These data indicate that, although the intratesticular temperatures in these groups were similar, electronic means of heat induction was more effective than the other methods of contraception. In comparing microwave and ultrasound, we found that ultrasound was more effective at a lower temperature because of its combined effect of heat and mechanics. This combined effect could cause an ion exchange between the fluid in the seminiferous tubules and rete testis, creating an environment not suitable for spermatogenesis. This point is currently under intensive investigation. 4) No pain was observed in ultrasound-treated animals while the animals were awake. 5)
Heat treatment did not affect libido.
6) The heat treatment did not affect testosterone levels. Also, we did not note any pathological occurrence in male accessory glands. 7) In progeny tests, no abortions were noted among the tested females. The number and health of the pups were similar to control animals. 8) Long-term effects of recurrent heat application by modified electronic equipment, with consideration of semen analysis, are currently under investigation in rats, dogs, cats, and monkeys. This pilot study suggests a means of producing reversible or irreversible sterilization in the male, with neither surgical nor pharmacological procedures.
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ACKNOWLEDGEMENTS This investigation was partly supported from the University of Missouri Research Council and MSRP Medical School Research Fund. The authors are grateful to Dr. C. Peterson, Chairman of Physical Medicine and Rehabilitation, for his interest and help in the study. Our thanks also go to Dr. Louis Holroyd, Chairman of the Department of Physics, for his consultation; Dr. Sam Dwyer, Professor of Biological Engineering; Mr. J. Gauchan for photography; and Mr. J.C. Turner and Mr. J.C. Loyd from the Medical School Experimental Shop for building the restr*aining table and the circulation pump utilized in this investigation. University of Missouri patent disclosure no. 74-P-UMC-020 REFERENCES 1.
Crew, F.A.F. A suggestion as to the cause of'the aspermatic condition of the imperfectly descended testis. J. Anat. 56398 (1922).
2.
Fukui, W. Action of body temperature on the testicle. Med. World 3:160-163 (1923).
3.
Moore, C.R. Heat application and testicular degeneration: function of scrotum. Am. J. Anat. 34:373-380 (1924).
4.
Young, W.C. The influence of high temperature on the guinea pig testes: histological changes and effects on reproduction. J. Exp. Zool. 49:459-463 (1927).
5.
Rock, J. and Robinson, D. Effect of induced intrascrotal hyperthermia on testicular function in man. Amer. J. Obst. Gynec. 93:793-798 (1965).
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Cordes, H. Untersuchungen uber einfluss acuter und chronischer allgemeinerkrankungen auf die testikel, speziell auf die spermatogenese, sourie beobachtugen uber das auftreten von fett in der hoden. Arch. Path. Anat. Physiol. 151:402-404 (1898).
7.
Kar, J.K. Azoospermia: 7:42-48 (1953).
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Watanabe, A. The effect of heat on the human spermatogenesis. J. Med. Sci. lO:lOl-106 (1959).
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Lamar, N.J. and Rodger, R. Season and human fertility in Galveston. Texas Anat. Rec. 87:453 (1943).
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an analysis of 42 cases.
Japan the
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10.
Robinson, D., Rock, J., and Menkin, M.F. Control of human spermatogenesis by induced changes of intrascrotal temperature. JAMA
204:290-298
(1968).
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Nickell, M.D., Russell, R.L., Fahim, Z., and Fahim, M.S. Long-term effect of vasectomy on reproduction and liver function. Fed. Proc. Vol. 33, No. 3, March 1974, part 1.
12.
Anderson, D.C. A simple and clinically useful method for plasma testosterone-like substances by competitive protein binding. Clin. Chim. Acta 29:513-522 (1970).
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