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In vitro immobilization of human spermatozoa with hydroxypropylmethylcellulose
CONTRACEPTION
IN VITRO IMMOBILIZATION OF HUMAN SPERMATOZOA WITH HYDROXYPROPYL-METHYLCELLULOSE
K.
Loewit,
M.D.
Institute for General and Experimental Pathology, University of Innsbruck, Austria, A-6020 Innsbruck, Fritz-Pregl-Strasse 3
ABSTRACT Hydroxypropylmethylcellulose (MC) in one to three per cent w/v aqueous solution caused immediate and permanent immobilization oi human spermatozoa in vitro. This spermicidal effect may open new ways of improving-i;uaginal contraception. It also accounts for the spermatotoxic effects of vaginal lubricants and other compounds containing MC as base vehicle.
Accepted for publication December 15, 1976
FEBRUARY
1977 VOL. 15 NO.2
233
CONTRACEPTION INTRODUCTION Metallic conwer and iron or salts of these heavy metals have been found in vitro to be potent sperm-immobilizing agents in several animalspecies includinq man (1,2,3,4). Correspondingly, iron chloride (Fe C13) in 0.153 M concentration significantly reduced fertility after vaginal application in rats (5). Thus, the question arose, whether these findings could be extended to human vaginal contraception which is still considered "an important segment of the general contraceptive armamentarium and needed to maximize individual choice in contraceptive methods" (6). For this purpose, several substances have been tested in search for a suitable vehicle, acting at the same time as a carrier for the spermicide and as a physical barrier to sperm migration (Loewit, unpublished results). Of the compounds studied, hydroxypropylmethylcelluloses (MCs) showed inherent sperm-immobilizing activity. This effect seems of considerable interest and shall be reported. MATERIAL AND METHODS Fresh ejaculates of normal motility were provided by healthy, fertile volunteers. The spermicidal effect was assessed microscopically as described before (4) by measuring the time interval between mixing equal parts of undiluted semen with test solution and complete sperm-immobilization. Air dried films of were Y?), Dow also tested. Hydroxypropylmethylcellulose (Methocel Chemical, Ges.m.b.H. Wien) was used in two variants, MC 65 HGpowder and MC 90 HG-granules. Solutions of different concentration from 0.1 % to 4.0 % w/v were prepared in distilled water. In order to obtain a clear solution MC first had to be dispersed in about one fourth of the final volume at 90°C by continuous stirring. This suspension then was cooled to about 5OC by adding the remaining amount of water in frozen form. Viscosimetry was done by means of a Haake Viscotester VT02 (Haake, Berlin-Lichterfelde) at different time intervals up to two and a half years. The solutions were kept at room temperature and tested for sperm-immobilizing capacity immediately after preparation and approximately two and a half years later. In addition to the earlier evaluation of the influence of solvents, temperature and pH on sperm motility (3,4), the effect of osmolarity was investigated. Equiosmolar control-solutions of polyvinyl-pyrrolidone (PVP) in distilled water were prepared using a Knauer Half-Micro-Osmometer (Knauer, Berlin 37, Holstweg 18, FRG).
234
FEBRUARY
1977 VOL. 15 NO. 2
CONTRACEPTION
RESULTS No influence on sperm motility could be detected after 15 minutes with 0.1 to 0.4 % w/v solutions of MC 65 HG or 0.5 % w/V solutions of MC 90 HG. However, higher concentrations increasingly led to rapid sperm immobilization. Thus, at and above concentrations of 1.5 8 w/v,complete sperm immobilization was observed under the microscope at the earliest possible readings within 5 to 15 seconds. Control experiments with PVP-solution proved ineffective in this regard. Thus, a 1 % PVP-solution which corresponded in viscosity (20 cP) and osmolarity (145 milliosmole) to a 1.5 % solution of MC 65 HG (30 CP, 150 milliosmole) showed no influence on sperm motility after 15 minutes. Similarly, concentrations of PVP up to 20 % w/v, even though mechanically impairing sperm migration, never led to immediate, complete and permanent immobilization. Data on 1.5 % and 1.7 % solutions of MC 65 HG as well as 3 % MC 90 HG are given in the Table. Similar results have been obtained with 1 % and 2 % solutions,respectively. Neither two and a half years storage nor marked decreases of viscosity affected this sperm immobilizing potency. Preliminary experiments with air dried films of MC gave less clear-cut results even though sperm motility was/abolished in the immediate contact zone. DISCUSSION Hydroxypropylmethylcellulose, originally considered as a vehicle for the spermicidal iron chloride, unequivocally exhibited a strong inherent sperm immobilizing effect itself. This effect does not seem to depend on osmolarity nor was it influenced by changes of pH and viscosity measured during the study. MC Connell (6) points out, that the clinical efficiency of any vaginal contraceptive depends at least as much on the vehicle, as on the spermicide selected. A satisfying base vehicle should not only display good carrier qualities, disperse the spermicide, prevent sperm access to the cervical canal, but also possess spermicidal properties itself. Thus, MCs, widely utilized in cosmetic and pharmaceutical products because of their physical qualities and physiological inertness, could be of interest to vaginal contraception from this double point of view. Indeed, MCs inadvertently have been shown to interfere with human fertility in vitro. Tagatz and coworkers in a study relating the useafmnal lubricants to human infertility have reported on the phenomenon of spermicidal effects of such lubricants. One of these consisted of "less than 1.5 per cent boric acid in a sterile suspension of approximately 1 to 5 per cent sodium carboxymethylcellulose" (7). Similarly, in a study
FEBRUARY
1977 VOL. 15 NO.2
235
CONTRACEPTION
N
236
FEBRUARY
1977 VOL. 15 NO. 2
CONTRACEPTION by Fox and coworkers dealing with the influence on fertility of vaginal pH during human coitus, sperm immobilization was found after the application of low pH buffer (pH 3.6). Again the vehicle used was 5 % methylcellulose and immobilization occurred before the vaginal pH was changed (8). In conclusion, the spermicidal effect of MCs, leading to immediate and permanent sperm immobilization in appropriate concentrations, should be taken into account as a cause of undesired side effects wherever MCs are applied in a respective way and as a possible new means of improvement of human vaginal contraception. ACKNOWLEDGEMENT We thank Austrowaren, bsterr. Warenhandelsgesellschaft m.b.H., Wien, for kindly supplying samples of Methocel-products. We are grateful to Ing. A. Kopenetz from K. Schwarzkopf & Co., Innsbruck,for providing facilities for Viscosimetry. REFERENCES 1. WHITE, I.G.: The toxicity of heavy metals to mammalian spermatozoa. Aust. J. Exp. Biol. -33: 359-366, 1955 2. SAITO, S., BUSH, I.M. and WHITMORE, W.F., Jr.: Effects of certain metals and chelating agents on rat and dog epididymal spermatozoan motility. Fertil. Steril. -18: 517-529, 1967 3. LOEWIT, K.: Immobilization of human spermatozoa with iron. Basis for a new contraceptive? Contraception 3: 219-244, 1971 In vitro Immo4. LOEWIT, K.: Eisen als Antikonzipiens? I. -bilisierung menschlicher Spermien durch Eisensalze. Exp. Path. 1: 198-201, 1972 5. LOEWIT, K., ZAMBELIS, N. and EGG, D.: Contraceptive effect of iron. Reduced fertility after vaginal application of iron chloride in rats. Contraception 4: 91-96, 1971 6. Mc CONNELL, R.F.: Special requirements for testing spermi25: 373-382, 1974 tides. Acta endocr. (Kbh) 7. TAGATZ, G.E., OGAKI, T. and SCIARRA, J.J.: The effect of vaginal lubricants on sperm motility and viability in vitro. Am. J. Obstet. Gynecol. 113: 88-90,lr 8. FOX, C.A., MELDRUM, S.J. and WATSON, B.W.: Continuous measurement by radio-telemetry of vagindpH during human coitus. J. Reprod Fert. 3: 69-75, 1973
FEBRUARY
1977 VOL. 15 NO.2
237
IN VITRO IMMOBILIZATION OF HUMAN SPERMATOZOA WITH HYDROXYPROPYL-METHYLCELLULOSE
K.
Loewit,
M.D.
Institute for General and Experimental Pathology, University of Innsbruck, Austria, A-6020 Innsbruck, Fritz-Pregl-Strasse 3
ABSTRACT Hydroxypropylmethylcellulose (MC) in one to three per cent w/v aqueous solution caused immediate and permanent immobilization oi human spermatozoa in vitro. This spermicidal effect may open new ways of improving-i;uaginal contraception. It also accounts for the spermatotoxic effects of vaginal lubricants and other compounds containing MC as base vehicle.
Accepted for publication December 15, 1976
FEBRUARY
1977 VOL. 15 NO.2
233
CONTRACEPTION INTRODUCTION Metallic conwer and iron or salts of these heavy metals have been found in vitro to be potent sperm-immobilizing agents in several animalspecies includinq man (1,2,3,4). Correspondingly, iron chloride (Fe C13) in 0.153 M concentration significantly reduced fertility after vaginal application in rats (5). Thus, the question arose, whether these findings could be extended to human vaginal contraception which is still considered "an important segment of the general contraceptive armamentarium and needed to maximize individual choice in contraceptive methods" (6). For this purpose, several substances have been tested in search for a suitable vehicle, acting at the same time as a carrier for the spermicide and as a physical barrier to sperm migration (Loewit, unpublished results). Of the compounds studied, hydroxypropylmethylcelluloses (MCs) showed inherent sperm-immobilizing activity. This effect seems of considerable interest and shall be reported. MATERIAL AND METHODS Fresh ejaculates of normal motility were provided by healthy, fertile volunteers. The spermicidal effect was assessed microscopically as described before (4) by measuring the time interval between mixing equal parts of undiluted semen with test solution and complete sperm-immobilization. Air dried films of were Y?), Dow also tested. Hydroxypropylmethylcellulose (Methocel Chemical, Ges.m.b.H. Wien) was used in two variants, MC 65 HGpowder and MC 90 HG-granules. Solutions of different concentration from 0.1 % to 4.0 % w/v were prepared in distilled water. In order to obtain a clear solution MC first had to be dispersed in about one fourth of the final volume at 90°C by continuous stirring. This suspension then was cooled to about 5OC by adding the remaining amount of water in frozen form. Viscosimetry was done by means of a Haake Viscotester VT02 (Haake, Berlin-Lichterfelde) at different time intervals up to two and a half years. The solutions were kept at room temperature and tested for sperm-immobilizing capacity immediately after preparation and approximately two and a half years later. In addition to the earlier evaluation of the influence of solvents, temperature and pH on sperm motility (3,4), the effect of osmolarity was investigated. Equiosmolar control-solutions of polyvinyl-pyrrolidone (PVP) in distilled water were prepared using a Knauer Half-Micro-Osmometer (Knauer, Berlin 37, Holstweg 18, FRG).
234
FEBRUARY
1977 VOL. 15 NO. 2
CONTRACEPTION
RESULTS No influence on sperm motility could be detected after 15 minutes with 0.1 to 0.4 % w/v solutions of MC 65 HG or 0.5 % w/V solutions of MC 90 HG. However, higher concentrations increasingly led to rapid sperm immobilization. Thus, at and above concentrations of 1.5 8 w/v,complete sperm immobilization was observed under the microscope at the earliest possible readings within 5 to 15 seconds. Control experiments with PVP-solution proved ineffective in this regard. Thus, a 1 % PVP-solution which corresponded in viscosity (20 cP) and osmolarity (145 milliosmole) to a 1.5 % solution of MC 65 HG (30 CP, 150 milliosmole) showed no influence on sperm motility after 15 minutes. Similarly, concentrations of PVP up to 20 % w/v, even though mechanically impairing sperm migration, never led to immediate, complete and permanent immobilization. Data on 1.5 % and 1.7 % solutions of MC 65 HG as well as 3 % MC 90 HG are given in the Table. Similar results have been obtained with 1 % and 2 % solutions,respectively. Neither two and a half years storage nor marked decreases of viscosity affected this sperm immobilizing potency. Preliminary experiments with air dried films of MC gave less clear-cut results even though sperm motility was/abolished in the immediate contact zone. DISCUSSION Hydroxypropylmethylcellulose, originally considered as a vehicle for the spermicidal iron chloride, unequivocally exhibited a strong inherent sperm immobilizing effect itself. This effect does not seem to depend on osmolarity nor was it influenced by changes of pH and viscosity measured during the study. MC Connell (6) points out, that the clinical efficiency of any vaginal contraceptive depends at least as much on the vehicle, as on the spermicide selected. A satisfying base vehicle should not only display good carrier qualities, disperse the spermicide, prevent sperm access to the cervical canal, but also possess spermicidal properties itself. Thus, MCs, widely utilized in cosmetic and pharmaceutical products because of their physical qualities and physiological inertness, could be of interest to vaginal contraception from this double point of view. Indeed, MCs inadvertently have been shown to interfere with human fertility in vitro. Tagatz and coworkers in a study relating the useafmnal lubricants to human infertility have reported on the phenomenon of spermicidal effects of such lubricants. One of these consisted of "less than 1.5 per cent boric acid in a sterile suspension of approximately 1 to 5 per cent sodium carboxymethylcellulose" (7). Similarly, in a study
FEBRUARY
1977 VOL. 15 NO.2
235
CONTRACEPTION
N
236
FEBRUARY
1977 VOL. 15 NO. 2
CONTRACEPTION by Fox and coworkers dealing with the influence on fertility of vaginal pH during human coitus, sperm immobilization was found after the application of low pH buffer (pH 3.6). Again the vehicle used was 5 % methylcellulose and immobilization occurred before the vaginal pH was changed (8). In conclusion, the spermicidal effect of MCs, leading to immediate and permanent sperm immobilization in appropriate concentrations, should be taken into account as a cause of undesired side effects wherever MCs are applied in a respective way and as a possible new means of improvement of human vaginal contraception. ACKNOWLEDGEMENT We thank Austrowaren, bsterr. Warenhandelsgesellschaft m.b.H., Wien, for kindly supplying samples of Methocel-products. We are grateful to Ing. A. Kopenetz from K. Schwarzkopf & Co., Innsbruck,for providing facilities for Viscosimetry. REFERENCES 1. WHITE, I.G.: The toxicity of heavy metals to mammalian spermatozoa. Aust. J. Exp. Biol. -33: 359-366, 1955 2. SAITO, S., BUSH, I.M. and WHITMORE, W.F., Jr.: Effects of certain metals and chelating agents on rat and dog epididymal spermatozoan motility. Fertil. Steril. -18: 517-529, 1967 3. LOEWIT, K.: Immobilization of human spermatozoa with iron. Basis for a new contraceptive? Contraception 3: 219-244, 1971 In vitro Immo4. LOEWIT, K.: Eisen als Antikonzipiens? I. -bilisierung menschlicher Spermien durch Eisensalze. Exp. Path. 1: 198-201, 1972 5. LOEWIT, K., ZAMBELIS, N. and EGG, D.: Contraceptive effect of iron. Reduced fertility after vaginal application of iron chloride in rats. Contraception 4: 91-96, 1971 6. Mc CONNELL, R.F.: Special requirements for testing spermi25: 373-382, 1974 tides. Acta endocr. (Kbh) 7. TAGATZ, G.E., OGAKI, T. and SCIARRA, J.J.: The effect of vaginal lubricants on sperm motility and viability in vitro. Am. J. Obstet. Gynecol. 113: 88-90,lr 8. FOX, C.A., MELDRUM, S.J. and WATSON, B.W.: Continuous measurement by radio-telemetry of vagindpH during human coitus. J. Reprod Fert. 3: 69-75, 1973
FEBRUARY
1977 VOL. 15 NO.2
237