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Rat Hyperemia Pregnancy Tests
Rat Hyperemia Pregnancy Tests Review, Evaluation, Comments, and Interpretation
Rose L. Berman, B.A.
IN
1940 A SERIES OF EXPERIMEN!f'S established the use of the female rat as a completely reliable test animal for pregnancy. This test was described by Frank and Berman13 and was the first successful pregnancy test utilizing rats with results obtainable in 8-24 hours. The test involves the subcutaneous injections of two 5-cc. quantities of the supernatant fluid from untreated, centrifuged urine into 2 immature 5O-Gm. female rats (weight range 45-60 Gm. ), the two injections being given 4-6 hours apart. Both animals are sacrificed with illuminating gas the next morning, 16-24 hours after the first injection. Examination of the ovaries is made by inspection with the naked eye. A characteristic hyperemia of the ovaries is produced by pregnancy urine. A full discussion and details of the test as well as indispensable precautions have been published recently.5 In the hands of the author over 9000 tests have yielded 100 per cent accurate results in the 16-24-hour period. Our published reports have stated that results obtained in 8 hours are reliable only when positive. We have studied all dosages of urine, untreated and extracted urines, the use of animals of various weights, all injection routes (intravenous, intraperitoneal, subcutaneous) and the sacrificing of animals at varying time intervals. In some instances we were able to obtain positive results within ~ hour after injection. In 1942,12 we briefly reported the results of a 4-hour test. Since the publication of our first paper, many modifications involving amount of urine (1.5 cc.-6.0 cc.), number of rats used (1-5), weight of rats (20 Cm.-adult), injection route (intravenous and intraperitoneal) From the Berman Clinical Laboratory, New York, N. Y. 276
..
,.
.
Vol. 7, No.3, 1956
RAT HYPEREMIA PREGNANCY TESTS
277
and time of sacrificing animals (1-24 hours) have been offered by a host of workers. The findings of other investigators 1 , 2, 3, 7, 8, 10, 14-20, 22-24, 2'6,28,30-34, 36-42 incorporating their particular modification have yielded inaccuracy of o to 50 per cent. It is apparent when reviewing the literature that the shorter the time interval between injection of the test urine and sacrifice of the rat and the smaller the dose of urine used, the greater the percentage of inaccurate results. TECHNICAL CONSIDERATIONS There have been many misconceptions about the technic involved in rat pregnancy testing. Collection of Urine Many authors 7 , 23, 24, 39 unnecessarily advocate taking the specific gravity of the urine specimen before use; some even vary the amount used on the basis of the specific gravity. It is fallacious to restrict the patient's fluid intake prior to collection of the specimen on the theory that this will produce a greater concentration of chorionic gonadotropin. Actually one only increases the salt concentration by limiting the intake. The purpose of concentration is therefore defeated and more concentrated urines are likely to kill the animals or make reading of the ovaries difficult since they may make them take on a pinkish tone which may be misread as positive. Evidence has been advanced that certain inorganic salts increase the action of pituitary extracts.l1 Apparently when salts are present in the urine in concentrated amounts, the "pink" ovary hyperemic reaction is induced. Sacrifice of Animals Some authors have substituted means other than illuminating gas for sacrificing animals, using ether, chloroform, or a blow to the head. 7 , 24, 28 The typical hyperemia reaction is caused by a marked multiplication and dilatation of ingrowing capillaries, and with the use of illuminating gas the typical carbon-monoxide effect on the ovary is produced which makes the anatomic changes more apparent. The use of other killing agents deprives the test of this advantage and may actually mask the hyperemia reaction . Preservation of Specimen Some claim that urine specimens for pregnancy testing can be sent to the
278
BERMAN
Fertility & Sterility
laboratory via the mail. 25 Such specimens can only be sent if properly packed with dry ice since the chorionic gonadotropin is destroyed at room temperature. Our laboratory has found complete hormone deterioration in as short a time as four hours. Treatment of Urine for Injection
Various modifications of the injected urine have been advocated: the use of urine with its sediment,24 filtering,39 icing,3 warming,24 addition of glucose to specimen,25 acidification. 20 . 31, 41 Frank and Berman directed the use of clear supernatant urine from a specimen which had been centrifuged. The sediment contains such extraneous matter as crystals, urates, W.B.C., epithelial cells, etc., which are not necessary for the test. Ice-cold urine injected into rats will either shock or kill them. There is no need to treat the specimen with warmth, acid, or glucose. Prescribing for the patient bicarbonate of soda the day before the urine is to be collected is an entirely unnecessary procedure. Interpretation of Response
Calling an ovarian response positive only when all four ovaries of the two animals are not only hyperemic but also enlarged will eliminate some true positives. 39 We reported that a positive ovary is usually, but not always, enlarged, and that occasionally one ovary in a rat will be negative while the other is positive. A response in one of two ovaries is considered a positive pregnancy test. The use of a color chart 10 with which to match the degree of redness of the rats' ovaries is an unnecessary complication since the degree of hyperemia has no practical import. Classification of ovarian reaction according to the degree of color reaction and size of ovarr4 is fallacious since the degree of reaction is meaningless. The statement35 that rat-test results are available only after 48 hours is untrue. Completely reliable results are available after 16-24 hours. We further disagree with the claim that the reading of rats' ovaries is a purely subjective matter dependent on distinguishing shades of pink. 6 The truth is that the positive ovary is not pink but a frank red. False-Positive and False-Negative Results
Many investigators have reported "doubtful" tests, false negatives, and
.-(
..
"
Vol. 7, No.3, 1956
RAT HYPEREMIA PREGNANCY TESTS
279
false positives. The reasons for these are easy to explain. It takes 16-24 hours to develop the full hyperemic effect. The definition of hyperemia is excess of blood in any part, and since other investigators sacrifice animals at times varying from 1-6 hours, this superabundance of blood in the ovaries has not had sufficient time to develop. These investigators are therefore reading gradations of pink and not the true-red reactions (the color of blood) in the ovaries. This accounts for "doubtful" or "false-positive" reactions. False positives are obtained by using mature, rather than immature rats. False negatives arise from the use of urine which has not been properly collected or stored, injecting insufficient quantities, killing animals incorrectly, or attempting to get a definitive reading before 16-24 hours. Instructions to Patients
To obtain proper specimens patients must be properly instructed. The instructions are: 1. No hormones, prostigmine, antihistamins, sulfonamides, quinine, or ergot for 96 hours prior to test. 2. No laxatives for 2 days prior to test. S. No alcoholic beverages for 1 day prior to test. All of the above substances are excreted in the urine and are toxic when injected into rats. 4. Save first urine on arising plus any urines obtained during the normal sleeping time. All specimens to be refrigerated immediately, and kept refrigerated until brought to the laboratory. GONADOTROPIC HORMONES
Much of the misinformation regarding pregnancy tests can be traced to a lack of knowledge of fundamentals concerning gonadotropins. In humans there are two gonadotropic hormones-the anterior pituitary gonadotropin ( APG ), consisting of a follicle-stimulating factor and a luteinizing factorand chorionic gonadotropin (CG), found only in pregnancy, hydatidiform mole, and chorionepithelioma. There are biological differences between the two hormones. Both are ovary-stimulating in the test animal, but the ovarystimulating effect of CG is more than lOO times that of APG per liter of urine. For example, with the Frank-Berman pregnancy test, a total of 10.0 cc. untreated centrifuged urine is injected into the test animal, and reliable
280
BERMAN
Fertility & Sterility
results are obtained. In order to demonstrate the presence of APG in menopausal and amenorrheic cases, it is necessary to extract the hormone from urine and inject the equivalent of 100-200 cc. urine into test animals for routine gonadotropin assay. In normal cyclic females, we can demonstrate APG only at the time of ovulation, between about Days 10 and 14 of the cycle. In order to demonstrate the gonadotropin in them we have to inject the equivalent of 250-300 cc. of urine into the test animal for routine assay. 3D ml. DOSE lNJECTED IN
FRANK .. BERIIAN TFST
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AMENORRHIA M I If 0 P A U S I
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roLLICLE STlIIULATlNG GOHAOO'lIIOPili
Vi!
LDTElNIZlNG GOIWX7.lIIOPIR
•
CIIlRIOIIIC GOIIA~
Fig. 1.
Gonadotropic hormone excretion levels.
Figure 1 demonstrates graphically the differences in excretion range between anterior pituitary gonadotropin and chorionic gonadotropin.
SCOPE OF PREGNANCY TEST The Frank-Berman pregnancy test is used to diagnose pregnancy or hydatidiform mole in the female, and chorionepithelioma in the male as well as the female. These are the only true functions of any reliable pregnancy test.
.
Vol. 7, No.3, 1956
RAT HYPEREMIA PREGNANCY TESTS
281
No pregnancy test can: (a) indicate the progress of the pregnancy, as claimed by Farris,4,lO on the basis of the degree of ovarian hyperemia since the degree of hyperemia has no significance. (b) tell the precise time of ovulation, as claimed by Farris,9, 27, 29, 41 since pregnancy tests are based on the presence of chorionic gonadotropin found only in pregnancy, hydatidiform mole, or chorionepithelioma. ( c) with certainty indicate whether the fetus is dead or alive. Depending on clinical symptoms, a negative test after repeated positive tests will aid in the establishment of fetal death. ( d) definitely establish the existence or nonexistence of an ectopic pregnancy, since the test will be positive only if the chorionic villi are well embedded and functioning . In brief, clinical conditions yield the following results:
1. 2. 3.
4. 5. 6.
7.
Positive pregnancy test Normal pregnancy Hydatidiform mole Chorionepithelioma Ectopic pregnancy, if chorionic villi are attached and functional. Postpartum up to 10-14 days. Dead fetus, when areas of placenta still remain viable. Abortion, if chorionic villi are attached and functional.
1.
2.
3.
4.
Negative pregnancy test Ectopic pregnancy, if villi are unattached. Dead fetus, placenta nonfunctional. Abortion, if chorionic villi are detached. All conditions other than the 7 outlined under positive pregnancy test.
SUMMARY 1. The Frank-Berman rat hyperemia pregnancy test has been briefly discussed. 2. The various rat hyperemia pregnancy tests that have been proposed as modifications of the Frank-Berman test have been noted. 3. The gonadotropic hormones involved in pregnancy, normal cycles, amenorrhea, and the menopause have been graphically presented. 4. The role of pregnancy tests has been elucidated.
282
BERMAN
Fertility & Sterility
REFERENCES 1. ALBERT, A. Evaluation of the hyperemia test for pregnancy as a routine clinical laborfltory procedure: Comparison of results with those of 1,000 consecutive Friedman tests. Proc. Staff Meet. Mayo GZin. 24:259, 1949. 2. AMORAL, C. D. Nossa experiencia com a modificar;ao de Frank e Berman ao teste de Aschheim e Zondek. An. brasil. ginec. 20:108, 1945. 3. BEHNKEN, E. W., LLOYD, C. W., and HUGHEs, E. C. The ovarian hyperemia reaction: Its use in qualitative and quantitative tests for urinary chorionic gonadotropin. Am. J. Obst. & Gynec. 56:930, 1948. 4. BERMAN, ROSE L. Correspondence. Fertil. & Steril. 2:87, 1951. 5. BERMAN, ROSE L. Notes on the 8 and 24 hour Frank-Berman pregnancy test. Am. J, Obst. & Gynec. 64:440, 1952. 6. BETTINGER, H. F. Pregnancy tests. M. J, Australia 1 :504, 1950. 7. BUNDE, C. A. An evaluation of the pregnancy test based on ovarian hyperemia in the immature rat. Am. J, Obst. & Gynec. 55:317, 1947. 8. FARRIS, E. J. Validity of the two-hour rat test for human pregnancy. Am. J, Obst. & Gynec. 48:200, 1944. 9. FARRIS, E. J. A test for determining the time of ovulation and conception in women. Am. J, Obst. & Gynec. 52:14, 1946. 10. FARRIS, E. J. A twenty-four-hour rat test for the diagnosis of early pregnancy and as an aid in predicting abortion. Fertil. & Steril. 1 :76, 1950. 11. FEVOLD, H. L., HISAW, F. L., and GREEP, R. Augmentation of the gonad stimulating action of pituitary extracts by inorganic substances, particularly copper salts. Am. J, Physiol. 117:68, 1936. 12. FRANK, R. T. Correspondence. Am. J, Obst. & Gynec. 43:729, 1942. 13. FRANK, R. T., and BERMAN, ROSE L. A twenty-four-hour pregnancy test. Am. J, Obst. & Gynec. 42:492, 1941. 14. FRIED, P. H. An evaluation of the two-hour rat test for pregnancy. Am. J, Obst. & Gynec. 54:689, 1947. 15. FRIED, P. H. The two-hour pregnancy test based on rat ovary hyperemia. West. J, Surg. 56:552, 1948. 16. FRIED, P. H. Factors influencing the rat ovary hyperemia reaction as a test for pregnancy. Am. J, Obst. & Gynec. 57:868, 1949. 17. FRIED, P. H., and RAKOFF, A. E. The one-hour rat ovary hyperemia pregnancy test with synergist. J,AM.A 141 :25, 1949. 18. FRIED, P. H., and RAKOFF, A. E. Improvement of the rat ovary hyperemia test for pregnancy by adding a pituitary synergist. J, Glin. Endocrinol. 10:423, 1950. 19. GREENBLATT, R. B., CLARK, S. L., and WEST, R. M. The male frog (Rana pipiens) compared to the female rat as a pregnancy test animal. Fertil. & Steril. 1 :533, 1950. 20. HOFFMAN, R. V., MARKEY, R. L., and GIORDANO, A. S. Comparison of the rat and Friedman tests for pregnancy. Am. J, Glin. Pathol. 21 :33, 1951. 21. HOFMAN, C. Modification au test d'hyperemie chez les rats. Rev. franQ. gyn. et l'obst. 47:265, 1944. 22. KAMNISTER, S. Experience with the six-hour test for pregnancy. Am. J, Obst. & Gynec. 47:265, 1944. 23. KELSEY, J. F., SALMON, A. A., DAVIS, C. D., and HAMBLEN, E. C. The Salmon modification of the ovarian hyperemia reaction in 1,042 consecutive pregnancy tests. Am. J, Obst. & Gynec. 60:904, 1950.
,
Vol. 7, No.3, 1956
24. KLINE, B. S.
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283
The Salmon, Geist, Salmon, Frank six-hour rat test for pregnancy.
Am. ]. Glin. Pathol. 14:557, 1944. 25. KOLMER, J. A., and BOERNER, F. Approved Laboratory Technique (ed. 4). 26. 27. 28. 29.
30. 31.
New York, Appleton, 1945, pp. 294-299. KUPPERMAN, H. S., and GREENBLATT, R. B. The two-hour pregnancy test. South. M.]. 39: 158, 1946. KUPPERMAN, H. S., and GREENBLATT, R. B. Further observations on the twohour pregnancy test. ].M.A. Georgia 36:58, 1947. KUPPERMAN, H. S., GREENBLATT, R. B., and NOBACK, C. S. A two- and six-hour pregnancy test. J. Glin. Endocrinol. 3:548, 1943. LEVIN, L., BUXTON, C. L., and ENGLE, E. T. On the validity of the hyperemia method for determining ovulation time in women. Am.]. Obst. & Gynec. 58:795, 1949. MASSENBACH, W. v. Kritische betrachtungen zur frage der schwangerschaftsreaktion an ratten. Zentralbl. Gyniik. 71 :849, 1949. O'HANRAHAN,1. Routine pregnancy tests with rats and frogs: Results in 593 cases.
West. J. Surg. 60:589, 1952.
32. RAMSEY, T. L., FALKENSTEIN, A. P., and SIYKOWSKI, R. J. The use of the albino rat in pregnancy hormone test. J. Lab. & Glin. Med. 29:419, 1944. 33. RIDDELL, R. J. Some observations on the 24-hour "hyperemia" biological test for pregnancy. M.]. Australia 2:382, 1949. 34. RILEY, G. M., SMITH, M. H., and BROWN, P. The rapid rat test for pregnancy: The ovarian hyperemia response as a routine diagnostic procedure. ]. Glin. Endocrinol. 8:233, 1948. 35. ROBBINS, S. L., PARKER, F., and DOYLE, W. C. The use of the South African frog (Xenopus laevis) in the diagnosis of pregnancy. New England J. Med. 234:784, 1946. 36. SALLES, A. DE AQUINO Teste de Frank para 0 diagn6stico precoce de gravidez. An. brasil. ginec. 15:467, 1943. 37. SALLES, A. DE AQUINO, DE CENZO, M. A., and BAIOCCHI, O. Teste de GalliMainini com 0 "Bufo marinus": Seu emprego na cHnici em conjunto com 0 teste de Frank. Med. cir. farm. 160:445, 1949. 38. SALMON, U. J., GEIST, S. H., SALMON, A. A., and FRANK, 1. L. A six-hour pregnancy test. ]. GUn. Endocrinol. 3:548, 1943. 39. SOMAN, D. W. Observations on the 24-hour rat test (Aschheim-Zondek modification) for the diagnosis of pregnancy. Indian M. Gaz. 79:68, 1944. 40. SOMAN, D. W. A preliminary study of six-hour rat test for pregnancy. Indian M. Gaz. 81:134,1946. 41. STURGIS, S. H., and HAOUR, P. The one-hour ovarian hyperemia response to intravenous injection of human urine in the immature rat. Fertil. & Steril. 2:347, 1951. 42. ZONDEK, B., SULMAN, F., and BLACK, R. The hyperemia effect of gonadotropins on the ovary and its use in a rapid pregnancy test. J.A.M.A. 128:939, 1945. 43. BERMAN, ROSE L. A critical evaluation of biological pregnancy tests. Am.]. Obst. & Gynec. (in press) .
Rose L. Berman, B.A.
IN
1940 A SERIES OF EXPERIMEN!f'S established the use of the female rat as a completely reliable test animal for pregnancy. This test was described by Frank and Berman13 and was the first successful pregnancy test utilizing rats with results obtainable in 8-24 hours. The test involves the subcutaneous injections of two 5-cc. quantities of the supernatant fluid from untreated, centrifuged urine into 2 immature 5O-Gm. female rats (weight range 45-60 Gm. ), the two injections being given 4-6 hours apart. Both animals are sacrificed with illuminating gas the next morning, 16-24 hours after the first injection. Examination of the ovaries is made by inspection with the naked eye. A characteristic hyperemia of the ovaries is produced by pregnancy urine. A full discussion and details of the test as well as indispensable precautions have been published recently.5 In the hands of the author over 9000 tests have yielded 100 per cent accurate results in the 16-24-hour period. Our published reports have stated that results obtained in 8 hours are reliable only when positive. We have studied all dosages of urine, untreated and extracted urines, the use of animals of various weights, all injection routes (intravenous, intraperitoneal, subcutaneous) and the sacrificing of animals at varying time intervals. In some instances we were able to obtain positive results within ~ hour after injection. In 1942,12 we briefly reported the results of a 4-hour test. Since the publication of our first paper, many modifications involving amount of urine (1.5 cc.-6.0 cc.), number of rats used (1-5), weight of rats (20 Cm.-adult), injection route (intravenous and intraperitoneal) From the Berman Clinical Laboratory, New York, N. Y. 276
..
,.
.
Vol. 7, No.3, 1956
RAT HYPEREMIA PREGNANCY TESTS
277
and time of sacrificing animals (1-24 hours) have been offered by a host of workers. The findings of other investigators 1 , 2, 3, 7, 8, 10, 14-20, 22-24, 2'6,28,30-34, 36-42 incorporating their particular modification have yielded inaccuracy of o to 50 per cent. It is apparent when reviewing the literature that the shorter the time interval between injection of the test urine and sacrifice of the rat and the smaller the dose of urine used, the greater the percentage of inaccurate results. TECHNICAL CONSIDERATIONS There have been many misconceptions about the technic involved in rat pregnancy testing. Collection of Urine Many authors 7 , 23, 24, 39 unnecessarily advocate taking the specific gravity of the urine specimen before use; some even vary the amount used on the basis of the specific gravity. It is fallacious to restrict the patient's fluid intake prior to collection of the specimen on the theory that this will produce a greater concentration of chorionic gonadotropin. Actually one only increases the salt concentration by limiting the intake. The purpose of concentration is therefore defeated and more concentrated urines are likely to kill the animals or make reading of the ovaries difficult since they may make them take on a pinkish tone which may be misread as positive. Evidence has been advanced that certain inorganic salts increase the action of pituitary extracts.l1 Apparently when salts are present in the urine in concentrated amounts, the "pink" ovary hyperemic reaction is induced. Sacrifice of Animals Some authors have substituted means other than illuminating gas for sacrificing animals, using ether, chloroform, or a blow to the head. 7 , 24, 28 The typical hyperemia reaction is caused by a marked multiplication and dilatation of ingrowing capillaries, and with the use of illuminating gas the typical carbon-monoxide effect on the ovary is produced which makes the anatomic changes more apparent. The use of other killing agents deprives the test of this advantage and may actually mask the hyperemia reaction . Preservation of Specimen Some claim that urine specimens for pregnancy testing can be sent to the
278
BERMAN
Fertility & Sterility
laboratory via the mail. 25 Such specimens can only be sent if properly packed with dry ice since the chorionic gonadotropin is destroyed at room temperature. Our laboratory has found complete hormone deterioration in as short a time as four hours. Treatment of Urine for Injection
Various modifications of the injected urine have been advocated: the use of urine with its sediment,24 filtering,39 icing,3 warming,24 addition of glucose to specimen,25 acidification. 20 . 31, 41 Frank and Berman directed the use of clear supernatant urine from a specimen which had been centrifuged. The sediment contains such extraneous matter as crystals, urates, W.B.C., epithelial cells, etc., which are not necessary for the test. Ice-cold urine injected into rats will either shock or kill them. There is no need to treat the specimen with warmth, acid, or glucose. Prescribing for the patient bicarbonate of soda the day before the urine is to be collected is an entirely unnecessary procedure. Interpretation of Response
Calling an ovarian response positive only when all four ovaries of the two animals are not only hyperemic but also enlarged will eliminate some true positives. 39 We reported that a positive ovary is usually, but not always, enlarged, and that occasionally one ovary in a rat will be negative while the other is positive. A response in one of two ovaries is considered a positive pregnancy test. The use of a color chart 10 with which to match the degree of redness of the rats' ovaries is an unnecessary complication since the degree of hyperemia has no practical import. Classification of ovarian reaction according to the degree of color reaction and size of ovarr4 is fallacious since the degree of reaction is meaningless. The statement35 that rat-test results are available only after 48 hours is untrue. Completely reliable results are available after 16-24 hours. We further disagree with the claim that the reading of rats' ovaries is a purely subjective matter dependent on distinguishing shades of pink. 6 The truth is that the positive ovary is not pink but a frank red. False-Positive and False-Negative Results
Many investigators have reported "doubtful" tests, false negatives, and
.-(
..
"
Vol. 7, No.3, 1956
RAT HYPEREMIA PREGNANCY TESTS
279
false positives. The reasons for these are easy to explain. It takes 16-24 hours to develop the full hyperemic effect. The definition of hyperemia is excess of blood in any part, and since other investigators sacrifice animals at times varying from 1-6 hours, this superabundance of blood in the ovaries has not had sufficient time to develop. These investigators are therefore reading gradations of pink and not the true-red reactions (the color of blood) in the ovaries. This accounts for "doubtful" or "false-positive" reactions. False positives are obtained by using mature, rather than immature rats. False negatives arise from the use of urine which has not been properly collected or stored, injecting insufficient quantities, killing animals incorrectly, or attempting to get a definitive reading before 16-24 hours. Instructions to Patients
To obtain proper specimens patients must be properly instructed. The instructions are: 1. No hormones, prostigmine, antihistamins, sulfonamides, quinine, or ergot for 96 hours prior to test. 2. No laxatives for 2 days prior to test. S. No alcoholic beverages for 1 day prior to test. All of the above substances are excreted in the urine and are toxic when injected into rats. 4. Save first urine on arising plus any urines obtained during the normal sleeping time. All specimens to be refrigerated immediately, and kept refrigerated until brought to the laboratory. GONADOTROPIC HORMONES
Much of the misinformation regarding pregnancy tests can be traced to a lack of knowledge of fundamentals concerning gonadotropins. In humans there are two gonadotropic hormones-the anterior pituitary gonadotropin ( APG ), consisting of a follicle-stimulating factor and a luteinizing factorand chorionic gonadotropin (CG), found only in pregnancy, hydatidiform mole, and chorionepithelioma. There are biological differences between the two hormones. Both are ovary-stimulating in the test animal, but the ovarystimulating effect of CG is more than lOO times that of APG per liter of urine. For example, with the Frank-Berman pregnancy test, a total of 10.0 cc. untreated centrifuged urine is injected into the test animal, and reliable
280
BERMAN
Fertility & Sterility
results are obtained. In order to demonstrate the presence of APG in menopausal and amenorrheic cases, it is necessary to extract the hormone from urine and inject the equivalent of 100-200 cc. urine into test animals for routine gonadotropin assay. In normal cyclic females, we can demonstrate APG only at the time of ovulation, between about Days 10 and 14 of the cycle. In order to demonstrate the gonadotropin in them we have to inject the equivalent of 250-300 cc. of urine into the test animal for routine assay. 3D ml. DOSE lNJECTED IN
FRANK .. BERIIAN TFST
.,'"
.. ..., '" .....'" .. o
o
...'"o
.." <
o o
...'"o ...'"
.. '"
~
''ii
300
,",~~L-_-£~'.I.I.I.I.LL._ _ NORMAL (10 _ 1h Do;rl)
AMENORRHIA M I If 0 P A U S I
III
roLLICLE STlIIULATlNG GOHAOO'lIIOPili
Vi!
LDTElNIZlNG GOIWX7.lIIOPIR
•
CIIlRIOIIIC GOIIA~
Fig. 1.
Gonadotropic hormone excretion levels.
Figure 1 demonstrates graphically the differences in excretion range between anterior pituitary gonadotropin and chorionic gonadotropin.
SCOPE OF PREGNANCY TEST The Frank-Berman pregnancy test is used to diagnose pregnancy or hydatidiform mole in the female, and chorionepithelioma in the male as well as the female. These are the only true functions of any reliable pregnancy test.
.
Vol. 7, No.3, 1956
RAT HYPEREMIA PREGNANCY TESTS
281
No pregnancy test can: (a) indicate the progress of the pregnancy, as claimed by Farris,4,lO on the basis of the degree of ovarian hyperemia since the degree of hyperemia has no significance. (b) tell the precise time of ovulation, as claimed by Farris,9, 27, 29, 41 since pregnancy tests are based on the presence of chorionic gonadotropin found only in pregnancy, hydatidiform mole, or chorionepithelioma. ( c) with certainty indicate whether the fetus is dead or alive. Depending on clinical symptoms, a negative test after repeated positive tests will aid in the establishment of fetal death. ( d) definitely establish the existence or nonexistence of an ectopic pregnancy, since the test will be positive only if the chorionic villi are well embedded and functioning . In brief, clinical conditions yield the following results:
1. 2. 3.
4. 5. 6.
7.
Positive pregnancy test Normal pregnancy Hydatidiform mole Chorionepithelioma Ectopic pregnancy, if chorionic villi are attached and functional. Postpartum up to 10-14 days. Dead fetus, when areas of placenta still remain viable. Abortion, if chorionic villi are attached and functional.
1.
2.
3.
4.
Negative pregnancy test Ectopic pregnancy, if villi are unattached. Dead fetus, placenta nonfunctional. Abortion, if chorionic villi are detached. All conditions other than the 7 outlined under positive pregnancy test.
SUMMARY 1. The Frank-Berman rat hyperemia pregnancy test has been briefly discussed. 2. The various rat hyperemia pregnancy tests that have been proposed as modifications of the Frank-Berman test have been noted. 3. The gonadotropic hormones involved in pregnancy, normal cycles, amenorrhea, and the menopause have been graphically presented. 4. The role of pregnancy tests has been elucidated.
282
BERMAN
Fertility & Sterility
REFERENCES 1. ALBERT, A. Evaluation of the hyperemia test for pregnancy as a routine clinical laborfltory procedure: Comparison of results with those of 1,000 consecutive Friedman tests. Proc. Staff Meet. Mayo GZin. 24:259, 1949. 2. AMORAL, C. D. Nossa experiencia com a modificar;ao de Frank e Berman ao teste de Aschheim e Zondek. An. brasil. ginec. 20:108, 1945. 3. BEHNKEN, E. W., LLOYD, C. W., and HUGHEs, E. C. The ovarian hyperemia reaction: Its use in qualitative and quantitative tests for urinary chorionic gonadotropin. Am. J. Obst. & Gynec. 56:930, 1948. 4. BERMAN, ROSE L. Correspondence. Fertil. & Steril. 2:87, 1951. 5. BERMAN, ROSE L. Notes on the 8 and 24 hour Frank-Berman pregnancy test. Am. J, Obst. & Gynec. 64:440, 1952. 6. BETTINGER, H. F. Pregnancy tests. M. J, Australia 1 :504, 1950. 7. BUNDE, C. A. An evaluation of the pregnancy test based on ovarian hyperemia in the immature rat. Am. J, Obst. & Gynec. 55:317, 1947. 8. FARRIS, E. J. Validity of the two-hour rat test for human pregnancy. Am. J, Obst. & Gynec. 48:200, 1944. 9. FARRIS, E. J. A test for determining the time of ovulation and conception in women. Am. J, Obst. & Gynec. 52:14, 1946. 10. FARRIS, E. J. A twenty-four-hour rat test for the diagnosis of early pregnancy and as an aid in predicting abortion. Fertil. & Steril. 1 :76, 1950. 11. FEVOLD, H. L., HISAW, F. L., and GREEP, R. Augmentation of the gonad stimulating action of pituitary extracts by inorganic substances, particularly copper salts. Am. J, Physiol. 117:68, 1936. 12. FRANK, R. T. Correspondence. Am. J, Obst. & Gynec. 43:729, 1942. 13. FRANK, R. T., and BERMAN, ROSE L. A twenty-four-hour pregnancy test. Am. J, Obst. & Gynec. 42:492, 1941. 14. FRIED, P. H. An evaluation of the two-hour rat test for pregnancy. Am. J, Obst. & Gynec. 54:689, 1947. 15. FRIED, P. H. The two-hour pregnancy test based on rat ovary hyperemia. West. J, Surg. 56:552, 1948. 16. FRIED, P. H. Factors influencing the rat ovary hyperemia reaction as a test for pregnancy. Am. J, Obst. & Gynec. 57:868, 1949. 17. FRIED, P. H., and RAKOFF, A. E. The one-hour rat ovary hyperemia pregnancy test with synergist. J,AM.A 141 :25, 1949. 18. FRIED, P. H., and RAKOFF, A. E. Improvement of the rat ovary hyperemia test for pregnancy by adding a pituitary synergist. J, Glin. Endocrinol. 10:423, 1950. 19. GREENBLATT, R. B., CLARK, S. L., and WEST, R. M. The male frog (Rana pipiens) compared to the female rat as a pregnancy test animal. Fertil. & Steril. 1 :533, 1950. 20. HOFFMAN, R. V., MARKEY, R. L., and GIORDANO, A. S. Comparison of the rat and Friedman tests for pregnancy. Am. J, Glin. Pathol. 21 :33, 1951. 21. HOFMAN, C. Modification au test d'hyperemie chez les rats. Rev. franQ. gyn. et l'obst. 47:265, 1944. 22. KAMNISTER, S. Experience with the six-hour test for pregnancy. Am. J, Obst. & Gynec. 47:265, 1944. 23. KELSEY, J. F., SALMON, A. A., DAVIS, C. D., and HAMBLEN, E. C. The Salmon modification of the ovarian hyperemia reaction in 1,042 consecutive pregnancy tests. Am. J, Obst. & Gynec. 60:904, 1950.
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24. KLINE, B. S.
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32. RAMSEY, T. L., FALKENSTEIN, A. P., and SIYKOWSKI, R. J. The use of the albino rat in pregnancy hormone test. J. Lab. & Glin. Med. 29:419, 1944. 33. RIDDELL, R. J. Some observations on the 24-hour "hyperemia" biological test for pregnancy. M.]. Australia 2:382, 1949. 34. RILEY, G. M., SMITH, M. H., and BROWN, P. The rapid rat test for pregnancy: The ovarian hyperemia response as a routine diagnostic procedure. ]. Glin. Endocrinol. 8:233, 1948. 35. ROBBINS, S. L., PARKER, F., and DOYLE, W. C. The use of the South African frog (Xenopus laevis) in the diagnosis of pregnancy. New England J. Med. 234:784, 1946. 36. SALLES, A. DE AQUINO Teste de Frank para 0 diagn6stico precoce de gravidez. An. brasil. ginec. 15:467, 1943. 37. SALLES, A. DE AQUINO, DE CENZO, M. A., and BAIOCCHI, O. Teste de GalliMainini com 0 "Bufo marinus": Seu emprego na cHnici em conjunto com 0 teste de Frank. Med. cir. farm. 160:445, 1949. 38. SALMON, U. J., GEIST, S. H., SALMON, A. A., and FRANK, 1. L. A six-hour pregnancy test. ]. GUn. Endocrinol. 3:548, 1943. 39. SOMAN, D. W. Observations on the 24-hour rat test (Aschheim-Zondek modification) for the diagnosis of pregnancy. Indian M. Gaz. 79:68, 1944. 40. SOMAN, D. W. A preliminary study of six-hour rat test for pregnancy. Indian M. Gaz. 81:134,1946. 41. STURGIS, S. H., and HAOUR, P. The one-hour ovarian hyperemia response to intravenous injection of human urine in the immature rat. Fertil. & Steril. 2:347, 1951. 42. ZONDEK, B., SULMAN, F., and BLACK, R. The hyperemia effect of gonadotropins on the ovary and its use in a rapid pregnancy test. J.A.M.A. 128:939, 1945. 43. BERMAN, ROSE L. A critical evaluation of biological pregnancy tests. Am.]. Obst. & Gynec. (in press) .