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Response by Drs. Levin, Buxton, and Engle
Correspondence The Rat Ovulation Test To the Editor: I recently had the opportunity to work with Dr. E ..T. Parris at the Wistar Institute when• I was able to observe the rat ovulation test performed many times. I was impressed by itH simplicity, validity, and excellent clinical results. It was therefore with great conct>m and assiduousness that I read thP article by Levin, Buxton, and Engle (AM .•T. 0BST. & GYN~;u. 58: 795-798, 1949) who attempt to disprove the test. After a careful perusal of their publication one notes the following salient features. 'l'he technique advocated by :Farris in previous communications is not adhered to in that tht~ strain of the rats used is not stated and fluorescent lamps were substituted for the special lamp suggested. Both of these requisites contribute immeasurably to the success of the test. Nevertheless, in two of the three cases in which laparotomy was performed (Cases of A. B. and A. S.) ovulation occurred or was absent as predicted in the criteria for reading the hyperemia response. In the third instance, the Case of B. P. where evidence of ovulation was found on laparotomy, incomplete testing is to be found. No urine collec· tions were made on important cycle days thirteen and fifteen. In the other four cases discussed by the authors one cannot argue with positive proof in either rlirection in view of the lack of true objective evidence. It must therefore he re-emphasized that the ability to interpret adequately these hyperemic responses in rat ovaries and to follow the prescribed techniques is of prime importanc·e before true comparative studies may be evaluated. ALVIN M. SIEGLER, M.D. 706 EASTERN PARKWAY, N. Y. NOVEMBER 14, 1949. BROOKLYN,
Response by Drs. Levin, Buxton, and Engle To the Editor: Dr. Siegler infers that our' inability to confirm the optimistic claims of Farris may be due to several factors. His first objeetion concerns the strain of animals we used. These werP ntts of the Long-Evans strain derived from a colony inbred in this laboratory for more than twenty years. The exact ages of the rats are known and only those 21 to 24 days old were used. 'l'here is a remote possibility that animals of this strain are lesH responsive than are those of other strains to gonadotrophin (if this is indeed the principle responsihle for the positive ''ovulatory'' reaction). However, this factor does not appear to t'nter into the discrepancy for, as stated in our paper, at least one "positive" eontrol rat was inrluded in eaeh day's test. The "positive" control was injected with urine from women known to be in the first trimester of pregnancy. Every one of these '·positive'' eontrols, without exception, showed a definite hyperemic reaction of the ovaries. This proved, at least to our own satisfaction, that the animals do unquestionably respond when adequately treated with gonadotrophin. The results obtained with the ''positive'' controls also appear to vitiate Dr. Siegler's objection relating to the type of light used. It is true that we examined the test animals' ovaries under a battery of fluorescent lights instead of the special lamp used by Farris. Nevertheless, when animals showed a positi,-e response, as did those injected with pregnancy urine, we had no difficulty in recognizing such a response. It seems reasonable to asHume that we eould as readily recognize a positive response caused by urine from an on1lntor~· woman. 470
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Dr. Siegler also attempts to make the point that our results are better than >YC claim. He suggests that the ovulation of subject A. B. was in actuality predicted by the hyperemia reaction while in subject B. P. the lack of tests on days 13 and 15 makes interpretation impossible. Dr. Siegler's point is correct only if there has been a further revision of Farris' criteria of a "positive" ovulatory response. Origina1Iy2 it was stated that a positive hyperemia response for at least three successive days is required to demonstrate a "normal" ovulation. Later, apparently on the basis of additional experience, Farrisa stated that four successive days of positive hyperemia are required to indicate a ''normal'' ovulatory pattern. In line with this latest requirement, neither subject A. B. nor B. P. showed a normal ovulatory pattern. Subject A. B. showed three days of doubtful positive reaction followed by one day of full positive reaction. A normal ovulation could not be diagnosed for B. P. even if positive responses had been obtained on days 13 and 15, for even in this event there would have been only two successive days of positive response, two days less than l!'arris' latest requirement. The response obtained from A. S. cannot be debated. It is true that a positive response was not obtained nor was any indication of ovulation found at laparotomy. 'l'he other four of our subjects were gynecologically normal. Although laparotomy was not done to examine the ovaries of these subjects, the basal body temperature curves showed the changes which many investigators now accept as indicative of ovulation. As suggested in our report, we also consider "the only certain means of proof is by appropriately timed laparotomy with subsequent microscopic examination of the ovaries.'' By the same token (and this we attempted to indicate in our original paper) Farris has not yet proved his point. Therefore his test still ''must remain as an interesting but unproved suggestion.'' LOLlS LEVIN, PH.D. C. L. BuxToN, M.D. E. T. ENGLE, Pu.D. NEW YORK.
NovEMBER 30, 1949.
References 1. Levin, L., Buxton, C. L., and Engle, E. T.: AM. J. 0BST. & GYXEC. 58: 795, 194fl. 2. Farris, E. J.: AM. J. 0BST. & GYNEC. 52: 14,1946. :3. liurphy, D.P., and Farris, E. J.: AM. J. OBST. & GYKEC. 54: 467, lD-!i.
Reply by Dr. Farris To the Editor: In the October, 1949, issue of this JoURNAL, an article by Louis Levin, C. L. Buxton, and Earl Engle appeared, which questioned the value of the hyperemia method for the determination of ovulation time. 'L'he technique recommended by me for ovulation timing was said by Levin, Buxton, and Engle to have been followed by them. In my early experiments, after consultation with General Electric Co. lighting engineers, it was decided that the specially selected fluorescent lights chosen for daylight color were unsatisfactory for reading hyperemia in the rats' ovaries. In our work, we use the Macbeth light No. ADP 20. This has proved to be quite satisfactory for reading of different shades of the same color. Since Levin and associates used fluorescent lights, and not a Macbeth light, their technique differed in this respect from mine. The essayists did not state whether they used the Wistar strain of rats. In our early testing we learned that other strains are usually less sensitive than the Wistar rat for this type of testing. This has been confirmed several times by other workers who were furnished our rats for testing of ovulation time, when the other strains employed failed to giye consistent reactions.
Response by Drs. Levin, Buxton, and Engle To the Editor: Dr. Siegler infers that our' inability to confirm the optimistic claims of Farris may be due to several factors. His first objeetion concerns the strain of animals we used. These werP ntts of the Long-Evans strain derived from a colony inbred in this laboratory for more than twenty years. The exact ages of the rats are known and only those 21 to 24 days old were used. 'l'here is a remote possibility that animals of this strain are lesH responsive than are those of other strains to gonadotrophin (if this is indeed the principle responsihle for the positive ''ovulatory'' reaction). However, this factor does not appear to t'nter into the discrepancy for, as stated in our paper, at least one "positive" eontrol rat was inrluded in eaeh day's test. The "positive" control was injected with urine from women known to be in the first trimester of pregnancy. Every one of these '·positive'' eontrols, without exception, showed a definite hyperemic reaction of the ovaries. This proved, at least to our own satisfaction, that the animals do unquestionably respond when adequately treated with gonadotrophin. The results obtained with the ''positive'' controls also appear to vitiate Dr. Siegler's objection relating to the type of light used. It is true that we examined the test animals' ovaries under a battery of fluorescent lights instead of the special lamp used by Farris. Nevertheless, when animals showed a positi,-e response, as did those injected with pregnancy urine, we had no difficulty in recognizing such a response. It seems reasonable to asHume that we eould as readily recognize a positive response caused by urine from an on1lntor~· woman. 470
Volume 59 r\umber 2
CORRESPONDENCE
471
Dr. Siegler also attempts to make the point that our results are better than >YC claim. He suggests that the ovulation of subject A. B. was in actuality predicted by the hyperemia reaction while in subject B. P. the lack of tests on days 13 and 15 makes interpretation impossible. Dr. Siegler's point is correct only if there has been a further revision of Farris' criteria of a "positive" ovulatory response. Origina1Iy2 it was stated that a positive hyperemia response for at least three successive days is required to demonstrate a "normal" ovulation. Later, apparently on the basis of additional experience, Farrisa stated that four successive days of positive hyperemia are required to indicate a ''normal'' ovulatory pattern. In line with this latest requirement, neither subject A. B. nor B. P. showed a normal ovulatory pattern. Subject A. B. showed three days of doubtful positive reaction followed by one day of full positive reaction. A normal ovulation could not be diagnosed for B. P. even if positive responses had been obtained on days 13 and 15, for even in this event there would have been only two successive days of positive response, two days less than l!'arris' latest requirement. The response obtained from A. S. cannot be debated. It is true that a positive response was not obtained nor was any indication of ovulation found at laparotomy. 'l'he other four of our subjects were gynecologically normal. Although laparotomy was not done to examine the ovaries of these subjects, the basal body temperature curves showed the changes which many investigators now accept as indicative of ovulation. As suggested in our report, we also consider "the only certain means of proof is by appropriately timed laparotomy with subsequent microscopic examination of the ovaries.'' By the same token (and this we attempted to indicate in our original paper) Farris has not yet proved his point. Therefore his test still ''must remain as an interesting but unproved suggestion.'' LOLlS LEVIN, PH.D. C. L. BuxToN, M.D. E. T. ENGLE, Pu.D. NEW YORK.
NovEMBER 30, 1949.
References 1. Levin, L., Buxton, C. L., and Engle, E. T.: AM. J. 0BST. & GYXEC. 58: 795, 194fl. 2. Farris, E. J.: AM. J. 0BST. & GYNEC. 52: 14,1946. :3. liurphy, D.P., and Farris, E. J.: AM. J. OBST. & GYKEC. 54: 467, lD-!i.
Reply by Dr. Farris To the Editor: In the October, 1949, issue of this JoURNAL, an article by Louis Levin, C. L. Buxton, and Earl Engle appeared, which questioned the value of the hyperemia method for the determination of ovulation time. 'L'he technique recommended by me for ovulation timing was said by Levin, Buxton, and Engle to have been followed by them. In my early experiments, after consultation with General Electric Co. lighting engineers, it was decided that the specially selected fluorescent lights chosen for daylight color were unsatisfactory for reading hyperemia in the rats' ovaries. In our work, we use the Macbeth light No. ADP 20. This has proved to be quite satisfactory for reading of different shades of the same color. Since Levin and associates used fluorescent lights, and not a Macbeth light, their technique differed in this respect from mine. The essayists did not state whether they used the Wistar strain of rats. In our early testing we learned that other strains are usually less sensitive than the Wistar rat for this type of testing. This has been confirmed several times by other workers who were furnished our rats for testing of ovulation time, when the other strains employed failed to giye consistent reactions.