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Correlation between histologic dating of human corpus luteum and the luteinizing hormone peak-biopsy interval
Correlation between histologic dating of human corpus luteum and the luteinizing hormone peak-biopsy interval Hl?CTOR-D.
CROXATTO,
MARIA-ELENA
ORTIZ,
HORACIO Santiago,
M.D. B.S.
B. CROXATTO,
M.D.
Chile
The purpose of this study was to assess the accuracy of retrospective dating of ovulation in women based on the histologic dating of the corpus luteum. Corpora lutea enucleated from the ovary of 39 women between one and six days following the LH peak in plasma were examined by routine histologic techniques and dated according to Corner’s criteria. The correlation between the luteinizing hormone (LH) peak-biopsy interval and histologic dating was assessed. Linear regression analysis of the data gave a correlation coefficient of 0.76. However, the dating of different corpora lutea obtained at the same LH peak-biopsy interval can differ by as much as four days. In addition, it was found that the stages named by Corner as days 1 and 2 seem to develop within the first 24 hdurs following the LH peak, while stages corresponding to days 4 and 5 each take two days to develop. It is concluded that the use of corpus luteum morphologic features for retrospective timing of ovulation is subject to an error of variable magnitude due to unequal duration of each stage, as well as considerable individual variation. (AM. J. 06s~~. GYNEOOL. 136:667, 1960.)
1956, Corner’ described a characteristic sequence of day-to-day histologic changes that can be used to establish morphologically the age of the human corpus luteum of menstruation. Corpus luteum dating in Corner’s work was based on the endometrial dating,’ clinical history, and menstrual data of each patient. Reports on corpus luteum dating in relation to parameters other than clinical history and endometrial dating have been missing in the literature until recently. Lundy and associates3 correlated corpus luteum dating with the timing of luteinizing hormone (LH) peak in eight patients. They found good correlation between the LH peak-biopsy interval and corpus luteum dating, according to Corner’s criteria. However, only three of the IN
eight patients were in the first week following the LH peak, a period in which the corpus luteum is undergoing rapid morphologic changes. Corpus luteum dating acording to Corner’s criteria has been used in several studies for retrospective dating of ovulation. 4-E Therefore, it seemed important to examine in greater depth the correlation between morphologic and hormonal parameters as they relate to the age of corpora lutea, in an attempt to validate such an approach. In this report, we present the correlation between corpus luteum dating according to Corner’s criteria with the LH peak-biopsy interval in 39 women within the first six days after the peak.
Material and methods From the Departamento de Anatomia Patol@ica, Facultad de Medic&, and thP LaboratMio de Endocrinoiogia, In&it&o de Ciencius BioQicas, Pontijicia Universidad Catdlica de Chile. Supported by World Health Organization Special Programme of Research, Development, and Research Training in Human Reproduction. Received for publication Accepted April
January 22, 1979.
23, 1979.
requests: Dr. Hector-D. Croxatto, Pontijicia Vniversidad Catolica de Chile, Institute a’e Cinecias Biologicas, Laboratorio de Endocrinologia, Casilla I1 4-D, Santiago, Chile.
Reprint
OOOZ-9378/80/050667+04$00.40/0
Subjects invited to participate in the study were selected among women who had their request for surgical sterilization approved by the Council of Family Planning of the J. J. Aguirre Hospital.* They were normal, healthy women menstruating regularly who had not used hormonal contraceptives for at least two cycles prior to the study. Those using an IUD had the
@ 1980
The
C. V. Mosby
Co.
*The investigators from the Pontificia Universidad Catblica de Chile did not participate in the selection or in the clinical and surgical management of patients undergoing surgical sterilization. 667
668
Croxatto.
Ortiz,
and Croxatto
March 1, 1980 Am. J. Obstet. Gynecol.
Table I. Distribution of cases according interval between LH peak and surgery
to the
ivo. of corpora lutea 24 48 72 96 120 144
3 5 13 8 6 4
Radioimmunoassay of LH was done by the doubleantibody technique.i Immunoreactive estrogens were measured as described by Edqvist and Johansson,R deleting the chromatographic step to separate estrone and estradiol. Progesterone was measured as described b!- Thornevcroft and Stone.’ Assay sensitivity, coefficients of variation, and antisera used in these RIA have been previously reported in detail.”
Results 1.
,,,,,,,.,, -6
l
-2 Days
Fig. I. Mean plasm LH. esrrogen. trarion in 39 ovulatory nrensrl-ual
0 from
+2 LH peak
and progesterone cycles. Vertical
porn-standard el-ror of rhe mean; number ntimber of samples iit each point.
+4
concen-
lines at each in parenthesis-
de\-it c rcmtrl c,tl at the time of’their last menses preceding \llt-ger\ Yone was lactating. l’rior to urger!, blood samples NYT~ drawn dail! beginning on clay 10 01. the cycle. fi)r determination of’ estrogen, LIi. and progesterone hp radioimmunoassay. Estrogen assa\ was done on the same day the sample N;LS tnken. 011 the basis of cervical mucus changes and p~eovula~r~estrogen peak. surgery was aimed at the da\ of ovulatiotl or any of the following five da>s. Al the time of thtl operation. the ovaries were inspected to ccrnllrm th;ti ovulation had occurred. The corpur luteum ~a& tnuclrated, sliced in half from the stigmata to thr hottonI, and both pic,c:es were immediately immerscri it, tormalin 10% fixative and processed fi)r IoutincL herll;ltox~lill-eosin stain two days latrl-. In each se< tion it was possible to examine the corpus lutcum from the stigmata to the base. Dating was done according to the findings of the most advanced area of rlrvelopmen t , usually toward the bottom and awa\ from the stigmata. Each specimen was dated according to Corner’s criteria by the same pathologist (H. D. Cl.) before he had access to the rest of the clinical and laborator!. data.
The number of corpora lutea examined at each interval after the LH peak is presented in Table I. All subjects were operated ott between one and six days following the LH peak with a greater proportion of cases being in the middle of this period. Difficulties in identifying an estrogen peak on the same day it occurs, as well as occupancy of surgical wards, precluded the inclusion of more subjects in the shorter intervals. In several instances, recovery of the ovum from the fallopian tube was to be attempted as part of another stud) and this also helped to bias the distribution of cases. Fig. 1 shows the mean plasma LH, estrogen, and progesterone concentrations of the 39 ovulatory menstrual cycles studied. Fig. 2 illustrates different stages of corpus luteum development. The correlation of corpus luteum dating with the LH peak-biopsy interval is presented in Fig. 3. A fitted curve obtained by leastsquares regression analysis gave an almost straight line intersecting the y axis on corpus luteum day 2. The correlation coefficient was 0.76. The three corpora lutea obtainted 24 hours following the LH peak were classified as corpus luteum day 2. Each stage of development was more frequently encountered in a given interval following the peak, but the same stage was also found in two, three, or four different intervals. Conversely, at each interval after the LH peak, one to four different stages of corpus luteum development were represented.
Comment In the present study, the LH peak determination was based on daily sampling; therefore, the margin of err01
Volume Number
Corpus luteum dating and LH peak-biopsy
136 5
interval
669
Fig. 2. Different stages of corpus luteum development. A, Theta and granulosa with radial arrangement of cells and incipient vascularization; corpus luteum day 2. (Hematoxylin and eosin. X200.)B, Granulosa layer with capillary vessels reaching the central cavity; corpus luteum day 3. (Hematoxylin and eosin. X 200.) C, Theta and granulosa with complete vascularization and advanced luteinization: corpus luteum day 4. (Hematoxylin and eosin. x80.) D, Partial involution of the theta and complete luteinization of the granulosa; vascularization and initial organization of the cavity: corpus luteum day 5. (Hematoxylin and eosin. x200.) in the estimation of time zero in individual cases is 2 12 hours. On the other hand, differences in the estimation of corpus luteum dating when several pathologists examine the same sample rarely exceed c 1 day. Therefore, dating or timing differences exceeding 60 hours among a group of corpora lutea obtained in the same interval or given the same dating, respectively, should be accounted for by individual variation. This was the case with specimens obtained 72 hours following the LH peak and with those dated day 4 or day 5. The greater variability observed in these three groups is probably a reflection of the larger number of specimens examined in these categories.
In spite of methodologic errors and individual variation, the morphologic changes described by Corner correlate well with time elapsed since the ovulatory stimulus. However, the present data suggest that the developmental stages named by Corner as day 1 and day 2 seem to occur within the first 24 hours which follow maximum levels of LH in plasma. On the other hand, progression from day 2 to day 3 takes only one day, but each subsequent stage up to day 6 takes two days to develop. The time interval between the ovulatory surge of LH
and follicular rupture is fairly constant in mammalsin which
this has been established.
In the human,
the av-
670
Croxatto, Ortiz, and Croxatto
24
4’e Hours
March I, 1980 Am. J. Obster. Gyd.
7.2 following
96 LH
li0
I&
peak.
Fig. 3. Correlation between histologic dating of corpus luteum and the LH peak-biopsy interval. Each point represents and individual case. erage interval is around 17 hours when the point of’ maximal circulating LH levels is taken as time zero.“-‘” However, becimse of the scarcity of data, the variability of- this interval among women remains to be established. The onset of luteinization in relation to the LH peak or to ovulation is even less well documented. A significant rise in plasma progesterone has been shown to
occur concomitantly with the LH peak.“’ This could he taken as a sign of luteinization taking place before follicular rupture. However, it has not been shown unequivocally that the preovulatory progesterone rise is associated with morphologic changes of the follicular wall corresponding to the early stages of luteinization. Moreover, other ovarian compartments can be the source of preovulatory progesterone.‘“. ” From the above discussion it appears that, presently, the LH peak is the most reliable single criterion to date ovulation in the human, more so when it is clearly preceded by an estrogen peak and followed by a sustained progesterone rise. We conclude that the sequence described by Corner is in agreement with an independent chronologic criterion such as hours following the LH peak, but the duration of each stage does not necessarily correspond to 24 hour periods. The use of corpus luteum morphologic age for the retrospective timing of ovulation is subjected to an error of variable magnitude, which is a function of unequal duration of each stage and considerable individual variation. We would like to thank Drs. J. Balmaceda and R. Hess and Mrs. M. Delgado, C. Llados, E. Nuhez, and A. Brandeis for their contributions to various phases of this work.
REFERENCES
1. Corner, G. W., Jr.: Am. J. Anat. 98:377, 1956. 2. Noyes, R. U’., Hertig. A. T.. and Rock, J.: Fertil. Steril. 1:3. 1950. 3. Lundy, E. L.., Lee, S. G.. Levy, W., Woodruff, I. D.. Wu. C., and Abdada, M.: Obstet. Gynecol. 44~14, 1974. 4. Yussman, M. A., and Taymor. M. L.: J. Clin. Endocrinol. 30:396, 1970. 5. Croxatto, H. B.: In Coutinho, E. M., and Fuchs, F.. editors: Physiology and genetics of reproduction, New York, 1974, Plenum Publishing Corporation, p. 159. 6. Pauerstein, C. J., Eddy, C. A., Croxatto, H-D., Hess, R., Siler-Khodr, T. M., and Croxatto, H. B.: AM. J. OBSTET. GYNECOL. 130~876, 1978. 7. Midgley, A. R., Jr.: Endocrinology 79:10, 1966. 8. Ed vist, L. E.. and Johansson, E. D. B.: Acta Endocrinol. (KbY, .) 71:716. 1972. 9. Thorneycroft. J. H.. and Stone, S. C.: Contraception 5:129, 1972. 10. Croxatto, H. B., Ortiz, M. E.. Diaz, S., Hess, R., Bal-
maceda, J., and Croxatto, H.-D.: AM. J. OBSTET. GYNE1X2:629, 1978. 11. Ferin, J., Thomas, K., and Bonte, J.: In Hafez, E. S. E., and Evans T. N., editors: Human reproduction, conception and contraception, Hagerstown, 1973, Harper & Row, Publishers, p. 267. 2. Croxatto, H. B., Carril, M., Cheviakoff, S., Patriti. N., Pedroza, E., Croxatto. H-D., Gbmez-Rogers, C., and Rosner, J.: In Ebling, F. J. G., and Anderson, I, W., editors: Biological and clinical aspects of reproduction. Amsterdam, 1976, Excerpta Medica, p. 282. 13. Schmidt-Gollwitzer, K., Schmidt-Gollwitzer, M., Scakmann, U.. and Eilets, J.: Int. J. Fertil. 22:232, 1977. 14. Johansson, E. D. B.. and Wide. L.: Acta Endocrinol. 62:82, 1969. 1.5. Leavitt, W. W.. Bosley, C. G., and Blaha, G. C.: Nature 234:283, 1971. 16. Resko, J. A., Koering, M. J., Coy, B. W.. and Phoenix, C. H. J. Clin. Endocrinol. 41:120, 1975. COL.
CROXATTO,
MARIA-ELENA
ORTIZ,
HORACIO Santiago,
M.D. B.S.
B. CROXATTO,
M.D.
Chile
The purpose of this study was to assess the accuracy of retrospective dating of ovulation in women based on the histologic dating of the corpus luteum. Corpora lutea enucleated from the ovary of 39 women between one and six days following the LH peak in plasma were examined by routine histologic techniques and dated according to Corner’s criteria. The correlation between the luteinizing hormone (LH) peak-biopsy interval and histologic dating was assessed. Linear regression analysis of the data gave a correlation coefficient of 0.76. However, the dating of different corpora lutea obtained at the same LH peak-biopsy interval can differ by as much as four days. In addition, it was found that the stages named by Corner as days 1 and 2 seem to develop within the first 24 hdurs following the LH peak, while stages corresponding to days 4 and 5 each take two days to develop. It is concluded that the use of corpus luteum morphologic features for retrospective timing of ovulation is subject to an error of variable magnitude due to unequal duration of each stage, as well as considerable individual variation. (AM. J. 06s~~. GYNEOOL. 136:667, 1960.)
1956, Corner’ described a characteristic sequence of day-to-day histologic changes that can be used to establish morphologically the age of the human corpus luteum of menstruation. Corpus luteum dating in Corner’s work was based on the endometrial dating,’ clinical history, and menstrual data of each patient. Reports on corpus luteum dating in relation to parameters other than clinical history and endometrial dating have been missing in the literature until recently. Lundy and associates3 correlated corpus luteum dating with the timing of luteinizing hormone (LH) peak in eight patients. They found good correlation between the LH peak-biopsy interval and corpus luteum dating, according to Corner’s criteria. However, only three of the IN
eight patients were in the first week following the LH peak, a period in which the corpus luteum is undergoing rapid morphologic changes. Corpus luteum dating acording to Corner’s criteria has been used in several studies for retrospective dating of ovulation. 4-E Therefore, it seemed important to examine in greater depth the correlation between morphologic and hormonal parameters as they relate to the age of corpora lutea, in an attempt to validate such an approach. In this report, we present the correlation between corpus luteum dating according to Corner’s criteria with the LH peak-biopsy interval in 39 women within the first six days after the peak.
Material and methods From the Departamento de Anatomia Patol@ica, Facultad de Medic&, and thP LaboratMio de Endocrinoiogia, In&it&o de Ciencius BioQicas, Pontijicia Universidad Catdlica de Chile. Supported by World Health Organization Special Programme of Research, Development, and Research Training in Human Reproduction. Received for publication Accepted April
January 22, 1979.
23, 1979.
requests: Dr. Hector-D. Croxatto, Pontijicia Vniversidad Catolica de Chile, Institute a’e Cinecias Biologicas, Laboratorio de Endocrinologia, Casilla I1 4-D, Santiago, Chile.
Reprint
OOOZ-9378/80/050667+04$00.40/0
Subjects invited to participate in the study were selected among women who had their request for surgical sterilization approved by the Council of Family Planning of the J. J. Aguirre Hospital.* They were normal, healthy women menstruating regularly who had not used hormonal contraceptives for at least two cycles prior to the study. Those using an IUD had the
@ 1980
The
C. V. Mosby
Co.
*The investigators from the Pontificia Universidad Catblica de Chile did not participate in the selection or in the clinical and surgical management of patients undergoing surgical sterilization. 667
668
Croxatto.
Ortiz,
and Croxatto
March 1, 1980 Am. J. Obstet. Gynecol.
Table I. Distribution of cases according interval between LH peak and surgery
to the
ivo. of corpora lutea 24 48 72 96 120 144
3 5 13 8 6 4
Radioimmunoassay of LH was done by the doubleantibody technique.i Immunoreactive estrogens were measured as described by Edqvist and Johansson,R deleting the chromatographic step to separate estrone and estradiol. Progesterone was measured as described b!- Thornevcroft and Stone.’ Assay sensitivity, coefficients of variation, and antisera used in these RIA have been previously reported in detail.”
Results 1.
,,,,,,,.,, -6
l
-2 Days
Fig. I. Mean plasm LH. esrrogen. trarion in 39 ovulatory nrensrl-ual
0 from
+2 LH peak
and progesterone cycles. Vertical
porn-standard el-ror of rhe mean; number ntimber of samples iit each point.
+4
concen-
lines at each in parenthesis-
de\-it c rcmtrl c,tl at the time of’their last menses preceding \llt-ger\ Yone was lactating. l’rior to urger!, blood samples NYT~ drawn dail! beginning on clay 10 01. the cycle. fi)r determination of’ estrogen, LIi. and progesterone hp radioimmunoassay. Estrogen assa\ was done on the same day the sample N;LS tnken. 011 the basis of cervical mucus changes and p~eovula~r~estrogen peak. surgery was aimed at the da\ of ovulatiotl or any of the following five da>s. Al the time of thtl operation. the ovaries were inspected to ccrnllrm th;ti ovulation had occurred. The corpur luteum ~a& tnuclrated, sliced in half from the stigmata to thr hottonI, and both pic,c:es were immediately immerscri it, tormalin 10% fixative and processed fi)r IoutincL herll;ltox~lill-eosin stain two days latrl-. In each se< tion it was possible to examine the corpus lutcum from the stigmata to the base. Dating was done according to the findings of the most advanced area of rlrvelopmen t , usually toward the bottom and awa\ from the stigmata. Each specimen was dated according to Corner’s criteria by the same pathologist (H. D. Cl.) before he had access to the rest of the clinical and laborator!. data.
The number of corpora lutea examined at each interval after the LH peak is presented in Table I. All subjects were operated ott between one and six days following the LH peak with a greater proportion of cases being in the middle of this period. Difficulties in identifying an estrogen peak on the same day it occurs, as well as occupancy of surgical wards, precluded the inclusion of more subjects in the shorter intervals. In several instances, recovery of the ovum from the fallopian tube was to be attempted as part of another stud) and this also helped to bias the distribution of cases. Fig. 1 shows the mean plasma LH, estrogen, and progesterone concentrations of the 39 ovulatory menstrual cycles studied. Fig. 2 illustrates different stages of corpus luteum development. The correlation of corpus luteum dating with the LH peak-biopsy interval is presented in Fig. 3. A fitted curve obtained by leastsquares regression analysis gave an almost straight line intersecting the y axis on corpus luteum day 2. The correlation coefficient was 0.76. The three corpora lutea obtainted 24 hours following the LH peak were classified as corpus luteum day 2. Each stage of development was more frequently encountered in a given interval following the peak, but the same stage was also found in two, three, or four different intervals. Conversely, at each interval after the LH peak, one to four different stages of corpus luteum development were represented.
Comment In the present study, the LH peak determination was based on daily sampling; therefore, the margin of err01
Volume Number
Corpus luteum dating and LH peak-biopsy
136 5
interval
669
Fig. 2. Different stages of corpus luteum development. A, Theta and granulosa with radial arrangement of cells and incipient vascularization; corpus luteum day 2. (Hematoxylin and eosin. X200.)B, Granulosa layer with capillary vessels reaching the central cavity; corpus luteum day 3. (Hematoxylin and eosin. X 200.) C, Theta and granulosa with complete vascularization and advanced luteinization: corpus luteum day 4. (Hematoxylin and eosin. x80.) D, Partial involution of the theta and complete luteinization of the granulosa; vascularization and initial organization of the cavity: corpus luteum day 5. (Hematoxylin and eosin. x200.) in the estimation of time zero in individual cases is 2 12 hours. On the other hand, differences in the estimation of corpus luteum dating when several pathologists examine the same sample rarely exceed c 1 day. Therefore, dating or timing differences exceeding 60 hours among a group of corpora lutea obtained in the same interval or given the same dating, respectively, should be accounted for by individual variation. This was the case with specimens obtained 72 hours following the LH peak and with those dated day 4 or day 5. The greater variability observed in these three groups is probably a reflection of the larger number of specimens examined in these categories.
In spite of methodologic errors and individual variation, the morphologic changes described by Corner correlate well with time elapsed since the ovulatory stimulus. However, the present data suggest that the developmental stages named by Corner as day 1 and day 2 seem to occur within the first 24 hours which follow maximum levels of LH in plasma. On the other hand, progression from day 2 to day 3 takes only one day, but each subsequent stage up to day 6 takes two days to develop. The time interval between the ovulatory surge of LH
and follicular rupture is fairly constant in mammalsin which
this has been established.
In the human,
the av-
670
Croxatto, Ortiz, and Croxatto
24
4’e Hours
March I, 1980 Am. J. Obster. Gyd.
7.2 following
96 LH
li0
I&
peak.
Fig. 3. Correlation between histologic dating of corpus luteum and the LH peak-biopsy interval. Each point represents and individual case. erage interval is around 17 hours when the point of’ maximal circulating LH levels is taken as time zero.“-‘” However, becimse of the scarcity of data, the variability of- this interval among women remains to be established. The onset of luteinization in relation to the LH peak or to ovulation is even less well documented. A significant rise in plasma progesterone has been shown to
occur concomitantly with the LH peak.“’ This could he taken as a sign of luteinization taking place before follicular rupture. However, it has not been shown unequivocally that the preovulatory progesterone rise is associated with morphologic changes of the follicular wall corresponding to the early stages of luteinization. Moreover, other ovarian compartments can be the source of preovulatory progesterone.‘“. ” From the above discussion it appears that, presently, the LH peak is the most reliable single criterion to date ovulation in the human, more so when it is clearly preceded by an estrogen peak and followed by a sustained progesterone rise. We conclude that the sequence described by Corner is in agreement with an independent chronologic criterion such as hours following the LH peak, but the duration of each stage does not necessarily correspond to 24 hour periods. The use of corpus luteum morphologic age for the retrospective timing of ovulation is subjected to an error of variable magnitude, which is a function of unequal duration of each stage and considerable individual variation. We would like to thank Drs. J. Balmaceda and R. Hess and Mrs. M. Delgado, C. Llados, E. Nuhez, and A. Brandeis for their contributions to various phases of this work.
REFERENCES
1. Corner, G. W., Jr.: Am. J. Anat. 98:377, 1956. 2. Noyes, R. U’., Hertig. A. T.. and Rock, J.: Fertil. Steril. 1:3. 1950. 3. Lundy, E. L.., Lee, S. G.. Levy, W., Woodruff, I. D.. Wu. C., and Abdada, M.: Obstet. Gynecol. 44~14, 1974. 4. Yussman, M. A., and Taymor. M. L.: J. Clin. Endocrinol. 30:396, 1970. 5. Croxatto, H. B.: In Coutinho, E. M., and Fuchs, F.. editors: Physiology and genetics of reproduction, New York, 1974, Plenum Publishing Corporation, p. 159. 6. Pauerstein, C. J., Eddy, C. A., Croxatto, H-D., Hess, R., Siler-Khodr, T. M., and Croxatto, H. B.: AM. J. OBSTET. GYNECOL. 130~876, 1978. 7. Midgley, A. R., Jr.: Endocrinology 79:10, 1966. 8. Ed vist, L. E.. and Johansson, E. D. B.: Acta Endocrinol. (KbY, .) 71:716. 1972. 9. Thorneycroft. J. H.. and Stone, S. C.: Contraception 5:129, 1972. 10. Croxatto, H. B., Ortiz, M. E.. Diaz, S., Hess, R., Bal-
maceda, J., and Croxatto, H.-D.: AM. J. OBSTET. GYNE1X2:629, 1978. 11. Ferin, J., Thomas, K., and Bonte, J.: In Hafez, E. S. E., and Evans T. N., editors: Human reproduction, conception and contraception, Hagerstown, 1973, Harper & Row, Publishers, p. 267. 2. Croxatto, H. B., Carril, M., Cheviakoff, S., Patriti. N., Pedroza, E., Croxatto. H-D., Gbmez-Rogers, C., and Rosner, J.: In Ebling, F. J. G., and Anderson, I, W., editors: Biological and clinical aspects of reproduction. Amsterdam, 1976, Excerpta Medica, p. 282. 13. Schmidt-Gollwitzer, K., Schmidt-Gollwitzer, M., Scakmann, U.. and Eilets, J.: Int. J. Fertil. 22:232, 1977. 14. Johansson, E. D. B.. and Wide. L.: Acta Endocrinol. 62:82, 1969. 1.5. Leavitt, W. W.. Bosley, C. G., and Blaha, G. C.: Nature 234:283, 1971. 16. Resko, J. A., Koering, M. J., Coy, B. W.. and Phoenix, C. H. J. Clin. Endocrinol. 41:120, 1975. COL.