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Effects of progesterone and norethindrone on experimental endometriosis in monkeys
Effects of progesterone and norethindrone on experimental endometriosis in monkeys ROGER Cleveland,
B.
LAWRENCE Baltimore,
SCOTT,
M.D.
Ohio R.
WHARTON,
JR.,
M.D.
Maryland
E x T E R N A L endometriosis, the disease entity due to the presence of endometrial tissue outside the uterus or invading the uterus from the serosal surface inward, is generally asymptomatic during pregnancy and seldom increases in size or has a return of symptoms until 2 to 5 years following a pregnancy. Therefore, pregnancy, if it can be achieved, is considered an excellent form of therapy for this common genital disease of women. Cyclic menses, involving constant estrogen and intermittent progesterone, when allowed to persist uninterrupted by preg-
nancy, predispose to the development and growth of endometriosis. This is generally explained on the basis of the more frequent chances of retrograde tubal flow of viable fragments of shed endometrium, capable of implanting, plus the local growth of areas of endometriosis cyclically subject to the same hormonal influences as the normally located endometrium. During pregnancy the levels of circulating estrogen and progesterone tend to increase and there is no intermittent withdrawal of progesterone to permit the uterine endometrium to shed and disseminate or the existing endometriosis to grow and infiltrate. The softening and lack of hemorrhage in preexisting endometriosis during pregnancy make the absence of symptoms understandable. The absence of symptoms for a period of time following pregnancy is less easily explained. Theoretically this has been attributed to either necrosis and atrophy or fibrosis of the decidual stromal tissue of the ectopic endometrium. In order to mimic the benefits of pregnancy various techniques of hormone administration have been suggested and tried on a clinical basis. Increasing doses of an estrogen, such as the synthetic one, diethylstilbestrol, were advocated; if used properly to produce amenorrhea and inhibit ovulation, menses would be absent and the ectopic endometrium would not show periodic hemorrhage. However, decidual transformation of the stroma and other effects secondary to
From the Department of Obstetricsand Gynecology, Western Reserve School of Medicine and University Hospitals of Cleveland, and the Department of Gynecology and Obstetrics, Johns Hopkins School of Medicine, and Johns Hopkins Hospital. This work was supported by United States Public Health Service, National Institutes of Health, Research Grant C-2892 (C 4 and 5). Some financial assistance for the purchase of a few of the animals, as well as the estrone, vaginal 50 mg. progesterone tablets (Colprosterone), and Go-fluoro17-a-brom progesterone (AY-59034) were provided by Ayerst Laboratories, New York, through the courtesy of the Medical Director, Dr. John B. Jewell. The I7o-ethinyl-19-nortestosterone or norethindrone (No&tin) for intramuscular i‘njection Iuas generously supplied by Parke, Davis B Co., Detroit, Michigan. Guest Speaker’s Address, presented at the Twenty-fourth Annual Meeting of the South Atlantic Association of Obstetricians and Gynecologists, Bal Harbor, Florida, Jan. 28-31, 1962. 867
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progesterone would not occur, negating this method as one mimicking pregnancy. The difficulty of administering increasing amounts of effective progesterone, together with its cost and rapid disappearance from the bloodstream and tissues, makes the use of injected progesterone prohibitive. Formerly, the oral progesterone derivatives were poorly or variably absorbed and therefore were not tested on a clinical basis in endometriosis. now Progesterone-like substances have been marketed. Although expensive, most of them can be administered orally, the dose can be increased as desired, and the end organ response is similar, although not identical, to injected progesterone. The endometrial stromal change is more marked than the glandular one. Clinical trials of “pseudopregnancy” are now in progress with these synthetic progestins. The most commonly used preparations are 19-norethynodrel with 1.5 per cent of ethinylestradiol-3-methyl ether (Enovid), norethisterone or norethindrone (Norlutin) , and 17a-hydroxyprogesterone caproate (Delalutin) . Clinical reports to this time, using long-term administration with initial increasing amounts for 4 to 12 months, have been reported with considerable enthusiasm.‘-4, 6, lo Rhesus (Macaca mulatta) monkeys are cyclically menstruating primates with patent uterotubal junctions and a menstrual and reproductive physiology similar to that of the human female. In addition, at least 5 instances of spontaneous endometriosis have been encountered in these animals. Autologous transplants of surgically excised endometrial tissue are highly successful in these animals and they then offer a superior laboratory mechanism for testing the effect of various hormones on endometriosis. The authors have previously reported their experiences with the effects of diethylstilbestrol,’ constant estrone, constant estrone and constant progesterone, and constant estrone and intermittent progesterone9 on the experimentally produced endometriosis in the animals. In the present experiments the effects of constant progesterone and continuous, although increasing, amount of norethindrone
will be studied. The difficulties of oral administration and the uncertainty of dosage levels by this route did not permit 11s to use norethynodrel. Norethindrone for intramuscular use allowed accurate control of the amount of drug given to each animal. Materials
and
methods
Progesterone experiments. Four
adult female rhesus monkeys (Nos. 901, 987, 988, and 990) were used. One animal (No. 901) had been used previously for experimental study; for 2 years it received intraperitoneal injections of peripheral venous blood at the time of menses and for another 2 years it was given twice weekly estrone injections to test the effect on autologous endometrial transplants. All animals had surgical transplants of endometrial tissue, secured by hysterotomy, to four areas---one ovary, tht serosal surface of the uterus, the rectosigmoid serosal surface, and the anterior abdominal wall beneath the rectus fascia. Prior to the injections of progesterone, the animals were re-explored in order to assure by gross inspection and biopsy that at least one transplant had viable endometriosis. Progesterone in oil, approximately 6 mg. CO.25 cc. of a solution in oil containing 25 mg. per cubic centimeter), was given intramuscularly 6 days each week for 31, 28, 19. and 12j/2 months, respectively. Surgical explorations with biopsies were done on these animals during the course of treatment and at the termination-the two shortest periods of administration were terminated by death of the animals, one by tuberculosis and one by freezing. Norethindrone experiments. Seven adult female rhesus monkeys were used (Nos. 839, 901, 949, 963, 964, 965, and 988). Monkey 839 was transferred to our colony from the regular Carnegie Institution of Washington, Department of Embryology colony when it was found to have a 4 by 3 cm. pelvic endometrial cyst several years after a hysterotomy during pregnancy. This animal was used for a 3 year study of the effect of testosterone prior to this present testing and a baseline biopsy of the cyst wall was taken
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Fig. 1. Monkey 901. Biopsy of endomctrial cyst wall after 31 months of progesterone in oil injections, 6 mg. each day for 6 days of each week. Note the closely packed glands with an atypical hyperplasia of the epithelium and the heavy inflammatory cell infiltration. (X70.)
one month after testosterone was stopped. Monkey 901 had been used for the intraperitoneal blood injections and estrone injections as mentioned above. Monkey 949 had been castrated, endometrium transplanted to the 4 areas noted above and used to test the effect of constant estrone and intermitent progesterone on these transplants. Monkey 963 had been previously castrated at the time of autologous endometrial transplantation and then used to study the effect of constant estrone and constant progesterone. Monkey 964 had been used in an unsuccessful attempt to shift the vagina into a gluteal region, following which endometrium was surgically transplanted to the areas mentioned and testosterone administered for 34f/’ months. Monkey 965 similar to Monkey 964 had experiments except the testosterone was administered for only 17 months. Monkey 988 was used previously for the progesterone study outlined above. Each of the animals had been proved by gross and microscopic examination to have at least one area of viable endometriosis before the present experiments were begun. Norethindrone was administered intramuscularly at a level of 5 to 10 mg. (1 C.C. equals 50 mg.) two to four times each week? increasing the dose over a 2 to 3 month interval in order that a maintenance dose of
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40 to 50 mg. each week was reached. The drug was given for a total period ranging from 5ya to 7 months. Monkeys 949 and 963 were castrates; therefore they received aqueous estrone, 0.25 mg. (1 C.C. equals 1 mg.), twice weekly prior to and during the use of norethindrone. Also these 2 animals were maintained for 6 months after discontinuation of norethindrone and estrone on estradiol valerate in sesame oil (Delestrogen; 1 C.C. equals 10 mg.), 2.5 mg. at the beginning of each interval of 4 weeks, and 17~hydroxyprogesterone caproate in sesame oil ( 1 C.C. equals 125 mg. ), 62.5 mg. at the midpoint of each interval of 4 weeks. All of the animals, except Nos. 964 and 965, were tested for vaginal bleeding during and following treatment. After 2 to 3 months and again at the end of treatment with norethindrone surgical explorations were done and selected biopsies of the endometriosis areas and on a few occasions endometrial biopsies by hysterotomy were taken. Five of the monkeys were again re-explored or autopsied 6 to 7 months after stoppage of norethindrone; 2 of the 7 monkeys died whiIe undergoing the test (61/z and 7 months after the initial injection), one complicated by an abscess in a pelvic endometrial cyst and one of tuberculosis. Results
Progesterone experiments. During the period of constant progesterone administration the vaginal bleeding pattern was very bizarre, ranging from 1 to 4 days of bleeding every 1 to 7 weeks to almost constant daily spotting. Ovulation was not inhibited in 2 animals at least, for a corpus luteum was identified in each of these 12j/2 and 19 months after the first injection. The areas of experimental endometriosis grossly seemed to increase slightly in size and all showed evidence of fairly recent hemorrhage. Microscopically the endometriosis was relatively unaffected; old and recent hemorrhage was evident, there was moderate stromal edema and slight enlargement of the stromal cells: a secretory glandular pattern was found on only one occasion and there was no excess
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Fig. 2. Monkey 965. Biopsy of endomrtriosis in rertus muscle after 69: months ethindronr. A, Low-power (~70) and B, high-power ( ,150) views show the drcidual tion in the stroma and the flattenrd epithelium of the glandb.
Fig. 3. Monkey A, Low-power cell cytoplasmic
of
11urrc’ac’-
839. Biopsy of an endometrial cyst wall after 7 months of norethindrone. views show the marked edema, decidual (x70) and B, high-power (x450) and infrequent endometrial glands. and nuclear disintegration,
Fig. 4. Monkey 901. Biopsy of endometrial Low-power (x70) and B, high-power (x450) cell disintegration and even some hyalinization rarity of endometrial glands.
cyst wall after 7 months of norethindrone. A, views show the stromal changes with decidual and inflammatory cell infiltration. Note the
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of fibrotic reaction or replacement. One 3 cm. endometrial cyst remained filled with bloody liquid during 31 months of treatment, the lining was thick and succulent, and a suggestive adenomatous hyperplasia pattern was present in the cyst wall (Fig. 1). Posttreatment biopsies after more than one month were not possible in this group. In summary, constant, not increasing, doses of progesterone over 12y2 to 31 months produced some stromal edema and increase in size of the stromal cells of the experimental endometriosis, but hemorrhage within the endometriosis continued and no unusual amount of fibrous tissue reaction or replacement occurred. Norethindrone experiments. In the 5 of the 7 monkeys which could be properly tested during treatment there was no evidence of vaginal bleeding. Within 1 to 2 months after treatment cyclic vaginal bleeding recurred spontaneously or was produced physiologic by hormones in estimated amounts in the castrated monkeys. After 2 to 3 months and at the end of the treatment periods gross inspection and biopsies of the areas of endometriosis revealed the usual changes reported clinically with the use of these progesterone-like substances. There was decidual stromal reaction with progressive intracellular edema; the decidual cells then disintegrated, cytoplasmic ghosts and “naked,” irregularly shaped and stained nuclei appeared, the edematous stroma had a moderate inflammatory cell infiltration, and there was a strong tendency with this necrosis for hyalinized areas to appear (Figs. 2-4). Endometrial glands were sparse, inactive, and flattened, resembling endothelial lined spaces. The arterioles became thickwalled. Two large endometrial cysts persisted, but gross evidence of blood in the cystic locules disappeared and the cyst wall became thickened and edematous. One cyst was found as a frank abscess after 7 months of treatment. In all instances the uterus became quite soft, enlarged two to four times normal size, and, when opened, contained a thick edematous endometrium similar in microscopic changes to those seen in the
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5. Monkey 901. Endometrial cyst wall 6 months after discontinuing norethindrone. There is rather extensive subepithelial fibrosis with only scattered areas of active endometrial stroma. (x100.)
ectopic endometrium, but without hyaline zones. When the ovaries were biopsied during treatment no old or recent corpora lutea were found and the tunicas were thickened and slightly more fibrotic than normal. In the 5 monkeys studied by gross and microscopic means 6 to 7 months after norethindrone was discontinued, the areas of endometriosis were practically all identifiable grossly. Microscopically in 4 of the 5 animals there was a definite fibrous tissue reaction about the endometriosis, as well as a replacement of some apparent stromal areas with young fibrous tissue (Figs. 5-8), This reaction varied in degree from minimal to moderate, although in general the gross size of the nodules was the same or slightly larger than observed before treatment. In addition all areas of endometriosis followed through each phase showed abundant active endometrial stroma and numerous endometrial glands, even at times secretory. The uterus diminished to normal size with normal appearing endometrium and no fibrous tissue reaction or replacement. In summary during 5f/4 to 7 months of norethindrone administration in monkeys with experimental endometriosis, stromal
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Fig. 6. Monkey 963. bowel endomctriosis 6 norethindrone. Note and scattered awns of
Patchy fibrosis in sigmoid months after discontinuing active endometrial glands active stroma. fX2OD.1
decidual reaction with edema and necrosis occurred in the ectopic and the normally located endometrium. Hyalinization of some necrotic stromal area was found in onIy the ectopic endometrium. Vaginal bleeding and intracystic hemorrhage disappeared. Sis to 7 months after such treatment the clndometriosis was still present without diminution in size. with abundant and active stroma and glands, and usually with increased fibrous tissue reaction about the areas, as well as some stromal replacement by young fibrouq tissue. Cyclic vaginal bleeding recurred, the uterine endometrium was normal, and hemorrhage within an endometrial cyst again became evident. Comment
Fig. 7. Monkey 963. Stromal fibrosis about endometrial glands in rectus muscle endometriosis 6 months after discontinuing norethindrone. (xl On. ’
Fig. 8. Monkey 964, Rectus muscle endometriosis 7 months after discontinuation of norethindrone. Note zones of fibrosis in the stroma plus active and persisting stroma and glands. (x100.)
The experiments with constant progesterone were planned to test the effect of this hormone on endometriosis when used on a long-term basis. They were not calculated to mimic the hormonal influences of pregnancy, for to reproduce such influences the amount of progesterone should have been progressively increased to practically prohibitive amounts. As performed, this constant dosage of hormone had little effect on the esperimental endometriosis except there was uniform evidence of irregular, at times almost constant, vaginal bleeding and hemorrhage within the external endometriosis. This hemorrhage within areas of endometriosis is also encountered clinically when continuous and constant small amounts of the newer progesterone-like substances are ,given. From this knowledge there evolves a useful preoperative application which will be mentioned later. During the 6 to 7 months of norethindrone on an increasing dosage schedule the areas of experimental endometriosis showed the changes reported in human clinical studies. The stromal change was marked, progressing from frank decidual reaction to intercellular edema, cytoplasmic disintegration and vacuolization, bizarre nuclear disruption and staining, and frequently even hyaline replacement of the necrotic stromal areas. A moderate inflammatory cell infil-
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Fig. 9. Posterior cul-de-sac endometriosis after 8 weeks of preoperative treatment with vaginal progesterone suppositories, 50 mg. daily. Interstitial and intraluminal hemorrhages are extensive. (x40.)
Fig. 10. hemorrhage gesterone endometrial (X50.)
Ovarian endometriosis highlighted by after 5 weeks of daily vaginal prosuppositories prior to operation. The glands showed secretory activity.
tration was noted. One cystic locule filled with blood before treatment became one filled with clear fluid and another such cyst became abscessed; the walls of the cysts were swollen and edematous. The epithelium did not share in the changes and the endometrial glands were sparse and lined with flattened cells, so that the glands resembled endothelium-lined channels. The uteri increased to four times in size, were very soft, and the endometrium thick and edematous with similar stromal decidual changes, but no hyalinization. Five of the animals (i.e., all of those so tested) had no vaginal bleeding and when endometrial biopsies were taken there was no evidence of recent tissue hemorrhage. The ovaries had no old or recent corpora lutea at the end of the treatment period and the tunicas were thicker and more fibrotic than normal. It was possible to study 5 of the 7 animals 6 to 7 months following treatment. The areas of endometriosis were still grossly evident and blood had replaced the clear fluid in one cyst. In 4 of the 5 animals there was definite fibrous tissue replacement of some of the stroma, yet numerous areas of normal endometrial glands and stroma remained. One had the impression that the over-a11 size of the areas remained about the same as before treatment and despite the repeated
biopsies-the fibrous tissue replacement may have been in the areas of stromal change which had increased the actual mass during treatment. From these experiments it can be fairly stated that hormonal “pseudopregnancy” state was produced for 1.57 to 210 days, about equal to or longer than a monkey’s normal gestational period (160 days), and portions of the stroma of the external endometriosis were replaced by fibrous tissue. However, the gross size of the lesions did not diminish and they contained abundant healthy and active endometrial glands and stroma. In no instance was a cure by complete fibrosis obtained in any of the areas of endometriosis by the long-term administration of norethindrone. Kistner,2’ 3 A n d r e w s and associates,l Thomas,lO Lebherz and Fobes,4 and Riva and co-worker9 have reported their clinical experiences in the treatment of external endometriosis with these new progestins. The improvement by relief of symptoms attributed to the endometriosis varied from 77.6 to 85 per cent with one author stating “nearly all benefited.“lO In many of the cases the diagnosis was based on symptoms and clinical findings without histologic proof. Riva and his group relied heavily upon culdoscopic examination as confirmatory, a
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Fig. 11. Ovarian surface endometriosis after 5 weeks of preoperative daily treatment with a synthetic progestin (AY-59034). The stroma edema and decidual reaction grossly enlarged the area and the hemorrhage further assisted gross identification. (x40.)
reliance open to serious question. All of the authors were in general agreement that dosages must be stepped up to prevent breakthrough bleeding, supplemental or incorporated estrogens (as in norethynodrel) were helpful, and the best results were with long-term use, such as 5 to 9 months. It was Kistner’s impression that pregnancies occurred sooner following this treatment than the 2.7 years average following conservative surgical treatment in his hospital. Scott,7 in his discussion of the paper by Andrews, on the other hand, was worried about the frequency of posttreatment delay in resumption of normal ovulatory cycles. The side effects of the drugs, such as nausea, emotional distress, growth of fibroids, breast tenderness, fluid retention, and breakthrough bleeding were particularly stressed by Lebherz and Fobes4 and others.*’ 5, 6 Some of these side effects could be alleviated by antiemetics and diuretics, making a treatment schedule a complex one indeed. The long-term use of expensive medication, fraught with a high incidence of annoying side effects and which has no apparent curative value, should be undertaken only after very serious consideration. Ovulation is prevented during the period of treatment, and posttreatment relief of symptoms and subsequent pregnancy rate cannot
be properly evaluated on the limited data available. Logically the treatment program should be limited to patients with maximum symptoms and minimal findings of external endometriosis and to those with recurrent endometriosis following conservative surgical treatment. A useful clinical application can be derived from the knowledge that constant small amounts of progesterone or the synthetic progestins produce hemorrhage in the endometriosis as well as softening of the pelvic agglutinations. For about 5 to 8 weeks prior to contemplated conservative surgical treatment of endometroisis, small amounts of the synthetic progestins (10 to 20 mg. daily) or vaginal suppositories of progcsterone (50 mg. daily) have been used in about 20 patients. These patients usually have a chocolate vaginal discharge for the last 2 to 3 weeks of treatment. ,4t the time of operation the areas of endometriosis have hemorrhagic “blossoms” (Figs. 9-I 1) , thus making them more easily identifiable for excision or fulguration and thereby lessening the chances of missing small foci which might flourish subsequently. In addition, the softening of the usually dense adhesions has been remarkable and cleavage planes were more easily established. Our results with vaginal suppositories of progesterone were at times good but quite variable, since absorption by this route is uncertain. The 6~ fluoro-I 7cY-brom progesterone (AY-59034) was not as effective as the other progestins in producing the desired changes in the endometriosis. Summary
and
conclusions
1. Four adult female rhesus monkeys with experimentally produced areas of external endometriosis were given daily progesterone in oil injections for periods ranging from 12vZ to 31 months. The animals had very irregular vaginal bleeding, at times almost constant, and the areas of endometriosis all showed evidence of recent or old hemorrhage, but no posttreatment fibrosis. 2. Seven adult female rhesus monkeys with experimentally produced areas of en-
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dometriosis were given parenteral norethindrone in increasing amounts for 5f/2 to 7 months. Gross and histologic studies during and 7 months following treatment revealed no posttreatment reduction in the size of the areas, but there was evidence of a patchy stromal fibrosis. Despite this, abundant active glands and stroma were still present. 3. From these experiments there is no evidence that this new progestin “cures” endometriosis and the clinical use of these agents for external endometriosis should be considered a temporizing measure for special selected cases. 4. Five to 8 weeks of preoperative small
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amounts of progesterone or one of the newer progestins on a constant, not increasing, dose schedule have been found useful before planned conservative surgical treatment. Hemorrhages within the endometriosis make their identification and complete excision easier and in addition the edema and softening of the pelvic structures enhance the establishment of proper cleavage planes. We wish to express our appreciation to Dr. James D. Ebert for his cooperation in this study and for the use of the facilities at the Carnegie Institution of Washington, Department of EmbryoIogy, Baltimore, Maryland.
REFERENCES
1. Andrews, M. C., Andrews, W. C., and Strauss, A. F.: AX J. OBST. & GYNEC. 78: 776, 1959. 2. Kistner, R. W.: AM. J. OBST. & GYNEC. 75: 264, 1958. 3. Kistner, R. W.: Fertil. & Steril. 10: 539, 1959. 4. Lebherz, T. B., and Fobes, C. D.: AM’. J. OBST. & GYNEC. 81: 102, 1961. 5. Mixson, W. T., and Hammond, D. 0.: AM. J. OBST. & GYNEC. 82: 754, 1961.
6. Riva, H. L., Wilson, J. H., and Kawasaki, D. M.: AM. J. OBST. & GYNEC. 82: 109, 1961. 7. Scott, J. W.: In discussion of paper by Andrews, Andrews, and Strauss.1 8. Scott, R. B., and Wharton, L. R., Jr.: AM. J. OBST. & GYNEC. 69: 573, 1955. 9. Scott, R. B., and Wharton, L. R., Jr.: AM. J. OBST. & GYNEC. 74: 852, 1957. 10. Thomas, H. H.: Obst. & Gynec. 15: 498, 1960.
B.
LAWRENCE Baltimore,
SCOTT,
M.D.
Ohio R.
WHARTON,
JR.,
M.D.
Maryland
E x T E R N A L endometriosis, the disease entity due to the presence of endometrial tissue outside the uterus or invading the uterus from the serosal surface inward, is generally asymptomatic during pregnancy and seldom increases in size or has a return of symptoms until 2 to 5 years following a pregnancy. Therefore, pregnancy, if it can be achieved, is considered an excellent form of therapy for this common genital disease of women. Cyclic menses, involving constant estrogen and intermittent progesterone, when allowed to persist uninterrupted by preg-
nancy, predispose to the development and growth of endometriosis. This is generally explained on the basis of the more frequent chances of retrograde tubal flow of viable fragments of shed endometrium, capable of implanting, plus the local growth of areas of endometriosis cyclically subject to the same hormonal influences as the normally located endometrium. During pregnancy the levels of circulating estrogen and progesterone tend to increase and there is no intermittent withdrawal of progesterone to permit the uterine endometrium to shed and disseminate or the existing endometriosis to grow and infiltrate. The softening and lack of hemorrhage in preexisting endometriosis during pregnancy make the absence of symptoms understandable. The absence of symptoms for a period of time following pregnancy is less easily explained. Theoretically this has been attributed to either necrosis and atrophy or fibrosis of the decidual stromal tissue of the ectopic endometrium. In order to mimic the benefits of pregnancy various techniques of hormone administration have been suggested and tried on a clinical basis. Increasing doses of an estrogen, such as the synthetic one, diethylstilbestrol, were advocated; if used properly to produce amenorrhea and inhibit ovulation, menses would be absent and the ectopic endometrium would not show periodic hemorrhage. However, decidual transformation of the stroma and other effects secondary to
From the Department of Obstetricsand Gynecology, Western Reserve School of Medicine and University Hospitals of Cleveland, and the Department of Gynecology and Obstetrics, Johns Hopkins School of Medicine, and Johns Hopkins Hospital. This work was supported by United States Public Health Service, National Institutes of Health, Research Grant C-2892 (C 4 and 5). Some financial assistance for the purchase of a few of the animals, as well as the estrone, vaginal 50 mg. progesterone tablets (Colprosterone), and Go-fluoro17-a-brom progesterone (AY-59034) were provided by Ayerst Laboratories, New York, through the courtesy of the Medical Director, Dr. John B. Jewell. The I7o-ethinyl-19-nortestosterone or norethindrone (No&tin) for intramuscular i‘njection Iuas generously supplied by Parke, Davis B Co., Detroit, Michigan. Guest Speaker’s Address, presented at the Twenty-fourth Annual Meeting of the South Atlantic Association of Obstetricians and Gynecologists, Bal Harbor, Florida, Jan. 28-31, 1962. 867
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progesterone would not occur, negating this method as one mimicking pregnancy. The difficulty of administering increasing amounts of effective progesterone, together with its cost and rapid disappearance from the bloodstream and tissues, makes the use of injected progesterone prohibitive. Formerly, the oral progesterone derivatives were poorly or variably absorbed and therefore were not tested on a clinical basis in endometriosis. now Progesterone-like substances have been marketed. Although expensive, most of them can be administered orally, the dose can be increased as desired, and the end organ response is similar, although not identical, to injected progesterone. The endometrial stromal change is more marked than the glandular one. Clinical trials of “pseudopregnancy” are now in progress with these synthetic progestins. The most commonly used preparations are 19-norethynodrel with 1.5 per cent of ethinylestradiol-3-methyl ether (Enovid), norethisterone or norethindrone (Norlutin) , and 17a-hydroxyprogesterone caproate (Delalutin) . Clinical reports to this time, using long-term administration with initial increasing amounts for 4 to 12 months, have been reported with considerable enthusiasm.‘-4, 6, lo Rhesus (Macaca mulatta) monkeys are cyclically menstruating primates with patent uterotubal junctions and a menstrual and reproductive physiology similar to that of the human female. In addition, at least 5 instances of spontaneous endometriosis have been encountered in these animals. Autologous transplants of surgically excised endometrial tissue are highly successful in these animals and they then offer a superior laboratory mechanism for testing the effect of various hormones on endometriosis. The authors have previously reported their experiences with the effects of diethylstilbestrol,’ constant estrone, constant estrone and constant progesterone, and constant estrone and intermittent progesterone9 on the experimentally produced endometriosis in the animals. In the present experiments the effects of constant progesterone and continuous, although increasing, amount of norethindrone
will be studied. The difficulties of oral administration and the uncertainty of dosage levels by this route did not permit 11s to use norethynodrel. Norethindrone for intramuscular use allowed accurate control of the amount of drug given to each animal. Materials
and
methods
Progesterone experiments. Four
adult female rhesus monkeys (Nos. 901, 987, 988, and 990) were used. One animal (No. 901) had been used previously for experimental study; for 2 years it received intraperitoneal injections of peripheral venous blood at the time of menses and for another 2 years it was given twice weekly estrone injections to test the effect on autologous endometrial transplants. All animals had surgical transplants of endometrial tissue, secured by hysterotomy, to four areas---one ovary, tht serosal surface of the uterus, the rectosigmoid serosal surface, and the anterior abdominal wall beneath the rectus fascia. Prior to the injections of progesterone, the animals were re-explored in order to assure by gross inspection and biopsy that at least one transplant had viable endometriosis. Progesterone in oil, approximately 6 mg. CO.25 cc. of a solution in oil containing 25 mg. per cubic centimeter), was given intramuscularly 6 days each week for 31, 28, 19. and 12j/2 months, respectively. Surgical explorations with biopsies were done on these animals during the course of treatment and at the termination-the two shortest periods of administration were terminated by death of the animals, one by tuberculosis and one by freezing. Norethindrone experiments. Seven adult female rhesus monkeys were used (Nos. 839, 901, 949, 963, 964, 965, and 988). Monkey 839 was transferred to our colony from the regular Carnegie Institution of Washington, Department of Embryology colony when it was found to have a 4 by 3 cm. pelvic endometrial cyst several years after a hysterotomy during pregnancy. This animal was used for a 3 year study of the effect of testosterone prior to this present testing and a baseline biopsy of the cyst wall was taken
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Fig. 1. Monkey 901. Biopsy of endomctrial cyst wall after 31 months of progesterone in oil injections, 6 mg. each day for 6 days of each week. Note the closely packed glands with an atypical hyperplasia of the epithelium and the heavy inflammatory cell infiltration. (X70.)
one month after testosterone was stopped. Monkey 901 had been used for the intraperitoneal blood injections and estrone injections as mentioned above. Monkey 949 had been castrated, endometrium transplanted to the 4 areas noted above and used to test the effect of constant estrone and intermitent progesterone on these transplants. Monkey 963 had been previously castrated at the time of autologous endometrial transplantation and then used to study the effect of constant estrone and constant progesterone. Monkey 964 had been used in an unsuccessful attempt to shift the vagina into a gluteal region, following which endometrium was surgically transplanted to the areas mentioned and testosterone administered for 34f/’ months. Monkey 965 similar to Monkey 964 had experiments except the testosterone was administered for only 17 months. Monkey 988 was used previously for the progesterone study outlined above. Each of the animals had been proved by gross and microscopic examination to have at least one area of viable endometriosis before the present experiments were begun. Norethindrone was administered intramuscularly at a level of 5 to 10 mg. (1 C.C. equals 50 mg.) two to four times each week? increasing the dose over a 2 to 3 month interval in order that a maintenance dose of
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norethindrone
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40 to 50 mg. each week was reached. The drug was given for a total period ranging from 5ya to 7 months. Monkeys 949 and 963 were castrates; therefore they received aqueous estrone, 0.25 mg. (1 C.C. equals 1 mg.), twice weekly prior to and during the use of norethindrone. Also these 2 animals were maintained for 6 months after discontinuation of norethindrone and estrone on estradiol valerate in sesame oil (Delestrogen; 1 C.C. equals 10 mg.), 2.5 mg. at the beginning of each interval of 4 weeks, and 17~hydroxyprogesterone caproate in sesame oil ( 1 C.C. equals 125 mg. ), 62.5 mg. at the midpoint of each interval of 4 weeks. All of the animals, except Nos. 964 and 965, were tested for vaginal bleeding during and following treatment. After 2 to 3 months and again at the end of treatment with norethindrone surgical explorations were done and selected biopsies of the endometriosis areas and on a few occasions endometrial biopsies by hysterotomy were taken. Five of the monkeys were again re-explored or autopsied 6 to 7 months after stoppage of norethindrone; 2 of the 7 monkeys died whiIe undergoing the test (61/z and 7 months after the initial injection), one complicated by an abscess in a pelvic endometrial cyst and one of tuberculosis. Results
Progesterone experiments. During the period of constant progesterone administration the vaginal bleeding pattern was very bizarre, ranging from 1 to 4 days of bleeding every 1 to 7 weeks to almost constant daily spotting. Ovulation was not inhibited in 2 animals at least, for a corpus luteum was identified in each of these 12j/2 and 19 months after the first injection. The areas of experimental endometriosis grossly seemed to increase slightly in size and all showed evidence of fairly recent hemorrhage. Microscopically the endometriosis was relatively unaffected; old and recent hemorrhage was evident, there was moderate stromal edema and slight enlargement of the stromal cells: a secretory glandular pattern was found on only one occasion and there was no excess
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Fig. 2. Monkey 965. Biopsy of endomrtriosis in rertus muscle after 69: months ethindronr. A, Low-power (~70) and B, high-power ( ,150) views show the drcidual tion in the stroma and the flattenrd epithelium of the glandb.
Fig. 3. Monkey A, Low-power cell cytoplasmic
of
11urrc’ac’-
839. Biopsy of an endometrial cyst wall after 7 months of norethindrone. views show the marked edema, decidual (x70) and B, high-power (x450) and infrequent endometrial glands. and nuclear disintegration,
Fig. 4. Monkey 901. Biopsy of endometrial Low-power (x70) and B, high-power (x450) cell disintegration and even some hyalinization rarity of endometrial glands.
cyst wall after 7 months of norethindrone. A, views show the stromal changes with decidual and inflammatory cell infiltration. Note the
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of fibrotic reaction or replacement. One 3 cm. endometrial cyst remained filled with bloody liquid during 31 months of treatment, the lining was thick and succulent, and a suggestive adenomatous hyperplasia pattern was present in the cyst wall (Fig. 1). Posttreatment biopsies after more than one month were not possible in this group. In summary, constant, not increasing, doses of progesterone over 12y2 to 31 months produced some stromal edema and increase in size of the stromal cells of the experimental endometriosis, but hemorrhage within the endometriosis continued and no unusual amount of fibrous tissue reaction or replacement occurred. Norethindrone experiments. In the 5 of the 7 monkeys which could be properly tested during treatment there was no evidence of vaginal bleeding. Within 1 to 2 months after treatment cyclic vaginal bleeding recurred spontaneously or was produced physiologic by hormones in estimated amounts in the castrated monkeys. After 2 to 3 months and at the end of the treatment periods gross inspection and biopsies of the areas of endometriosis revealed the usual changes reported clinically with the use of these progesterone-like substances. There was decidual stromal reaction with progressive intracellular edema; the decidual cells then disintegrated, cytoplasmic ghosts and “naked,” irregularly shaped and stained nuclei appeared, the edematous stroma had a moderate inflammatory cell infiltration, and there was a strong tendency with this necrosis for hyalinized areas to appear (Figs. 2-4). Endometrial glands were sparse, inactive, and flattened, resembling endothelial lined spaces. The arterioles became thickwalled. Two large endometrial cysts persisted, but gross evidence of blood in the cystic locules disappeared and the cyst wall became thickened and edematous. One cyst was found as a frank abscess after 7 months of treatment. In all instances the uterus became quite soft, enlarged two to four times normal size, and, when opened, contained a thick edematous endometrium similar in microscopic changes to those seen in the
and
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5. Monkey 901. Endometrial cyst wall 6 months after discontinuing norethindrone. There is rather extensive subepithelial fibrosis with only scattered areas of active endometrial stroma. (x100.)
ectopic endometrium, but without hyaline zones. When the ovaries were biopsied during treatment no old or recent corpora lutea were found and the tunicas were thickened and slightly more fibrotic than normal. In the 5 monkeys studied by gross and microscopic means 6 to 7 months after norethindrone was discontinued, the areas of endometriosis were practically all identifiable grossly. Microscopically in 4 of the 5 animals there was a definite fibrous tissue reaction about the endometriosis, as well as a replacement of some apparent stromal areas with young fibrous tissue (Figs. 5-8), This reaction varied in degree from minimal to moderate, although in general the gross size of the nodules was the same or slightly larger than observed before treatment. In addition all areas of endometriosis followed through each phase showed abundant active endometrial stroma and numerous endometrial glands, even at times secretory. The uterus diminished to normal size with normal appearing endometrium and no fibrous tissue reaction or replacement. In summary during 5f/4 to 7 months of norethindrone administration in monkeys with experimental endometriosis, stromal
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Fig. 6. Monkey 963. bowel endomctriosis 6 norethindrone. Note and scattered awns of
Patchy fibrosis in sigmoid months after discontinuing active endometrial glands active stroma. fX2OD.1
decidual reaction with edema and necrosis occurred in the ectopic and the normally located endometrium. Hyalinization of some necrotic stromal area was found in onIy the ectopic endometrium. Vaginal bleeding and intracystic hemorrhage disappeared. Sis to 7 months after such treatment the clndometriosis was still present without diminution in size. with abundant and active stroma and glands, and usually with increased fibrous tissue reaction about the areas, as well as some stromal replacement by young fibrouq tissue. Cyclic vaginal bleeding recurred, the uterine endometrium was normal, and hemorrhage within an endometrial cyst again became evident. Comment
Fig. 7. Monkey 963. Stromal fibrosis about endometrial glands in rectus muscle endometriosis 6 months after discontinuing norethindrone. (xl On. ’
Fig. 8. Monkey 964, Rectus muscle endometriosis 7 months after discontinuation of norethindrone. Note zones of fibrosis in the stroma plus active and persisting stroma and glands. (x100.)
The experiments with constant progesterone were planned to test the effect of this hormone on endometriosis when used on a long-term basis. They were not calculated to mimic the hormonal influences of pregnancy, for to reproduce such influences the amount of progesterone should have been progressively increased to practically prohibitive amounts. As performed, this constant dosage of hormone had little effect on the esperimental endometriosis except there was uniform evidence of irregular, at times almost constant, vaginal bleeding and hemorrhage within the external endometriosis. This hemorrhage within areas of endometriosis is also encountered clinically when continuous and constant small amounts of the newer progesterone-like substances are ,given. From this knowledge there evolves a useful preoperative application which will be mentioned later. During the 6 to 7 months of norethindrone on an increasing dosage schedule the areas of experimental endometriosis showed the changes reported in human clinical studies. The stromal change was marked, progressing from frank decidual reaction to intercellular edema, cytoplasmic disintegration and vacuolization, bizarre nuclear disruption and staining, and frequently even hyaline replacement of the necrotic stromal areas. A moderate inflammatory cell infil-
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Fig. 9. Posterior cul-de-sac endometriosis after 8 weeks of preoperative treatment with vaginal progesterone suppositories, 50 mg. daily. Interstitial and intraluminal hemorrhages are extensive. (x40.)
Fig. 10. hemorrhage gesterone endometrial (X50.)
Ovarian endometriosis highlighted by after 5 weeks of daily vaginal prosuppositories prior to operation. The glands showed secretory activity.
tration was noted. One cystic locule filled with blood before treatment became one filled with clear fluid and another such cyst became abscessed; the walls of the cysts were swollen and edematous. The epithelium did not share in the changes and the endometrial glands were sparse and lined with flattened cells, so that the glands resembled endothelium-lined channels. The uteri increased to four times in size, were very soft, and the endometrium thick and edematous with similar stromal decidual changes, but no hyalinization. Five of the animals (i.e., all of those so tested) had no vaginal bleeding and when endometrial biopsies were taken there was no evidence of recent tissue hemorrhage. The ovaries had no old or recent corpora lutea at the end of the treatment period and the tunicas were thicker and more fibrotic than normal. It was possible to study 5 of the 7 animals 6 to 7 months following treatment. The areas of endometriosis were still grossly evident and blood had replaced the clear fluid in one cyst. In 4 of the 5 animals there was definite fibrous tissue replacement of some of the stroma, yet numerous areas of normal endometrial glands and stroma remained. One had the impression that the over-a11 size of the areas remained about the same as before treatment and despite the repeated
biopsies-the fibrous tissue replacement may have been in the areas of stromal change which had increased the actual mass during treatment. From these experiments it can be fairly stated that hormonal “pseudopregnancy” state was produced for 1.57 to 210 days, about equal to or longer than a monkey’s normal gestational period (160 days), and portions of the stroma of the external endometriosis were replaced by fibrous tissue. However, the gross size of the lesions did not diminish and they contained abundant healthy and active endometrial glands and stroma. In no instance was a cure by complete fibrosis obtained in any of the areas of endometriosis by the long-term administration of norethindrone. Kistner,2’ 3 A n d r e w s and associates,l Thomas,lO Lebherz and Fobes,4 and Riva and co-worker9 have reported their clinical experiences in the treatment of external endometriosis with these new progestins. The improvement by relief of symptoms attributed to the endometriosis varied from 77.6 to 85 per cent with one author stating “nearly all benefited.“lO In many of the cases the diagnosis was based on symptoms and clinical findings without histologic proof. Riva and his group relied heavily upon culdoscopic examination as confirmatory, a
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Fig. 11. Ovarian surface endometriosis after 5 weeks of preoperative daily treatment with a synthetic progestin (AY-59034). The stroma edema and decidual reaction grossly enlarged the area and the hemorrhage further assisted gross identification. (x40.)
reliance open to serious question. All of the authors were in general agreement that dosages must be stepped up to prevent breakthrough bleeding, supplemental or incorporated estrogens (as in norethynodrel) were helpful, and the best results were with long-term use, such as 5 to 9 months. It was Kistner’s impression that pregnancies occurred sooner following this treatment than the 2.7 years average following conservative surgical treatment in his hospital. Scott,7 in his discussion of the paper by Andrews, on the other hand, was worried about the frequency of posttreatment delay in resumption of normal ovulatory cycles. The side effects of the drugs, such as nausea, emotional distress, growth of fibroids, breast tenderness, fluid retention, and breakthrough bleeding were particularly stressed by Lebherz and Fobes4 and others.*’ 5, 6 Some of these side effects could be alleviated by antiemetics and diuretics, making a treatment schedule a complex one indeed. The long-term use of expensive medication, fraught with a high incidence of annoying side effects and which has no apparent curative value, should be undertaken only after very serious consideration. Ovulation is prevented during the period of treatment, and posttreatment relief of symptoms and subsequent pregnancy rate cannot
be properly evaluated on the limited data available. Logically the treatment program should be limited to patients with maximum symptoms and minimal findings of external endometriosis and to those with recurrent endometriosis following conservative surgical treatment. A useful clinical application can be derived from the knowledge that constant small amounts of progesterone or the synthetic progestins produce hemorrhage in the endometriosis as well as softening of the pelvic agglutinations. For about 5 to 8 weeks prior to contemplated conservative surgical treatment of endometroisis, small amounts of the synthetic progestins (10 to 20 mg. daily) or vaginal suppositories of progcsterone (50 mg. daily) have been used in about 20 patients. These patients usually have a chocolate vaginal discharge for the last 2 to 3 weeks of treatment. ,4t the time of operation the areas of endometriosis have hemorrhagic “blossoms” (Figs. 9-I 1) , thus making them more easily identifiable for excision or fulguration and thereby lessening the chances of missing small foci which might flourish subsequently. In addition, the softening of the usually dense adhesions has been remarkable and cleavage planes were more easily established. Our results with vaginal suppositories of progesterone were at times good but quite variable, since absorption by this route is uncertain. The 6~ fluoro-I 7cY-brom progesterone (AY-59034) was not as effective as the other progestins in producing the desired changes in the endometriosis. Summary
and
conclusions
1. Four adult female rhesus monkeys with experimentally produced areas of external endometriosis were given daily progesterone in oil injections for periods ranging from 12vZ to 31 months. The animals had very irregular vaginal bleeding, at times almost constant, and the areas of endometriosis all showed evidence of recent or old hemorrhage, but no posttreatment fibrosis. 2. Seven adult female rhesus monkeys with experimentally produced areas of en-
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dometriosis were given parenteral norethindrone in increasing amounts for 5f/2 to 7 months. Gross and histologic studies during and 7 months following treatment revealed no posttreatment reduction in the size of the areas, but there was evidence of a patchy stromal fibrosis. Despite this, abundant active glands and stroma were still present. 3. From these experiments there is no evidence that this new progestin “cures” endometriosis and the clinical use of these agents for external endometriosis should be considered a temporizing measure for special selected cases. 4. Five to 8 weeks of preoperative small
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amounts of progesterone or one of the newer progestins on a constant, not increasing, dose schedule have been found useful before planned conservative surgical treatment. Hemorrhages within the endometriosis make their identification and complete excision easier and in addition the edema and softening of the pelvic structures enhance the establishment of proper cleavage planes. We wish to express our appreciation to Dr. James D. Ebert for his cooperation in this study and for the use of the facilities at the Carnegie Institution of Washington, Department of EmbryoIogy, Baltimore, Maryland.
REFERENCES
1. Andrews, M. C., Andrews, W. C., and Strauss, A. F.: AX J. OBST. & GYNEC. 78: 776, 1959. 2. Kistner, R. W.: AM. J. OBST. & GYNEC. 75: 264, 1958. 3. Kistner, R. W.: Fertil. & Steril. 10: 539, 1959. 4. Lebherz, T. B., and Fobes, C. D.: AM’. J. OBST. & GYNEC. 81: 102, 1961. 5. Mixson, W. T., and Hammond, D. 0.: AM. J. OBST. & GYNEC. 82: 754, 1961.
6. Riva, H. L., Wilson, J. H., and Kawasaki, D. M.: AM. J. OBST. & GYNEC. 82: 109, 1961. 7. Scott, J. W.: In discussion of paper by Andrews, Andrews, and Strauss.1 8. Scott, R. B., and Wharton, L. R., Jr.: AM. J. OBST. & GYNEC. 69: 573, 1955. 9. Scott, R. B., and Wharton, L. R., Jr.: AM. J. OBST. & GYNEC. 74: 852, 1957. 10. Thomas, H. H.: Obst. & Gynec. 15: 498, 1960.