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Transplacental effects of diethylstilbestrol on the genital tract of hamster offspring
Cancer Letters, 1 (1976) 139--146
139
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
TRANSPLACENTAL EFFECTS OF DIETHYLSTILBESTROL ON THE GENITAL TRACT OF HAMSTER OFFSPRING*
M. RUSTIA and P. SHUBIK
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105 (U.S.A.) (Received 22 September 1975)
SUMMARY
The effect of diethylstilbestrol (DES) was investigated in Syrian golden hamster progeny exposed during the terminal stages of gestation. Eleven pregnant hamsters were treated with 40 or 20 mg/kg body weight (b.w.) of DES. This treatment led to hyperplastic and neoplastic lesions in the reproductive system of most female progeny and was associated with continuous estrogenic stimulation. Seventy per cent of the male progeny developed spermatic granulomas of the epididymis and testis. The incidence and severity of these lesions were proportional to the dose of DES.
INTRODUCTION
Estrogens are well known as inducers of cancer in several animal species [10,11,25]. It is estimated that between 500,000--2,000,000 young women in the United States have been exposed in utero to diethylstilbestrol (DES) [13], since Smith [24] introduced the drug for prevention and treatment of complications of pregnancy. Recent reports of vaginal adenocarcinomas i~ young women, whose mothers received this therapy during pregnancy [ 12--16], and the discovery of a variety of potentially malignant epithelial lesions in the genital tract of exposed subjects [1,5,26] led us to attempt to reproduce the treatment experimentally in hamsters. This preliminary communication describes the findings in reproductive organs of hamsters exposed in utero to DES.
*Supported by U.S. Public Health Service Contract NO1 CP33278 from the National Cancer Institute, NIH. Address correspondence to: Dr. Mario Rustia, Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105
(U.S.A.)
140 MATERIALS AND METHODS Eleven pregnant hamsters (Syrian golden) from the Eppley Colony were given one or t w o consecutive daily doses of DES, which was suspended in an aqueous solution with 0.1% gelatin and administered by intragastric intubatiol at dose levels of 40 mg/kg or 20 mg/kg b.w. Single doses were given on day 15 of gestation to groups 1 and 3, respectively, and groups 2 and 4 received two doses of 40 mg/kg or 20 mg/kg DES, respectively, on days 14 and 15 of gestation (Table 1). The animals were allowed to die spontaneously or were killed when moribund. Complete autopsies were performed on all animals. RESULTS From 11 litters, there were 115 newborn hamsters, of which 70 (60%) were weaned. The number of weaned animals in each group and the incidence of lesions and tumors in the tissues of the reproductive tract of female or male offspring are summarized in Table 1. Microscopically the ovaries revealed follicular cysts. Corpora lutea were absent. Nodular hyperplasia of granulosa cells was seen in three instances. The oviducts were dilated and in 25 instances contained large quantities of pus (pyosalpinx) and/or eosinophilic material. Large areas of the luminal surface were covered by cuboidal nonciliated epithelium. Focal or more extensive zonal squamous cell metapiasia with keratinization and parakeratosis of luminal epithelium was seen in over 50% of all instances. The most prominent changes in the reproductive system were observed in the uterus and cervix. Uterine lesions included hyperplasia of endometrial epithelium and glands, sometimes associated with features analogous to adenomyosis and squamous cell metaplasia. There was one adenocarcinoma. Hyperplasia of the endometrial epithelium was first observed in a group 3 female offspring at 15 weeks of age and was characterized b y an increased number of endometrial glands, which were frequently cystically dilated. In 50% of the cases the endometrial hyperplasia presented itself as abnormal villous projections (polypoid projections) containing stroma and glands. Twenty-five percent of these projections showed inflammation and necrosis and focal squamous cell metaplasia with cornification, and they occasionally protruded into the dilated cervix. Glandular proliferation through the myometrium, similar to adenomyosis in humans was seen (groups 1 and 2) in 5 instances. The glands appeared tortuous and dilated; they expanded through the muscular coat, and penetrated deeply into the subserosa (Fig. 2). Squamous cell metaplasia occurred in 18 instances and showed keratinization and stratification of squamous cells (epidermization) with parakeratosis. In 4 instances, foci of epithelial dysplasia were observed. Histologically, the cervix exhibited increased fibromuscular tissue, hypertrophy, hyalinization, and sometimes mucoid transformation of the stroma. The endocervical canals were usually dilated and obstructed by accumulation of inflammatory material and debris.
141
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The squamous epithelium covering the cervical canal was keratinized, had marked parakeratosis, some dyskeratosis with pearl formation, and simple downgrowths. In 4 instances, cells and nuclear atypia were irregularly arranged; they tended to infiltrate the stroma and were designated as dysplasia. Glandular downgrowths (adenosis) extending deeply into the cervical stroma were found in 8 instances (Fig. 3). Tumors Four of fourteen (28%) female progeny in group 1, and four of eight (50%) in group 3 had reproductive tract tumors. The polyps developed at the endocervix as pedunculated excrescences protruding into the cervical canal. Two occurred in group 1 and two in group 2 (Table 1). Two squamous cell papillomas developed, one each in groups 1 and 2, respectively. The mixed mesodermal t u m o r was firm in consistency, well encapsulated 35 x 30 x 30 mm, and extended from the cervix and upper vaginal wall subcutaneously into the perianal region. The t u m o r presented features of mesenchymal sarcomas (myxosarcoma) and contained heterologous elements of tissue, as cartilage and fibers suggestive of cross-striations (Fig. l), and an epithelial c o m p o n e n t with a patch of squamous epithelium. The vaginal mucosa manifested changes of a continuous estrus state with keratinization. Hyperplasia and keratinization of stratified squamous epithelia with parakeratosis were found in 19 cases. Two squamous cell papillomas, one each in groups 1 and 2, were found at 29 and 50 weeks of age, respectively. Lesions in males Over 7 0 ~ of the male progeny had spermatic granulomas of the epididymis, while the testes were affected by this type of lesion in 40% of cases. The typical granulomas consisted of histiocytic cells of an epithelioid type with eosinophilic, frequently vacuolated cytoplasm, varying amounts of pigment and occasionally with blue-gray staining sphericles analogous to Michaelis-G u t m a n n bodies. These changes were observed intraductally, where the
Fig. 1. Mixed Miillerian tumor which originated in the cervix. Note chondromatous ar~as and rhabdomyoblastic fibrils suggesting cross-striation. Hematoxylin and eosin (H & E), x 86. Inset: x 337. Fig. 2. Invagination and proliferation of uterine glands below muscular layer to subserosa indicating conditions suggestive of adenomyosis. H & E, x 135.
Fig. 3. Dilatation of the cervical canal, squamous cell metaplasia with keratinization and parakeratosis, and glandular downgrowths (adenosis) into the stroma. H & E, x 28. Fig. 4. Hyperplasia of muscular coat, inflammatory infiltrate, and advanced granulomatous extraductal lesion containing epithelioid cellular elements and foci of calcification. H & E, x 86.
144
granulomas probably originate, and extraductally in the epididymis or testicular interstitia (Fig. 4). Calcifications in the form of dense, basophilic foci (concretions) were present intra- and extraductally in both the epididymis and the testes. An adenoma of Cowper's gland and a leiomyosarcoma of the seminal vesicles were recorded at 73 and 61 weeks of age, respectively (Table 1). A report on the frequency of tumors in untreated control hamsters was published recently [22,23]. The changes described here in the reproductive organs were not seen in untreated animals, although these organs were histopathologically examined and may, therefore, serve as comparative controls. DISCUSSION
A causal relationship between transplacental exposure to DES and the observed changes in the genital tract is suggested by the frequency and complexity of the lesions. It also appears that DES treatment in the late stages of pregnancy induced in female offspring a permanent hormonal derangement, which is manifested as continuous estrogenic stimulation. Dunn et al. [4] reported the occurrence of epidermoid carcinomas and precancerous lesions in old mice treated neonatally with a single dose of DES. Similarly, precancerous lesions characterized by extensive adenosis of the cervix and vagina with epidermization were produced after multiple postnatal injections of DES in mice [6]. The tumor spectrum in female hamster progeny, however, differs from that recorded in mice. The squamous cell papillomas and polyps of the cervix and vagina are considered benign neoplasms and their progression to malignancy under continuous estrogenic stimulation has not been established. The occurrence of the mixed mesenchymal tumor of the cervix and the adenocarcinoma of the uterus are of particular interest, since the former neoplasm contains cellular elements that develop from the Miillerian system by heteroplasia [29] and to our best knowledge has not been previously described in this species, while the adenocarcinoma was recorded at a very early period of life (35 weeks). The other epithelial lesions, the metaplastic, dysplastic, hyperplastic abnormalities, and the glandular downgrowths (analogous to adenosis and adenomyosis) in the cervix, uterine horns, and vagina are thought to be the result of continuous estrogenic secretion by follicular cysts of the ovary, which in turn developed as a consequence of an altered pattern of gonadotrophins due to disturbances in the hypothalamic--hypophyseal system after exposure to estrogen during the critical period of development [2,6,8,9]. The direct action of DES on tissues of the reproductive tract, however, cannot be excluded. In this context it is important that experimental findings indicate that epithelial abnormalities of certain segments of the genital tract, which are produced under the influence of persistent estrogenic stimulation, are irreversible [6,9,18,20,21,28] and may progress into overt malignancy [3,6--9,11,17,19,27]. Clear cell vaginal adenocarcinomas have not yet been observed in this experimental system. However, the findings of certain cervical and vaginal alterations, such as squamous cell metaplasia with
145
keratinization, occasional dysplasia and adenosis, and neoplasia, are similar to the changes described in young women whose mothers received DES during pregnancy. ACKNOWLEDGEMENTS
We thank Dr. David Clayson for constructive suggestions regarding the manuscript, Mara Payich for excellent technical assistance, Mardelle Susman for editorial assistance, and Andrew Washington and Walter Williams for photography. REFERENCES 1 Barber, H.R.K. and Sommers, S.C. (1974) Vaginal adenosis, dysplasia, clear cell adenocarcinoma after diethylstilbestrol treatment in pregnancy. Obstet. Gyn., 43, 645--652. 2 Barraclough, C.A. (1968) Alterations in reproductive function following prenatal and early postnatal exposure to hormones. Adv. Repro. Physiol., 3, 81--112. 3 Dunn, T.B. (1969) Cancer of the uterine cervix in mice fed a liquid diet containing an antifertility drug, J. Natl. Cancer Inst., 43, 671--692. 4 Dunn, T.B. and Green, A.W. (1963) Cysts of the epididymis, cancer of the cervix, granular cell myoblastoma, and other lesions after estrogen injection in newborn mice. J. Natl. Cancer Inst., 31,425--455. 5 Fetherston, W.C. (1975) Squamous neoplasia of vagina related to DES syndrome. Am. J. Obstet. Gynecol., 122, 176--180. 6 Forsberg, J.G. (1975) Late effects in the vaginal and cervical epithelia after injections of diethylstilbestrol into neonatal mice. Am. J. Obstet. and Gynecol., 121,101--104. 7 Forsberg, J.G. (1972) Estrogen, vaginal cancer, and vaginal development. Am. J. Obstet. Gynecol., 113, 83--87. 8 Forsberg, J.G. (1973)Cervicovaginal epithelium: Its origin and development. Am. J. Obstet. Gynecol., 115, 1025--1043. 9 Forsberg, J.G. (1974): Induction of condition leading to cancer in the genital tract by estrogen during the differentiation phase of the genital epithelium. Adv. in the Biosciences, 13, 139--151. 10 Gardner, W.U. (1941): The effect of estrogen on the incidence of mammary and pituitary tumors in hybrid mice. Cancer Res., 11,345--358. 11 Gardner, W.U., Pfeiffer, C.A. and Trentin, J.J. (1959) Hormonal factors in experimental carcinogenesis. In: The Physiopathology of Cancer, pp. 153--237. Editor: F. Homburger. Harper and Bros., New York - - London. 12 Greenwald, P. and Nasca, P.C. (1973) Transplacental induction of vaginal cancer by synthetic estrogens. Prog. Clin. Cancer, 5, 13--19. 13 Herbst, A.L. (1975) Personal communication. 14 Herbst, A.L., Robboy, S.J., Scully, R.E. and Foskanzer, D.C. (1974) Clear-cell adenocarcinoma of the vagina and cervix in girls: Analysis of 170 registry cases. Am. J. Obstet. Gynecol., 119, 713--724. 15 Herbst, A.L., Poskanzer, D.C., R o b b o y , S.J., Friedlander, L., and Scully, R.E. (1975) ~ Prenatal exposure to stilbestrol - - A prospective comparison of exposed female offspring with unexposed controls. N. Engl. J. Med., 292,334--339. 16 Herbst, A.L., Unfelder, H. and Poskanzer, D.C. (1971) Adenocarcinoma of the vagina: Association of maternal stilbestrol therapy with tumor appearance in y o u n g w o m e n . N. Engl. J. Med., 284, 878--881.
146 17 Heston, W.E., Vlahakis, G. and Desmukes, B. (1973) Effects of the antifertility drug Enovid in five strains of mice, with particular regard to carcinogenesis. J. Natl. Cancer Inst., 51,209--224. 18 Kimura, T. and Nandi, S. (1967) Nature of the induced persistent vaginal cornification in mice. IV. Changes in the vaginal epithelium of old mice treated neonatally with estradiol or testosterone. J. Natl. Cancer Inst., 39, 75--93. 19 Meissner, W.A., Sommers, S.C. and Sherman, G. (1957) Endometrial hyperplasia, endometrial carcinoma, and endometriosis produced experimentally by estrogen. Cancer, 10, 500--509. 20 McLachlan, J.A. and Dixon, R.L. (1973) Effect of gestational exposure to DDT or diethylstilbestrol on the reproductive capacity of offspring. Pharmacologist, 15, 199. 21 McLachlan, J.A., Shah, H.C. and Gipson, S. Abstract 172. The fate of [14C]diethylstilbestrol in the pregnant mouse and McLachlan, J.A., Shah, H.C., Newbold, R.R. and Bullock, B.C2.'Abstract 173. Effect of prenatal exposure of mice to diethylstilbestrol on the reproductive tract function in the offspring. Soc. Tox., 14th annual meeting, March 9--13, 1975. 22 Rustia, M. (1974) Multiple carcinogenic effects of the ethylnitrosourea precursors ethylurea and sodium nitrite in hamsters. Cancer Res., 34, 3232--3244. 23 Rustia, M. The effect of gonadal ablation on transplacentally induced neurogenic tumors in hamsters. Cancer Res., in press. 24 Smith, O.W. (1948) Diethylstilbestrol in the prevention and treatment of complications of pregnancy. Am. J. Obstet. Gynecol., 56, 821--834. 25 Shubik, P. and Hartwell, J.L. Survey of compounds which have been tested for carcinogenic activity. PHS 149, U.S. Dept. Health, Education, and Welfare, 1969. 26 Stafl, A. and Mattingly, R.F. (1974) Vaginal adenosis: A precancerous lesion? Amer. J. Obstet. Gynecol., 120, 666--673. 27 Taylor, H.C. (1947) Endocrine factors in the origin of tumors of the uterus. In: Endocrinal and Neurological Diseases, pp. 119--137. Oxford University Press, New York. 28 Takasugi, N., Bern, H.A. and DeOme, K.B. (1962) Persistent vaginal cornification in mice. Science, 128, 438--439. 29 Williamson, E.O. and Christopherson, W.M. (1972) Malignant mixed Miillerian tumors of the uterus. Cancer, 29, 585--592.
139
© Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
TRANSPLACENTAL EFFECTS OF DIETHYLSTILBESTROL ON THE GENITAL TRACT OF HAMSTER OFFSPRING*
M. RUSTIA and P. SHUBIK
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105 (U.S.A.) (Received 22 September 1975)
SUMMARY
The effect of diethylstilbestrol (DES) was investigated in Syrian golden hamster progeny exposed during the terminal stages of gestation. Eleven pregnant hamsters were treated with 40 or 20 mg/kg body weight (b.w.) of DES. This treatment led to hyperplastic and neoplastic lesions in the reproductive system of most female progeny and was associated with continuous estrogenic stimulation. Seventy per cent of the male progeny developed spermatic granulomas of the epididymis and testis. The incidence and severity of these lesions were proportional to the dose of DES.
INTRODUCTION
Estrogens are well known as inducers of cancer in several animal species [10,11,25]. It is estimated that between 500,000--2,000,000 young women in the United States have been exposed in utero to diethylstilbestrol (DES) [13], since Smith [24] introduced the drug for prevention and treatment of complications of pregnancy. Recent reports of vaginal adenocarcinomas i~ young women, whose mothers received this therapy during pregnancy [ 12--16], and the discovery of a variety of potentially malignant epithelial lesions in the genital tract of exposed subjects [1,5,26] led us to attempt to reproduce the treatment experimentally in hamsters. This preliminary communication describes the findings in reproductive organs of hamsters exposed in utero to DES.
*Supported by U.S. Public Health Service Contract NO1 CP33278 from the National Cancer Institute, NIH. Address correspondence to: Dr. Mario Rustia, Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 42nd and Dewey Avenue, Omaha, Nebraska 68105
(U.S.A.)
140 MATERIALS AND METHODS Eleven pregnant hamsters (Syrian golden) from the Eppley Colony were given one or t w o consecutive daily doses of DES, which was suspended in an aqueous solution with 0.1% gelatin and administered by intragastric intubatiol at dose levels of 40 mg/kg or 20 mg/kg b.w. Single doses were given on day 15 of gestation to groups 1 and 3, respectively, and groups 2 and 4 received two doses of 40 mg/kg or 20 mg/kg DES, respectively, on days 14 and 15 of gestation (Table 1). The animals were allowed to die spontaneously or were killed when moribund. Complete autopsies were performed on all animals. RESULTS From 11 litters, there were 115 newborn hamsters, of which 70 (60%) were weaned. The number of weaned animals in each group and the incidence of lesions and tumors in the tissues of the reproductive tract of female or male offspring are summarized in Table 1. Microscopically the ovaries revealed follicular cysts. Corpora lutea were absent. Nodular hyperplasia of granulosa cells was seen in three instances. The oviducts were dilated and in 25 instances contained large quantities of pus (pyosalpinx) and/or eosinophilic material. Large areas of the luminal surface were covered by cuboidal nonciliated epithelium. Focal or more extensive zonal squamous cell metapiasia with keratinization and parakeratosis of luminal epithelium was seen in over 50% of all instances. The most prominent changes in the reproductive system were observed in the uterus and cervix. Uterine lesions included hyperplasia of endometrial epithelium and glands, sometimes associated with features analogous to adenomyosis and squamous cell metaplasia. There was one adenocarcinoma. Hyperplasia of the endometrial epithelium was first observed in a group 3 female offspring at 15 weeks of age and was characterized b y an increased number of endometrial glands, which were frequently cystically dilated. In 50% of the cases the endometrial hyperplasia presented itself as abnormal villous projections (polypoid projections) containing stroma and glands. Twenty-five percent of these projections showed inflammation and necrosis and focal squamous cell metaplasia with cornification, and they occasionally protruded into the dilated cervix. Glandular proliferation through the myometrium, similar to adenomyosis in humans was seen (groups 1 and 2) in 5 instances. The glands appeared tortuous and dilated; they expanded through the muscular coat, and penetrated deeply into the subserosa (Fig. 2). Squamous cell metaplasia occurred in 18 instances and showed keratinization and stratification of squamous cells (epidermization) with parakeratosis. In 4 instances, foci of epithelial dysplasia were observed. Histologically, the cervix exhibited increased fibromuscular tissue, hypertrophy, hyalinization, and sometimes mucoid transformation of the stroma. The endocervical canals were usually dilated and obstructed by accumulation of inflammatory material and debris.
141
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The squamous epithelium covering the cervical canal was keratinized, had marked parakeratosis, some dyskeratosis with pearl formation, and simple downgrowths. In 4 instances, cells and nuclear atypia were irregularly arranged; they tended to infiltrate the stroma and were designated as dysplasia. Glandular downgrowths (adenosis) extending deeply into the cervical stroma were found in 8 instances (Fig. 3). Tumors Four of fourteen (28%) female progeny in group 1, and four of eight (50%) in group 3 had reproductive tract tumors. The polyps developed at the endocervix as pedunculated excrescences protruding into the cervical canal. Two occurred in group 1 and two in group 2 (Table 1). Two squamous cell papillomas developed, one each in groups 1 and 2, respectively. The mixed mesodermal t u m o r was firm in consistency, well encapsulated 35 x 30 x 30 mm, and extended from the cervix and upper vaginal wall subcutaneously into the perianal region. The t u m o r presented features of mesenchymal sarcomas (myxosarcoma) and contained heterologous elements of tissue, as cartilage and fibers suggestive of cross-striations (Fig. l), and an epithelial c o m p o n e n t with a patch of squamous epithelium. The vaginal mucosa manifested changes of a continuous estrus state with keratinization. Hyperplasia and keratinization of stratified squamous epithelia with parakeratosis were found in 19 cases. Two squamous cell papillomas, one each in groups 1 and 2, were found at 29 and 50 weeks of age, respectively. Lesions in males Over 7 0 ~ of the male progeny had spermatic granulomas of the epididymis, while the testes were affected by this type of lesion in 40% of cases. The typical granulomas consisted of histiocytic cells of an epithelioid type with eosinophilic, frequently vacuolated cytoplasm, varying amounts of pigment and occasionally with blue-gray staining sphericles analogous to Michaelis-G u t m a n n bodies. These changes were observed intraductally, where the
Fig. 1. Mixed Miillerian tumor which originated in the cervix. Note chondromatous ar~as and rhabdomyoblastic fibrils suggesting cross-striation. Hematoxylin and eosin (H & E), x 86. Inset: x 337. Fig. 2. Invagination and proliferation of uterine glands below muscular layer to subserosa indicating conditions suggestive of adenomyosis. H & E, x 135.
Fig. 3. Dilatation of the cervical canal, squamous cell metaplasia with keratinization and parakeratosis, and glandular downgrowths (adenosis) into the stroma. H & E, x 28. Fig. 4. Hyperplasia of muscular coat, inflammatory infiltrate, and advanced granulomatous extraductal lesion containing epithelioid cellular elements and foci of calcification. H & E, x 86.
144
granulomas probably originate, and extraductally in the epididymis or testicular interstitia (Fig. 4). Calcifications in the form of dense, basophilic foci (concretions) were present intra- and extraductally in both the epididymis and the testes. An adenoma of Cowper's gland and a leiomyosarcoma of the seminal vesicles were recorded at 73 and 61 weeks of age, respectively (Table 1). A report on the frequency of tumors in untreated control hamsters was published recently [22,23]. The changes described here in the reproductive organs were not seen in untreated animals, although these organs were histopathologically examined and may, therefore, serve as comparative controls. DISCUSSION
A causal relationship between transplacental exposure to DES and the observed changes in the genital tract is suggested by the frequency and complexity of the lesions. It also appears that DES treatment in the late stages of pregnancy induced in female offspring a permanent hormonal derangement, which is manifested as continuous estrogenic stimulation. Dunn et al. [4] reported the occurrence of epidermoid carcinomas and precancerous lesions in old mice treated neonatally with a single dose of DES. Similarly, precancerous lesions characterized by extensive adenosis of the cervix and vagina with epidermization were produced after multiple postnatal injections of DES in mice [6]. The tumor spectrum in female hamster progeny, however, differs from that recorded in mice. The squamous cell papillomas and polyps of the cervix and vagina are considered benign neoplasms and their progression to malignancy under continuous estrogenic stimulation has not been established. The occurrence of the mixed mesenchymal tumor of the cervix and the adenocarcinoma of the uterus are of particular interest, since the former neoplasm contains cellular elements that develop from the Miillerian system by heteroplasia [29] and to our best knowledge has not been previously described in this species, while the adenocarcinoma was recorded at a very early period of life (35 weeks). The other epithelial lesions, the metaplastic, dysplastic, hyperplastic abnormalities, and the glandular downgrowths (analogous to adenosis and adenomyosis) in the cervix, uterine horns, and vagina are thought to be the result of continuous estrogenic secretion by follicular cysts of the ovary, which in turn developed as a consequence of an altered pattern of gonadotrophins due to disturbances in the hypothalamic--hypophyseal system after exposure to estrogen during the critical period of development [2,6,8,9]. The direct action of DES on tissues of the reproductive tract, however, cannot be excluded. In this context it is important that experimental findings indicate that epithelial abnormalities of certain segments of the genital tract, which are produced under the influence of persistent estrogenic stimulation, are irreversible [6,9,18,20,21,28] and may progress into overt malignancy [3,6--9,11,17,19,27]. Clear cell vaginal adenocarcinomas have not yet been observed in this experimental system. However, the findings of certain cervical and vaginal alterations, such as squamous cell metaplasia with
145
keratinization, occasional dysplasia and adenosis, and neoplasia, are similar to the changes described in young women whose mothers received DES during pregnancy. ACKNOWLEDGEMENTS
We thank Dr. David Clayson for constructive suggestions regarding the manuscript, Mara Payich for excellent technical assistance, Mardelle Susman for editorial assistance, and Andrew Washington and Walter Williams for photography. REFERENCES 1 Barber, H.R.K. and Sommers, S.C. (1974) Vaginal adenosis, dysplasia, clear cell adenocarcinoma after diethylstilbestrol treatment in pregnancy. Obstet. Gyn., 43, 645--652. 2 Barraclough, C.A. (1968) Alterations in reproductive function following prenatal and early postnatal exposure to hormones. Adv. Repro. Physiol., 3, 81--112. 3 Dunn, T.B. (1969) Cancer of the uterine cervix in mice fed a liquid diet containing an antifertility drug, J. Natl. Cancer Inst., 43, 671--692. 4 Dunn, T.B. and Green, A.W. (1963) Cysts of the epididymis, cancer of the cervix, granular cell myoblastoma, and other lesions after estrogen injection in newborn mice. J. Natl. Cancer Inst., 31,425--455. 5 Fetherston, W.C. (1975) Squamous neoplasia of vagina related to DES syndrome. Am. J. Obstet. Gynecol., 122, 176--180. 6 Forsberg, J.G. (1975) Late effects in the vaginal and cervical epithelia after injections of diethylstilbestrol into neonatal mice. Am. J. Obstet. and Gynecol., 121,101--104. 7 Forsberg, J.G. (1972) Estrogen, vaginal cancer, and vaginal development. Am. J. Obstet. Gynecol., 113, 83--87. 8 Forsberg, J.G. (1973)Cervicovaginal epithelium: Its origin and development. Am. J. Obstet. Gynecol., 115, 1025--1043. 9 Forsberg, J.G. (1974): Induction of condition leading to cancer in the genital tract by estrogen during the differentiation phase of the genital epithelium. Adv. in the Biosciences, 13, 139--151. 10 Gardner, W.U. (1941): The effect of estrogen on the incidence of mammary and pituitary tumors in hybrid mice. Cancer Res., 11,345--358. 11 Gardner, W.U., Pfeiffer, C.A. and Trentin, J.J. (1959) Hormonal factors in experimental carcinogenesis. In: The Physiopathology of Cancer, pp. 153--237. Editor: F. Homburger. Harper and Bros., New York - - London. 12 Greenwald, P. and Nasca, P.C. (1973) Transplacental induction of vaginal cancer by synthetic estrogens. Prog. Clin. Cancer, 5, 13--19. 13 Herbst, A.L. (1975) Personal communication. 14 Herbst, A.L., Robboy, S.J., Scully, R.E. and Foskanzer, D.C. (1974) Clear-cell adenocarcinoma of the vagina and cervix in girls: Analysis of 170 registry cases. Am. J. Obstet. Gynecol., 119, 713--724. 15 Herbst, A.L., Poskanzer, D.C., R o b b o y , S.J., Friedlander, L., and Scully, R.E. (1975) ~ Prenatal exposure to stilbestrol - - A prospective comparison of exposed female offspring with unexposed controls. N. Engl. J. Med., 292,334--339. 16 Herbst, A.L., Unfelder, H. and Poskanzer, D.C. (1971) Adenocarcinoma of the vagina: Association of maternal stilbestrol therapy with tumor appearance in y o u n g w o m e n . N. Engl. J. Med., 284, 878--881.
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