Reduced fertility in female mice exposed transplacentally to diethylstilbestrol (DES)

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1 FERTILITY AND STERILITY Copyright c 1982 The American Fertility Society Printed in U.SA. Reduced fertility in female mice exposed transplacentally to diethylstilbestrol (DES) John A. McLachlan, Ph.D.* Retha R. Newbold, CT(ASCP)t Hasmukh C. Shah, Ph.D.t Michael D. Hogan, Ph.D. Robert L. Dixon, Ph.D.11 Transplacental Toxicology Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina Prenatal exposure to diethylstilbestrol (DES), a synthetic estrogen, has been associated with a low incidence of vaginal adenocarcinoma as well as a variety of more numerous benign abnormalities in the reproductive tract of human beings and. experimental animals. For the purpose of assessing the effects of prenatal exposure to DES on postnatal reproductive tract function, timed pregnant CD~l mice were treated subcutaneously with doses of DES ranging from 0.01 to 100 jj.{j/kg/day on days 9 through 16 of gestation. Thefertility of the female offspring was determined postnatally by a repetitive forced breeding technique. The most striking effect observed was a dose-related decrease in reproductive capacity ranging from minimal subfertility at the lower DES doses to a high frequency of total sterility at the highest DES doses. Reduced reproductive capacity appeared to be a reflection of both a decrease in the total number of litters and smaller litter sizes. A major component of the sterility seen in those females given higher doses of DES was oviductal/ovarian, since the number of ova recovered from the oviductal ampullae after induced ovulation was less than 30% that of controls. In addition, structural abnormalities of the oviduct, uterus, cervix, and vagina were observed, and contributed to infertility. These data suggest that in utero exposure to DES results in permanent impairment of female mouse reproductive capacity. Recent reports of altered pregnancy outcomes in young women who were exposed in utero to DES demonstrate the clinical importance of the findings obtained in mice. Fertil Steril 38:364, 1982 Received March 10, 1981; revised and accepted May 7,1982. *Reprint requests: John A. McLachlan, Ph.D., Transplacental Toxicology Group, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina ttransplacental Toxicology Group, National Institute of Environmental Health Sciences. :j:present address: Chemical Manufacturing Association, 182 Connecticut Avenue, NW, Washington, D.C Biometry and Risk Assessment Program, National Institute of Environmental Health Sciences. IIReproductive Toxicology Group, National Institute of Environmental Health Sciences. In 1971, Herbst and his colleagues described the latent appearance of a previously rare genital tract tumor, vaginal adenocarcinoma, in young women whose mothers had been given diethylstilbestrol (DES) during gestation. 1 Transplacental effects of DES on the female fetus of both humans and experimental animals have since been confirmed by many investigators. 2-7 Although the incidence of carcinoma appeared to be low, benign abnormalities of the reproductive tract, including a variety of macroscopic structural changes,8-9 have been reported. The relationship of these structural anomalies to fertility is unclear; how- 364 McLachlanet ai. Fertility in DES-exposed mice Fertility and Sterility

2 ever, a possible association between upper genital tract changes and sub fertility has been suggested. 8 In 1973, Pomerance lo reported the results of his examination of 1 young women exposed to DES who appeared to exhibit a high incidence of anovulatory bleeding and infertility. His findings, which were not developed from a case-controlled study, were supported by a preliminary communication 11 from our laboratory in 1977, which noted our observance of reduced fertility in female mice exposed prenatally to DES. DES-associated reproductive difficulties have recently received renewed attention in the clinical literature, with reports of an increased incidence of menstrual irregularities, fetal wastage, and infertility in women exposed to DES in utero Although data obtained from women in the National Cooperative Diethylstilbestrol Adenosis (DESAD) project 1 suggest few differences in the percentages of women who became pregnant, the age distribution at which first pregnancy occurred, or the number of pregnancies, the risk of unfavorable pregnancy outcome did rise markedly in women exposed to DES during gestation. In light of the current interest in fertility regarding the DES-exposed offspring, the importance of an animal model to study the mechanisms of the prenatal effects of DES is obvious. Previous reports from this laboratory have described the transplacental toxicity of DES 6, 11 as well as other common chemicals. 16 We undertook the current study to determine mechanisms responsible for the subfertility in DES-exposed offspring; it should provide additional information for the evaluation of reproductive effects in women. ANIMALS MATERIALS AND METHODS Adult female CD-1 mice were obtained from Charles River Breeding Laboratories, Wilmington, MA, and bred to male mice of the same strain in the animal quarters at the National Institute of Environmental Health Sciences (NIEHS). Vaginal plug detection was considered day 0 of pregnancy. Pregnant mice were housed individually in cages under controlled lighting (14 hours of light and 10 hours of darkness) and temperature (21 0 to 22 0 C) conditions. DES (Sigma Chemical Co., St. Louis, MO) dissolved in corn oil or corn oil alone was administered as a subcutaneous injec- tion to pregnant females on days 9 through 16 of gestation at daily doses of 0.01, 1, 2.,, 10, or 100 J.Lglkg maternal body weight. These groups of animals will be referred to as DES-O (control), DES-0.01, DES-1, DES-2., DES-, DES-10, and DES-100, respectively. Pregnant mice delivered their young, and the litters were weighed, reduced to 8, and examined for gross abnormalities. Litter weights were recorded weekly until 1 month of age, and then mice were weaned and segregated by sex. FORCED BREEDING EXPERIMENT Randomly selected female offspring from the various dose groups were assigned to a forced breeding study consisting of two separate replicates. The first replicate included female mice prenatally exposed at either the 0.01, 1, 10, or 100 J.Lglkg dose level, while the second replicate was made up of mice from the DES-2., DES-, and DES-10 dose groups. Both replicates had their own matched controls. Typically, one female from each of the designated dose groups was housed in the same cage with a sexually mature, untreated male of proven fertility. The breeding cages were examined daily, and when a female was observed to be pregnant, she was placed in an isolation cage. After the female delivered, the number of young in the litter were counted and examined for gross malformations. Following this examination, the female was immediately returned to the breeding cage. This forced breeding regimen was maintained for 8 months (32 weeks), during which time the cumulative number of live young delivered by each female was recorded, along with other data, including time to first litter, litters per mouse, neonates per litter, and number of mice having litters. At the termination of the forced breeding study, the animals were sacrificed by cervical dislocation, and reproductive tissues were examined for structural abnormalities. CROSS-FOSTERING EXPERIMENT At birth, prenatally exposed offspring (DES-O, DES-10, and DES-100 only) were cross-fostered to either control mothers or mothers given subcutaneous injections of DES on days 9 through 16 of their pregnancy. At 1 month of age, the offspring were weaned and placed in a forced breeding study for 9 weeks. The cumulative number of live young per mouse was determined by a protocol similar to that described in the preceding McLachlan et ai. Fertility in DES-exposed mice 36

3 Table 1. Fertility in Female Mice Exposed Prenatally to DEsa Total no. of mice having Prenatal No. of Time (in days) to Litters Live neonates x litters Total no. live treatment females b first litterc, d per mouseb, d per litterd,, ;;.4 y~~:efel s! ± 2,2(88~ 6,7 ± ,0 ± 0.3(83)8" ,8 ± 2, DES-O,Ol 64 34,9 ± 2,6(6).9 ± ± 0.2(64) ± 2,7 DES ± 2,0(6),6 ± ,1 ± 0,3(61) ± 2,6 DES-2, ± 2,9(23).6 ± ± 0,(21) ,9 ± 4.3 DES ± 9.6(21) 3.2 ± 0.,1 ± 0,8(18) ,3 ± 4.4 DES-lot 67 83,2 ± 29,3(23) 0. ± 0,1 7,2 ± 1.8(23) ± 0.8 DES ,6 ± 18,6(lD) 0,3 ± 0.1 6,3 ± 1.4(9) ± 0.7 amice were females exposed prenatally to DES (0.01, 1, 2.,, 10, or 100 fj-g/kg/day) on days 9 to 16 of gestation. bbased on all females surviving for entire 32-week period and determined by repetitive forced breeding techniques, cbased on all females littering during the 32-week period, dmean ± standard error, Simple mean unadjusted for potential cage effects. ebased on all females surviving for and having litters during the 32-week period, 'Means from the two replicates have been combined to yield an unweighted, overall average, Individual control values were: 39.4 ± 4,0(64) days and 29,0 ± 2.0(24) days, 6,3 ± 0.2(62) litters and 7.1 ± 0.2 (21) litters, 11.1 ± 0.3(62) and ld.9 ± 0.(21) neonates per litter, and 69,6 ± 2.9 and 77.9 ± 4.0 total live offspring, respectively, Corresponding figures for DES-10 females were: 82.6 ± 10.6(20) and 83,7 ± 7.7(3), 0,6 ± 0,1() and 0,3 ± 0.1(12), 7.1 ± 0,9(20) and 7.3 ± 3.(3), and 4.4 ± 1.2 and 1.8 ± 1.2. KEffective sample size, section. Animals were sacrificed at the termination of this experiment. VAGINAL OPENING DETERMINATION Female offspring (ten DES-O and ten DES-lOa, only) were weaned 23 days after birth and housed five animals per cage. They were checked visually on a daily basis for vaginal opening from 21 to 3 days of age. ESTROUS CYCLE DETERMINATION (1) Eight or more l-month-old female offspring from each treatment group were housed two to three per cage, and vaginal smears were obtained daily for 9 weeks. (2) Animals approximately 8 months old were observed daily for 2 weeks for changes in the appearance of the vagina as described by Champlin et al.17 In some cases determination of the estrous cycle was made by vaginal flushings. RADIOIMMUNOASSAY TECHNIQUES Eight-month-old female offspring described in the estrous cycle determination experiment were sacrificed by decapitation. Blood samples from individual animals selected without regard to time of day or stage of estrous cycle were collected in heparinized tubes and centrifuged at 600 x g. Radioimmunoassay (RIA) for total plasma estrogens was performed as described previously McLachlan et al. Fertility in DES-exposed mice OVARIAN MORPHOLOGY Two-month-old female offspring (five per dose group DES-a, 0.01, 1, and 10, and four per dose group DES-laO) were sacrificed, and their ovaries were fixed in Bouin's solution. Tissue was then processed by routine histologic procedures. A quantitative estimate of the number of ovarian follicles and corpora lutea was made by a modification of the serial sectioning techniques of Pedersen and Peters.19 Three consecutive 6-J.L sections were cut at three different levels in the ovary. Thus, nine representative sections were used to obtain relative follicle values for the entire ovary. The data obtained from this method of evaluation are merely representative and do not indicate actual follicle numbers. The follicles were classified as follows: small follicles with one layer of granulosa cells (Pedersen's class l-3b); intermediate follicles with more than one layer of granulosa cells but no antrum (Pedersen's class 4-B); and large follicles with an antrum (Pederson's class 6-8). OVIDUCTAL FUNCTIONAL ANATOMY Two-month-old female offspring (five DES-O and five DES-lOa) were sacrificed by cervical dislocation. A polyethylene cannula (PE 0 gauge) attached to a l-ml syringe was inserted into the cervical os, and a solution of Coomassie blue dye (approximately 0 to 100 J.Ll) was injected into the cranial portion of the uterine lumen. The course of the dye was visualized under a dissecting microscope by transverse illumination. Fertility and Sterility

4 QI <Il :J 0 E "- 0 c> c: :J 0 >. QI ~ -0 0 c: QI > :;: 2.!:! :J E :J U Prenatal DES dose Cug/kg) a v I o Figure 1 Total reproductive capacity of mice exposed prenatally to DES. Mice were CD-1 female offspring exposed prenatally to DES on days 9 to 16 of gestation. Postnatal fertility was determined by a repetitive forced breeding technique and expressed as the total number of live young born per mouse over an 8-month (32-week) interval. The cumulative number of young per mouse is plotted on the y-axis, while the duration of forced breeding is plotted on the x-axis. This figure is based on a representative subset of the total experimental data and is a schematic representation of breeding characteristics in mice exposed prenatally to DES. SUPEROVULATION EXPERIMENTS Pregnant mare's serum gonadotropin (PMSG) obtained from Sigma Chemical Co., St. Louis, MO, was injected intraperitoneally ( IU/mouse) into 2-month-old control (six DES-O) and DESexposed female offspring (six DES 0.01, seven DES-I, two DES-10, and five DES-100). Fortyeight hours later, human chorionic gonadotropin (hcg) purchased from Sigma Chemical Co., St. Louis, MO, was administered intraperitoneally ( IU/mouse). Four animals from each dose group (except the DES-lO group, which had only two females available for study) were immediately placed with 2-month-old untreated males of pro v en fertility. The following morning, mating was determined by the presence of a vaginal plug. All female mice were sacrificed by cervical dislocation 16 hours after hcg injections, and their reproductive tracts were removed and examined for gross abnormalities. The oviducts were dis- sected free from the ovary and fat pad. Ovaries were fixed in Bouin's solution and processed for histologic examination. Oocytes were collected from the oviductal ampullae by previously described procedures. 2o Cumulus cells were dispersed by placement of the oocytes in a 1% hyaluronidase solution (Worthington Biochemical Corp., Freehold, NJ). Denuded ova were counted under the dissecting microscope and then examined for structural abnormalities by phase contrast microscopy before and after staining with methylene blue. STATISTICAL ANALYSIS For the forced breeding study, the natural pairing introduced by caging was taken into account by using Page's L-test 21 to assess the significance of the observed dose-related trends in the total number of live offspring. Dose levels DES-10 and DES-100 were not included in the analysis, since not all cages contained animals exposed at these levels. (Exclusion of these dose groups would obviously make the test more conservative, since so many of these treated animals were infertile.) Furthermore, the analysis was restricted to those cages in which all of the animals under consideration survived for the entire 32 weeks of forced breeding. (For the summary data reported in Table 1, however, all surviving animals in a given dose group were included.) In analyzing the data from the ovarian morphology experiment both a Kruskal-Wallis nonparametric one-way analysis of variance and a J onckeere trend test were performed, followed by a Mann-Whitney U test of pairwise treatmentcontrol differences where indicated. Data from the RIA and superovulation experiments were Table 2. Fertility of Female Mice Following Prenatal Exposure to DES and Cross-Fostering Mothers DES-10 d DES-lOO d Young" DES-10c DES-lOOc Total reproductive capacityb 17 6 o 1 19 afive females per group. bexpressed as cumulative number of live young per mouse after 9 weeks of forced breeding. cfemale offspring exposed prenatally to diethylstilbestrol (10 or 100 flg/kg) on days 9 to 16 of gestation. dpregnant CD-1 mice exposed to diethylstilbestrol (10 or 100 flglkg) on days 9 to 16 of pregnancy. McLachlan et a1. Fertility in DES-exposed mice 367

5 Table 3. Levels of Total Plasma Estrogens in Mice Exposed Prenatally to DEsa Prenatal treatment No. of offspring DES-O 8 DES- 9 DES-lOO 10 Estrogen pglml 9.0 ± b ± b ± 8.9 amice were 8-month-old females exposed prenatally to DES ( or 100 f.lglkg) on days 9 to 16 of gestation; values determined by RIA and refer to mean ± standard error of the mean of determinations made on plasma samples obtained from individual animals. bnot significantly different from controls. analyzed by analysis of variance procedures in a multiple-comparison framework. RESULTS When DES (DES-O, DES-0.01, DES-1, DES-2., and DES-) was injected subcutaneously into pregnant mice on days 9 through 16 of gestation, newborn offspring appeared normal, and body weights during the first 4 weeks of postnatal life were similar. Toxic effects of the drug exposure were characterized by a decrease in the number of litters and in the size of litters in the DES-10 and DES-100 dose groups. Severe embryotoxicity was associated with the highest dose (DES-100); more than half the mothers aborted or resorbed their litters. However, by 1 month of age all surviving offspring attained body weights comparable to those of control animals. At 1 month of age, when the female offspring were placed in a repetitive forced breeding study, the most striking effect observed was a doserelated decrease in reproductive capacity. The results of 8 months (32 weeks) of forced breeding experiments are shown in Figure 1 (the cumulative number of young per mouse is plotted on the y-axis, and the duration of forced breeding is plotted on the x-axis). Minimal subfertility was observed at DES-0.01, while essential sterility was often reached at DES-10 and DES-100. The data in Table 1 suggest that the dose-related decrease in cumulative number of offspring, which is significant at the 1 % level in each replicate, appears to be a reflection of both a reduced number of litters and smaller live litter sizes. In a separate experiment, we cross-fostered DES-exposed and control offspring to control and treated mothers to determine whether the observed decrease in fertility was a prenatal effect or contained a neonatal component. Table 2 summarizes the results from this experiment. The decrease in fertility apparently is observed only in mice exposed prenatally to DES. The time of vaginal opening was determined in control and DES-100 female offspring only. On day 21, no animals from either the control or DES-exposed group had vaginal openings. At 29 days of age, vaginal opening had occurred in 0% of the control females, compared with 100% ofthe DES-100 offspring. Vaginal opening occurred in 100% of the controls by day 3. Estrous cycles of the 1-month-old animals exposed prenatally to DES- or less did not differ markedly in duration or frequency from comparable controls. However, increased incidences of female hypospadias and extensive vaginal keratinization in DES-100 animals made determination of estrous cycles by vaginal cytologic study difficult in the 1- and 8-month-old animals. Therefore, circulating plasma estrogen levels were determined by RIA. Table 3 shows that at 8 months of age the plasma estrogen levels in the DES-O (controls) or DES- and DES-100 groups were not significantly different. Ovaries of non breeding 2-month-old female offspring exposed prenatally to either corn oil (control) or DES were examined histologically. The results are shown in Table 4. No significant overall effect or trend was seen for either follicles or corpora lutea. This absence of a dose-related Table 4. Ovarian Morphology of Female Mice Exposed Prenatally to DEsa Prenatal treatment DES-O DES-O.Ol DES-1 DES-I0 DES-lOO No. of offspring 4 Small 16. ± 4.3 b 13.4 ± ± ± ± 7.4 Intermediate 13.4 ± ± ± ± ± 3.9 No. of follicles Large 22.0 ± ± ± ± ±.2 Corpora lutea 6.7 ± ± ± ± ± 3.8 amice were 2-month-old female offspring treated prenatally with DES (0.01, 1, 10, or 100 f.lglkg/day) on days 9 to 16 of gestation. The quantitative estimate of the number of follicles was made by examining three consecutive 6-f.L sections cut at three different levels in the ovary. The data on this chart are representative and do not indicate actual follicle numbers. bmean ± standard deviation. 368 McLachlan et al. Fertility in DES-exposed mice Fertility and Sterility

6 Table. Ovarian Function in Mice Exposed Prenatally to DEsa Prenatal treatment DES-O DES-O.Ol DES-l DES-lO DES-I00 Mice ovulating Total eggs collected from oviduct No. of eggs/mouse d 4.1e 13.0d 14.6d ± 13.3 (100%)b ± 6.6 (69.7%) ± 8.9 (88.1%) ± 7.1 (2.4%) ± 3.6 (28.%) No. of mice mating" 4/4 4/4 4/4 2/2 4/4 amice were 2-month-old female offspring exposed prenatally to DES (0, 0.01, 1, 10, or 100 flg/kg/day) on days 9 to 16 of gestation and postnatally to PMSG ( IU) followed by hcg ( IU). bnumber of eggs per mouse expressed mean value ± standard deviation and as a percentage of that of controls. CRatio of number of females with vaginal plugs to the number of females mated. Four animals from each dose group were mated the morning after hcg injection (except the DES-I0 group, in which only two females ovulated). dp < 0.01, when compared with controls. enot significantly different from controls. trend for corpora lutea does not reflect the fact that two of the five DES-lO females and three of the four DES-lOO females failed to exhibit corpora lutea in their 6-f.L cross-sections. In addition to these differences, DES-exposed animals induced to ovulate by treatment with PMSG followed by hcg tended to have fewer oocytes recovered from the oviductal ampullae than corresponding control animals (Table ). All the mice that ovulated also mated, as determined by the presence of a vaginal plug or vaginal sperm the morning after exposure to a male. However, microscopic examination of the ova after superovulation revealed gross differences in their appearance. ova with the first polar body (Fig. 2) had sharp, distinct borders and were surrounded by an intact zona pellucida; whereas all the ova obtained from DES-lOO females were degenerating (Fig. 3). Malformations of the oviduct ofdes-loo animals were common structural alterations and posed problems in the ova collection (Fig. 4). Following instillation of dye into the upper uterine cavity, uterine dye retention was observed in of control female mice. However, in of females exposed in utero to DES-lOO, dye was observed to readily pass the uterotubal junction, transverse the oviduct, and fill the ovarian bursa. Another gross difference between control and DES-exposed animals at 8 months was the increased size of the cervix and the upper third of the vagina (Fig. ). This was particularly noticeable in the DES-lO and DES-lOO dose groups. animals,6,7 attention has been focu~ed on common benign structural abnormalities and their relationship to fertility in prenatally exposed offspring. 8, 9, 12-1 Previous reports from this laboratory described a mouse model for the transplacental effects of DES on the developing fetal reproductive tract and subsequent associated abnormalities. 6 The data reported in the present study demonstrate that prenatal exposure to relatively low doses of DES can adversely affect the fertility of female offspring. DES exposure resulted in a reduced number of total live offspring. Extrapolation of animal data to human beings is al- DISCUSSION Initial concern with the transplacental toxicity of DES was centered on the development of vaginal adenocarcinoma at puberty. Since this lesion is apparently rare in humans l - and experimental Figure 2 Photomicrograph of an ovum and the first polar body recovered from the ampulla of the oviduct of a control 2-month-old CD-l mouse receiving PMSG and hcg treatment. The zona pellucida is still intact, and the ovum has distinct borders. McLachlan et ai. Fertility in DES-exposed mice 369

7 Figure 3 Photomicrograph of an ovum recovered from the oviduct of a 2-month-old CD-l female offspring exposed prenatally to DES on days 9 through 16 of gestation at a daily dose of 100 f..lg/kg maternal body weight. PMSG and hcg were injected before the collection of ova. Blebs of various sizes and shapes inside the zona indicate a degenerating ovum. ways difficult. However, it should be noted that the highest dose used in this study (100 J..Lg/kg) did not differ markedly from that often administered therapeutically to pregnant women. The exact mechanisms responsible for decreased fertility are still unclear. Vaginal cytologic studies of l-month-old animals exposed to doses of DES- or lower demonstrated normal estrous cycles. Although puberty, as determined by vaginal opening date, was attained earlier in / high-dose DES animals, plasma estrogen levels and mating behavior suggested that ovarian estrogen production was within the control range of average values when assessed at a single time point. In experimental systems, estrogen has been shown to give rise to a disturbance in the hypothalamic differentiation during a critical state of development, resulting in a changed gonadotropin pattern Alterations in the hypothalamus resulting in irregular cycling cannot be ruled out as a contributing factor to infertility in offspring maintained in the forced breeding experiments.. Irregular cycling could be responsible for the early loss and lower numbers of corpora lutea in the high-dose animals. It should be pointed out that the method used to quantitate the various 370 McLachlan et al. Fertility in DES -exposed mice ovarian classes underestimates the total number of 'small follicles in the ovary. However, since comparisons are being made between ovaries of control and treated animals, the correlation should be valid. Further studies of ovarian follicular dynamics and pathologic alterations are necessary for determination of the relationship of the ovary to subfertility in the DES-exposed offspring. Preliminary data from this laboratory suggest an early depletion of oocytes from the ovary.2 Structural abnormalities in the reproductive tract certainly represent a major component of infertility in the DES-exposed offspring. In a previous study,6 Wolffian duct as well as Mullerian duct tissues were stimulated in utero by DES exposure. Often Wolffian remnants were cystic and hyperpl~stic in DES-IOO animals. 6 Thus, abnormally developing W olffian ducts may contribute to the abnormal oviductal and uterine architecture as well as to cervical enlargement in mice exposed prenatally to DES. Radiographic studies of women exposed prenatally to DES revealed similar upper genital tract abnormalities, particularly in the cervix and uterus. 8, 9 Cervical abnormali ties and T -shaped uteri appear to be frequently associated with DES exposure. Considering the oviductal malformations observed previ0usly in mice,6 and the current observation that dye injected into the uterine lumen of DES-treated mice readily passed the uterotubal junction, while in control mice it did not, the role of this anatomic region in observed ectopic pregnancies in DES-exposed women merits further investigation. In fact, the lower number of ova collected Figure 4 Photomicrograph of oviductal malformation in a CD-l mouse exposed prenatally to DES-IOO. The oviduct is wrapped around the ovary and appears to be dilated (hematoxylin and eosin, original magnification x 2). Fertility and Sterility

8 CONTROL DES-100 Figure Illustration of control and prenatally DES-exposed female reproductive tracts. Notice the structural alterations including cervicovaginal enlargement and oviductal malformations in the DES-exposed tissue. from the oviducts ofdes-loo mice may represent early transport of ova into the uterine lumen. In the present study, the uterine contents were not examined for ova. Acknowledgments. The authors gratefully acknowledge the technical assistance of Mr. Tracy Hanner and the excellent manuscript typing of Ms. Lauren King and Ms. Vickie Englebright. Mr. Thomas A. Clemmer ably contributed to the statistical analysis of the data. The estrogen radioimmunoassays were performed by Dr. David Schomberg, Duke University Medical Center, Durham, North Carolina. REFERENCES 1. Herbst AL, Ulfelder H, Poskanzer DC: Adenocarcinoma of the vagina: association of maternal stilbestrol therapy with tumor appearance in young women. N Engl J Med 284:878, Greenwald P, BarlowJJ, Nasca PC, Burnett WS: Vaginal cancer after maternal treatment with synthetic estrogens. N Engl J Med 28:390, Tsukada Y, Hewett WJ, Barlow JJ, Pickren JW: Clearcell adenocarcinoma (mesonephroma) of the vagina. Cancer 29:1208, Gilson MD, DiBona DD, Knab DR: Clear cell adenocarcinoma in young females. Obstet Gynecol 41:494, Herbst AL, Robboy SJ, Scully RE, Poskanzer DC: Clearcell adenocarcinoma of the vagina and cervix in young females: an analysis of 170 registry cases. Am J Obstet GynecoI119:713, McLachlan JA, Newbold RR, Bullock BC: Long-term effects on the female mouse genital tract associated with prenatal exposure to diethylstilbestrol. Cancer Res 40:3988, Walker B: Reproductive tract abnormalities in mice after prenatal exposure to DES. Teratology 21:313, Kaufman RH, Binder GL, Gray PM Jr, Adam E: Upper genital tract changes associated with exposure in utero to diethylstilbestrol. Am J Obstet Gynecol 128:1, Haney AF, Hammond CB, Soules MR, Creasman WT: Diethylstilbestrol-induced upper genital tract abnormalities. Fertil Steril 31:142, Pomerance W: Post-stilbestrol secondary syndrome. Obstet Gynecol 42:12, McLachlan JA: Prenatal exposure to diethylstilbestrol in mice: toxicological studies. J Toxicol Environ Health 2:27, Herbst AL, Hubby MM, Blough RR, Azizi F: A comparison of pregnancy experience in DES-exposed and DES-unexposed daughters. J Reprod Med 24:62, Berger MJ, Goldstein DP: Impaired reproductive performance in DES-exposed women. Obstet Gynecol :2, Schmidt G, Fowler WC, Talbert LM, Edelman DA: Reproductive history of women exposed to diethylstilbestrol in utero. Fertil Steril 33:21, Barnes AB, Colton T, Gundersen J, Noller KL, Tilley BC, Strama T, Townsend DE, Hatab P, O'Brien PC: Fertility and outcome of pregnancy in women exposed in utero to diethylstilbestrol. N Engl J Med.302:609, McLachlan JA, Newbold RR, Korach KS, Lamb JC IV, Suzuki Y: Transplacental toxicology: prenatal factors influencing postnatal fertility. In Developmental Toxicity, Edited by CA Kimmel, J Buelke-Sam. New York, Raven Press, 1981, p Champlin AK, DOIT DL, Gates AH: Determining the state of the estrous cycle in the mouse by the appearance of the vagina. BioI Reprod 8:491, Aksel S, Schomberg DW, Tyrey L, Hammond CB: Vasomotor symptoms, serum estrogens and gonadotropic levels in surgical menopause. Am J Obstet GynecoI126:16, Pedersen T, Peters H: Proposal for a classification of 00- cytes and follicles in the mouse ovary. J Reprod Fertil 17:, Rafferty KA: In Methods in Experimental Embryology of the Mouse. Baltimore, John Hopkins Press, 1970, p Hollander M, Wolfe DA: Nonparametric Statistical Methods. New York, John Wiley and Sons, Forsberg JG: Induction of conditions leading to cancer in the genital tract by estrogen during the differentiation phase of the genital epithelium. In Hormones and Embryonic Development, Advances in Biosciences. New York, Pergamon Press, 1974, p Takasugi N, Bern HA: Tissue changes in mice with persistent vaginal cornification induced by early postnatal treatment with estrogen. J Nat! Cancer Inst 33:8, Barraclough CA: Alterations in reproductive function following prenatal and early postnatal exposure to hormones. In Advances in Reproductive Physiology, Edited by A McLaren. London, Logos Press, 1968, p Newbold RR, Bullock BC, McLachlan JA: Ovarian and oviductal abnormalities in mice exposed prenatally to diethylstilbestrol. Work in preparation McLachlan et ai. Fertility in DES-exposed mice 371