Effects on Females

As described for the prenatally DES-exposed males, outbred CD-1 mice were treated with subcutaneous injections of DES on days 9-16 of gestation at doses ranging from 0.01 to 100 |g/kg during pregnancy. Mice were born on day 19 and female offspring were followed for effects on fertility and incidence of reproductive tract neoplasia later in life. As seen in Table 2, the effects of prenatal DES exposure on female mice and humans are comparable. Abnormalities such as reproductive tract dysfunction, structural malformations, and increased tumors in the reproductive tract were observed in both species.

Table 2. Similar developmental effects of prenatal exposure to DES in mice and humans -female offspring

Reproductive tract dysfunction Subfertility/infertility Structural malformations

Oviduct, uterus, cervix, vagina; paraovarian cysts of mesonephric origin Increased reproductive tract tumors Vaginal adenosis and adenocarcinoma

Fig. 1. Total reproductive capacity of mice exposed prenatally to DES. Mice were CD-1 female offspring exposed prenatally to DES on days 9-16 of gestation. Postnatal fertility was determined by a repetitive breeding technique and expressed as the total number of live young born per mouse over an 8 months (32 weeks) interval. The cumulative number of young per mouse is plotted on the x-axis. Note that there is a dose-related decrease in fertility at all DES doses and that even the mice exposed to low doses of DES exhibit decreased fertility as compared to corresponding control mice. This figure is reproduced from [27] with permission

Fig. 1. Total reproductive capacity of mice exposed prenatally to DES. Mice were CD-1 female offspring exposed prenatally to DES on days 9-16 of gestation. Postnatal fertility was determined by a repetitive breeding technique and expressed as the total number of live young born per mouse over an 8 months (32 weeks) interval. The cumulative number of young per mouse is plotted on the x-axis. Note that there is a dose-related decrease in fertility at all DES doses and that even the mice exposed to low doses of DES exhibit decreased fertility as compared to corresponding control mice. This figure is reproduced from [27] with permission

Fertility

To assess the effects of prenatal DES exposure on postnatal reproductive tract function, the fertility of DES female mice was determined using a continuous breeding protocol [27]. The most striking effect observed was a dose-related decrease in reproductive capacity, ranging from minimal subfertility at the lower DES doses to total sterility at the highest DES doses (Fig. 1). It is interesting to note that over the course of the breeding schedule, even the low doses of DES (0.01 ^g/kg/day) exhibited subfertility. The observed reduced reproductive capacity appeared to be a reflection of a decrease in the total number of litters and in litter sizes. A major component of the sterility seen in females that were given high doses of DES was in the oviduct and ovary; the number of ova recovered from the ampulla of the oviduct after induced ovulation was less than 30% that of the controls. Also, structural abnormalities were observed in the oviduct, uterus, cervix, and vagina, which contributed to subfertility. Together these data suggested that in utero exposure to DES, even at low doses, results in the permanent impairment of female mouse reproductive capacity. Numerous reports of altered pregnancy outcomes and decreased fertility in young women exposed in utero to DES [10], as well as accidental DES exposure to wildlife resulting in infertility, further point out the importance of these findings in mice, and demonstrate that environmental estrogens play an important role in decreased female fertility.

Cancer

To assess the long-term effects of prenatal DES exposure on the female, mice were sacrificed at 12 to 18 months of age, and reproductive tract tissues were studied for histological alterations. Histological examination revealed lesions throughout the reproductive tracts [28]. The vagina of DES-exposed mice was characterized by excessive keratinization and female hypospadias (urethra opens into the vagina rather than the vulva), and at the highest dose (100 |g/kg), 25% had epidermoid tumors of the vagina. Vaginal adenocarcinoma was seen in the 2.5, 5, and 10 |g/kg DES dose groups although its frequency was rare. The cervix of DES-exposed offspring was often enlarged, but the size of cervical lumen was not different from control untreated mice. Stromal stimulation was responsible for the enlargement in the cervical region. Further evidence of stromal alterations were documented by a low prevalence of benign (leiomyomas) and malignant (stromal cell sarcomas and leiomyosar-comas) tumors in the cervix. In the uterus, epithelial and stromal stimulation were also observed, and cystic endometrial hyperplasia was common even in the lower DES-dose animals; a low incidence of benign (leiomyomas) and malignant (stromal cell sarcomas and leiomyosarcomas) uterine tumors was also observed. The ovaries of prenatally DES-treated females were more cystic than controls; at the highest dose (100 |g/kg), ovarian tumors were noted and 100% of the oviducts were inflamed and congenitally malformed. Taken together, these data suggested that in utero exposure to DES results in not only subfertil-ity/infertility and reproductive tract abnormalities including malformation, but also an increase in reproductive tract tumors. While this increase was low, it was still a significant increase as compared to untreated mice.

Since data from the literature suggested that developmental exposure to DES just during neonatal life resulted in a high incidence of vaginal abnormalities [29-31], we compared prenatal and neonatal DES exposure and resulting abnormalities. Outbred CD-1 mice were treated neonatally with DES (2 |g/pup/ day) on days 1-5. This treatment resulted in a high incidence (90-95%) of uterine cancer when the mice aged to 18 months [32,33]. Other species including rats and hamsters [34, 35] neonatally exposed to DES have also been reported to have a high incidence of reproductive tract abnormalities including uterine tumors. Thus, the neonatal mouse model replicates tumors seen in other experimental models and therefore may be predictive of the carcinogenic potential of estrogens in the adult human uterus as it ages. A representative his-tological picture of the murine uterine carcinoma associated with neonatal treatment with DES is shown in Fig. 2. These tumors rarely metastasized but, in aged animals (24 months of age or older), the lesions sometimes showed spread

Fig. 2. A, B Uterine carcinoma - the most common pattern seen is irregularly shaped glands that vary from dilated structures to almost solid nests of cells (T) (x10). B Uterine carcinoma - high power view of A showing solid nests of cells (T) and columnar and cuboidal cells lining glandular elements of the tumor (x20)

Fig. 2. A, B Uterine carcinoma - the most common pattern seen is irregularly shaped glands that vary from dilated structures to almost solid nests of cells (T) (x10). B Uterine carcinoma - high power view of A showing solid nests of cells (T) and columnar and cuboidal cells lining glandular elements of the tumor (x20)

to para-aortic lymph nodes or direct extension to contiguous organs. It is significant that the mouse tumors progress through the same morphological and biological continuum of hyperplasia to atypical hyperplasia to neoplasm as seen in women. Uterine carcinoma were not observed in the uterus of untreated control CD-1 mice at corresponding ages, nor after similar adult short-term exposure to estrogens, suggesting that the developmental stage of the uterus and the time of estrogen exposure were important factors in the development of the lesions.

Birth

Human Fetus

Reproductive Tract Differentiation

Birth

Birth

Birth

Mouse Fetus

Reproductive Tract Differentiation

Reproductive Tract Differentiation

Teratogenic Carcinogenic

Fig. 3. Comparative developmental events in reproductive tract differentiation

Teratogenic Carcinogenic

Fig. 3. Comparative developmental events in reproductive tract differentiation

For the female, perinatal exposure (prenatal or neonatal) to DES resulted in subfertility/infertility and increased incidences of reproductive tract tumors (benign and malignant). For the most part, tumor incidence was increased, but still low, after prenatal treatment, whereas neonatal exposure caused a higher incidence of tumors. Teratogenic (malformations) abnormalities were higher in animals prenatally exposed rather than neonatally exposed. Since developmental events in the reproductive tract occurring in early neonatal life for the mouse occur entirely prenatally in humans, the perinatal mouse (prenatal and neonatal) is useful to model human fetal development. This is schematically explained in Fig. 3. This figure illustrates, as previously stated, that the timing of exposure or the stage of tissue differentiation determines the subsequent resulting abnormalities.

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