Prevalence of Premature Ovarian Failure in Women with Tuberous Sclerosis

Tuberous Sclerosis Complex (TSC) is an autosomal dominant tumor suppressor disorder characterized by hamartomas, or benign growths, in various organ systems. Inactivating mutations in either the TSC1 or the TSC2 gene cause most cases of TSC. Recently, the use of ovarian specific conditional knock-out mouse models has demonstrated a crucial role of the TSC genes in ovarian function. Mice with complete deletion of Tsc1 or Tsc2 showed accelerated ovarian follicle activation and subsequent premature follicular depletion, consistent with the human condition premature ovarian failure (POF). POF is defined in women as the cessation of menses before the age of 40 and elevated levels of follicle stimulating hormone (FSH). The prevalence of POF is estimated to be 1%, affecting a substantial number of women in the general population. Nonetheless, the etiology of most cases of POF remains unknown. Based on the mouse model results, we hypothesized that the human TSC1 and TSC2 genes are likely to be crucial for ovarian development and function. Moreover, since women with TSC already have one inactivated TSC gene, we further hypothesized that they may show a higher prevalence of POF. To test this hypothesis, we surveyed 1000 women with TSC belonging to the Tuberous Sclerosis Alliance, a national support organization. 182 questionnaires were analyzed for information on menstrual and reproductive function, as well as TSC. This self-reported data revealed 8 women (4.4%) with possible POF, as determined by menstrual history report and additional supportive data. This prevalence is much higher than 1% in the general population. Data from all women suggested other reproductive pathology associated with TSC such as a high rate of miscarriage (41.2%) and menstrual irregularity of any kind (31.2%). These results establish a previously unappreciated effect of TSC on women’s reproductive health. Moreover, these data suggest that perturbations in the cellular pathways regulated by the TSC genes may play an important role in reproductive function.

The association between maternal use of spermicides, condoms, intra-uterine devices or progesterone and major structural birth defects

Birth defects occur in 1 of every 33 babies born in the United States, and are the leading cause of infant death. Mothers using contraceptives that become pregnant may continue to use their contraceptives after their first missed menstrual period, thus exposing their baby in utero to the contraceptive product. Progesterone is also sometimes prescribed during the first trimester of pregnancy to mothers with a history of miscarriages or infertility problems. To ensure the safety of these products, it is important to investigate whether there is an increased occurrence of babies born with birth defects to mothers using various contraceptive methods or progesterone in early pregnancy. Using data from the National Birth Defects Prevention Study (NBDPS), an ongoing multi-state, population based case-control study, this study assessed maternal exposures to IUDs, spermicides, condoms and progesterone in early pregnancy. ^ Progesterone used for threatened miscarriage during the first three months of pregnancy was associated with an increased occurrence of hypoplastic left heart (adjusted odds ratios (OR) 2.24, 95% CI 1.13-4.21), perimembranous ventricular septal defects (OR 1.64, 95% CI 1.10-2.41), septal associations (OR 2.52, 95% CI 1.45-4.24), esophageal atresia (OR 1.82, 95% CI 1.04-3.08), and hypospadias (OR 2.12, 95% CI 1.41-3.18). Mothers using progesterone for injectable contraception had increased (OR > 2.5), but insignificant odds ratios for anencephaly, septal associations, small intestinal atresias and omphalocel. Progesterone used for fertility was not associated with an increased occurrence of any birth defects examined. ^ Mothers using progesterone for fertility assistance and threatened miscarriage were very similar with respect to their demographics and pregnancy history. They also both reported similar types of progesterone. Thus, if progesterone was a causal risk factor for birth defects we would have expected to observe similar increases in risk among mothers using progesterone for both indications. Because we predominantly observed increased associations among mothers using progesterone for threatened miscarriage but not fertility assistance, it is possible the increased associations we observed were confounded by indication (i.e. progesterone was administered for vaginal bleeding which occurred as a sequelae to the formation of a congenital anomaly. ^ No significant increased associations were observed between maternal spermicide use during pregnancy and 26 of 27 types of structural malformations. While multiple statistical tests were performed we observed first trimester maternal spermicide use to be associated with a significant increased occurrence of perimembranous ventricular septal defects (OR 2.21, 95% CI 1.16-4.21). A decreased occurrence (OR < 1.0) was observed for several categories of birth defects among mothers who conceived in the first cycle after discontinuing the use of spermicides (22 of 28) or male condoms (23 of 33). ^ Overall the percent of IUD use was similar between mothers of controls and mothers of all cases in aggregate (crude OR 1.05, 95% CI 0.61-1.84). Power was limited to detect significant associations between IUD use and birth defects, however mothers using an IUD in the month immediately prior to conception or during pregnancy were not associated with an increase of birth defects. Limb defects and amniotic band sequence previously reported to be associated with IUD use during pregnancy were not found to occur among any mothers reporting the use of an IUD during pregnancy.^

Monitoring of industrial exposure for chloracne

This study (1) established comedogenicity dose response curves for the pure compounds of 3,3$\sp\prime$,4,4$\sp\prime$-tetrachloroazobenzene (TCAB) and 3,3$\sp\prime$,4,4$\sp\prime$-tetrachloroazoxybenzene (TCAOB) individually and as a couple-compound using a rabbit ear model; (2) used a rabbit ear model to establish comedogenicity potential for TCAB and TCAOB as they existed in a given industrial herbicide manufacture process; (3) evaluated actual environmental contamination in a herbicide industrial setting by air monitoring and wipe sampling; (4) biologically monitored potentially exposed workers for alterations in follicular orifice size as an index of actual exposure to chloracnegenic compounds; and (5) biologically monitored potentially exposed workers for changes in weight, cholesterol, triglycerides and blood sugar.^ A silastic monomer mold (an objective measure) was used to measure change in follicular orifice size over time. This required taking impressions of (1) skin of the forehead and right and left malar crescents of workers and (2) the skin of the external ear of the rabbit. Molds were stained using a solution of hematoxylin and digitized using a Nikon UFX microscope (magnification 300 X), a drawing tube and a digitizing tablet attached to an IBM Personal Computer. Comedogenicity assays were used to establish dose-response curves for TCAB, TCAOB and the couple-compound TCAB + TCAOB.^ No evidence of chloracne or toxicity was observed in any of the workers. Nor, was there a statistically significant increase in size of follicular orifice means measured over time. This was attributed to extensive personal and environmental hygiene programs along with teaching the workers about chloracne, its cause and its prevention. These programs may have been the greatest factor in preventing the development of chloracne in this group of workers. Monitoring of the plant environment showed relatively high concentrations of the couple-compound (TCAB + TCAOB). Comedogenicity assays showed a linear dose-response relationship over time for TCAB, TCAOB and the couple-compound. An antagonistic action was found for the TCAB/TCAOB of the couple-compound; such action may provide some protection to workers in this type of setting. It is speculated that the observed antagonistic action may be due to the difference in binding affinities of TCAB/TCAOB for receptor sites. ^

Apoptosis and the molecular complementation of bcl-2, myc and p53 in multistep lymphomagenesis

Follicular lymphoma is the most common lymphoid malignancy in humans. The bcl-2 transgenic mice, which mimic the human follicular lymphoma, initially exhibit a polyclonal hyperplasia due to the overriding of apoptosis by deregulated bcl-2. After a latency period of 15 month 20% of the animals developed clonal lymphomas. Approximately, 50% of these high grade lymphomas presented chromosomal translocations involving c-myc, suggesting that deregulation of this gene is important in the complementation with bcl-2. E$\mu$-myc x bcl-2 double transgenic mice were generated to assess the ability of this two genes to complement in an in vivo system. The double transgenic mice presented a shortened latency (3-4 weeks) and higher incidence of tumor development. Quantification of the extent of programmed cell death indicated that bcl-2 can abrogate the high rate of apoptotic cell death that results from myc deregulation. Bcl-2-Ig, E$\mu$-myc, and bcl-2/E$\mu$-myc lymphomas were examined using PCR-SSCP to detect the presence of p53 mutations in exons 5-9. A high incidence of p53 mutations in E$\mu$-myc lymphomas suggested that inactivating lesions of p53 may represent an important step in the genetic complementation of c-myc in lymphomagenesis. Surprisingly, p53 mutations were quite uncommon in bcl-2 lymphomas suggesting that inactivating mutations of p53 and overexpression of bcl-2 may not cooperate in lymphoma progression. To assess this question, we generated mice that contained a deregulated bcl-2 gene and were nullizygous for p53 (p53KO). No reduction in the tumor latency was observed in the p53KO/bcl-2-Ig hybrid mice when compared with p53 KO mice. Using splenic mononuclear cells isolated from p53KO mice and bcl-2 transgenic mice we demonstrated that bcl-2 suppresses p53 mediated apoptosis in response to DNA damage initiated by $\gamma$-radiation even though p53 protein is induced normally in the bcl-2 overexpressing cells. Western analysis of the expression of p53 target proteins after $\gamma$-radiation showed a correlation between the p53-dependent induction of bax protein after radiation and the ability of p53 to mediate apoptosis. ^

Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^