What’s your diagnosis ?
32 year-old patient presents for an ultrasound scan at an infertility clinic. The patient has a 6 year history of infertility.
What’s your diagnosis ?
32 year-old patient presents for an ultrasound scan at an infertility clinic. The patient has a 6 year history of infertility.
A small T-shaped uterus with cavity constrictions is common in DES daughters
A rational systematic evaluation is essential to the management of a couple with repeated early pregnancy wastage. Psychologic support in the form of frequent discussions and sympathetic counseling are crucial to the successful evaluation and treatment of the anxious couple. A prompt and orderly evaluation will relieve anxiety. When no etiologic factor is identified, a 60% to 80% fetal salvage rate may be expected. Once a patient conceives, serial ultrasonography, beta-hCG determination, and estradiol determination may be useful in detecting the stage of the embryonic death if subsequent abortion occurs. A karyotypic analysis of the products of conception should be performed if fetal loss occurs.
PIP: This review of the current literature on the clinical management of repeated early pregnancy wastage focuses on several etiologic factors (i.e., genetic, medical, immunologic, endocrine, psychogenic, environmental, occupational, infectious, and uterine) which have been noted to result in repeated pregnancy wastage. Suggestions for further clinical study are outlined where appropriate, and a rational approach to clinical evaluation and management is provided, based on the interpretation of the state of the art. The frequency of clinically recognized spontaneous abortion in the general population has been estimated to range between 15-20%. The actual spontaneous abortion rate is difficult to determine due to the fact that some patients do not seek medical services and abort completely at home. Despite the present uncertainty concerning the actual risk of recurrent abortion, most clinicians agree that repeated early spontaneous pregnancy wastage (i.e., repeated pregnancy loss) is defined as the occurrence of 3 or more pregnancy losses prior to the 20th week of gestation. From cytogenetic studies of aborted products of conception, chromosomal abnormalities account for between 50-60% of spontaneous abortions in the 1st trimester of pregnancy. Most of the chromosomal aberrations involved in spontaneous abortions have been presumed to be due to random events that are not necessarily repetitious. Sporadic chromosomal errors account for approximately 30% of spontaneous pregnancy losses, and repeated pregnancy loss under these conditions would therefore occur as a matter of chance and would not be predictive of future pregnancy loss. Several medical diseases have been implicated in causing habitual abortion, and these include systemic lupus erythematosus, congenital cardiac disease, and renal disease. The severity of the disease correlates best with fetal wastage. An absence of blocking antibodies within the serum of women with repeated abortions was reported by Rocklin et al. A review of the literature shows that only an association exists between psychologic disturbances and habitual abortion. Intrauterine infection may result in early pregnancy wastage, and fetal death may result from an acute overwhelming infection. It has long been recognized that congenital anomalies of the uterus have been responsible in some instances for reproductive failure. The gynecologist must consider the time of initiation of an evaluation of a patient with reproductive loss. Any evaluation must include a complete history and a karyotypic analysis with fluorescent banding of both partners, a hysterogram, and a properly timed endometrial biopsy. In the authors’ experience, about 50% of patients with repeated pregnancy loss have no discernible etiologic factor. Subsequent early pregnancy should be carefully monitored in these patients. When no etiologic factor is identfied, a 60-80% fetal salvage rate may be expected.
Kaufman and associates performed hysterosalpingographic examinations of 267 women who had been exposed in utero to diethylstilbestrol (DES). One hundred
eighty-five (69%) of these women demonstrated an abnormality, and 82 women had normal findings. The pregnancy outcome among 93 women who conceived was significantly worse than that found in a control population of 87 women. Abnormalities on the hysterogram included a small T-shaped uterus with cavity constrictions.
The T-shaped uterus is the most commonly noted abnormality. Haney and Hammond recorded various uterine measurements, which revealed a reduced surface area of the endometrium of these patients, as compared with a suitable control population. Nevertheless, there was no increased incidence of pregnancy loss in patients enrolled in the National Cooperative Diethylstilbestrol Adenosis Project. Rosenfeld and Bronson noted that 3 of 25 DES-exposed women reported repeated abortion of three or more pregnancies. One of these patients conceived without therapy and carried to term. Thus, there is little evidence that the uterine defects associated with DES exposure in utero are associated with recurrent early pregnancy wastage.
Some epigenetic alterations that influence cancer risk are inherited through the germline from the DES-exposed to offspring and are observed in multiple DES generations of victims
Epigenetic alterations of the genome such as DNA promoter methylation and chromatin remodeling play an important role in tumorigenesis. These modifications take place throughout development with subsequent events occurring later in adulthood. Recent studies, however, suggest that some epigenetic alterations that influence cancer risk are inherited through the germline from parent to child and are observed in multiple generations. Epigenetic changes may be inherited as Mendelian, non-Mendelian, or environmentally induced traits. Here, we will discuss Mendelian, non-Mendelian, and environmentally induced patterns of multigenerational epigenetic alterations as well as some possible mechanisms for how these events may be occurring.
One example of multiple generations in families showing effects of an environmental agent are daughters of mothers who were exposed to diethylstilbestrol (DES) during the first trimester.
The daughters show developmental abnormalities and an increased risk of developing a rare type of clear-cell adenocarcinoma. DES daughters also show a 2.5-fold increase in breast cancer risk after 40 years of age. To prove that this indeed is an inherited transgenerational effect, granddaughters and great granddaughters of the exposed mothers will need to show a DES phenotype. This analysis has not yet been completed.
Mouse studies have shown that the F2 generation from a DES-exposed pregnant female had strikingly similar effects as the F1 generation, including abnormal uterine development and uterine cancer. The proposed mechanism of action of DES is aberrant CpG methylation of key uterine cancer genes. The changes in CpG methylation may be stable throughout gametogenesis, providing insight into the transgenerational effects of DES.
Long-term Effects of Exposure to Diethylstilbestrol
The present survey represents a preliminary look at possible health effects of prenatal exposure to DES in humans beyond those already known. As such, it should be considered as a guide to areas needing further investigation in less biased samples. The findings, however, are consistent with those of experimental studies in animals and indicate a need for follow-up in the human cohort. Of particular note are conditions that suggest impaired immune function, such as infectious illness, allergic and autoimmune conditions, and malignant tumors.
Studies of certain genetic strains of mice with exposure to DES during the critical neonatal period of immune system ontogeny-analogous to the first trimester of human pregnancy-show persistent, lifelong immunosuppression. The main DES effect is a reduced number of Thelper cells, important for the induction and regulation of many immune responses. B-cell response is impaired in assays requiring T-cell mediation but is normal if corrected for T-helper numbers. Experiments with varying combinations of B and T cells in vitro show that the defect is in T-cell number but not function.
Mice with neonatal exposure also have a reduced number of natural killer cells thought to recognize and kill certain tumor cells. Following injection with a classic carcinogen (3-methylcholanthrene [MCA]), DES-exposed mice show a reduced ability to resist tumors; MCA-induced sarcomas appear in greater numbers and at a faster rate.
Our findings regarding asthma, arthritis, and lupus are also consistent with those of a small study of human peripheral blood lymphocytes suggesting a hyperreactive immune response in women with in utero exposure to DES. Another small study of the daughters suggested possible functional alterations of natural killer cells. Even more suggestive, our findings are consistent with those of a recent preliminary report from the largest ongoing follow-up of DES daughters, the federally funded Diethylstilbestrol Adenosis Project. This report indicates about a twofold increase in autoimmune conditions in women with prenatal exposure compared with controls. One small study of daughters who had had DES associated cancer or reproductive problems showed no consistent increase in the rates of infectious disease but a suggestive increase in the rates of autoimmune disease compared with controls.
Evaluating immunologic consequences in women and men with DES exposure will be complicated by possible genetic contributions and by varying dosage and timing of the prenatal exposure. Furthermore, health consequences might become detectable only as the population ages, when the immune system generally declines in competence.
Possible pathologic disorders of the prostate, as noted in our survey, were also noted in experimental models of DES effects in rodents.In preliminary studies of mice with neonatal DES exposure, evidence of cytologic malignancy in the area of the prostate appeared only in the experimentally treated animals. Recent experiments in which human fetal prostate tissue was grafted into DES-treated and untreated athymic nude mice and then allowed to continue growing revealed ductal dilation and persistent distortion of ductal architecture; these conditions could contribute to early or increased development of prostatic neoplasms (S. Mee, G.R. Cunha, C.V. Yonemura, et al, “The Effects of Diethylstilbestrol on Human Prostate Development,” unpublished data, 1987).
Further inquiry should also focus on a possible increased prevalence of elevated prolactin levels among DES daughters. Substantial endocrine alterations occur in rodents; more specifically, experimental studies show that perinatal DES exposure results in a disruption of hypothalamic-pituitary feedback systems, including the regulation and production of prolactin. While endocrine effects of a similar magnitude are not apparent within the human cohort, there could be an increase of more subtle functional alterations in daughters or sons (or both) with exposure to DES. Although difficult to measure, abnormal endocrine function could be contributing to a diminished reproductive capacity in ways beyond the more apparent and well-documented structural anomalies. Two small studies of plasma hormones in the daughters suggest abnormalities that may reflect a disturbance of hypothalamic-pituitary-ovarian function. An additional study suggests that hyperprolactinemia may be a significant factor in infertility in daughters with DES exposure.
Comprehensive follow-up of DES-exposed daughters and sons is required to answer questions about the long-term consequences of prenatal exposure. As the cohort ages and reaches new “milestones” of increased health risks, such follow-up can contribute to a more adequate assessment of this population’s risks and to the early detection of various health conditions that may be affected by prenatal DES exposure and be responsive to treatment. In addition, an increased knowledge of the health consequences associatedwith DES exposure addresses a broader scientific need to examine fully the results of this reproductive exposure. Women and men with in utero exposure to DES constitute a unique, identified cohort from which much can be learned about the hormonal effects on both normal and abnormal human development. This cohort can provide a greater understanding of developmental biology, sex differentiation, and pathologic processes in humans. Beyond the basic knowledge to be gained are implications for therapeutic substances in use currently or considered for the future.
In utero DES exposure associated with a hyper-reactive immune response during the reproductive years
In order to study the effect of in utero diethylstilbestrol (DES) exposure on the immune system of adult women, the blastogenic response of peripheral blood lymphocytes to two mitogens was compared in eight DES-exposed patients and in eight age-matched controls with normal menstrual cycles and proven fertility.
As measured by the uptake of 3H-thymidine (mean [+/- standard error]), response to the T-cell mitogen phytohemagglutin (PHA) was significantly higher (P less than 0.002) in cells of DES-exposed women (88.6 +/- 5.7 X 10(3) cpm) than in controls (44.0 +/- 8.9 X 10(3) cpm) at the lowest dose of mitogen tested (0.125 microgram/ml). Moreover, lymphocytes of DES-exposed subjects showed maximal blastogenic response to PHA at a concentration (0.125 microgram/ml) two to four times lower (P less than 0.002) than controls (0.25 microgram/ml to 0.5 microgram/ml). Cells of both DES-exposed subjects and controls were maximally responsive to pokeweed mitogen (PWM) at the lowest dose tested (0.625 microgram/ml).
These findings suggest that in utero DES exposure is associated with a hyper-reactive immune response during the reproductive years.
DES effect on stimulation of the reticuloendothelial system
The observations that limited neonatal diethylstilbesterol (DES) exposure in mice produces persistent natural killing defects and that natural killer (NK) cells have an origin early in gestation suggested the possibility that NK abnormalities may exist in in utero DES-exposed women.
However, when compared to controls, these women showed slightly higher NK activity with no evidence of stimulation by accessory mononuclear cells. Altered natural killing cannot be invoked in this population as a contributing factor to increased cancer risk.
DES Daughters have alterations in T-cell-mediated immunity
Between 1940 and 1970, 1.5 million female fetuses were exposed to diethylstilbestrol in utero. Numerous deleterious effects on reproductive anatomic and physiologic characteristics have been documented in these women. However, the effects of this exposure on nonreproductive systems, which may have lifelong consequences as this cohort of women progresses beyond the childbearing years, have received little attention. On the basis of an earlier preliminary observation of altered immune reponse, we hypothesized that diethylstilbestrol-exposed women may show abnormalities in T-cell-mediated immune response.
Thirteen women exposed to diethylstilbestrol in utero were compared with 13 age- and menstrual cycle phase-matched control subjects with respect to the in vitro T-cell response to the mitogens phytohemagglutinin, concanavalin A, and interleukin 2.
As compared with controls, tritiated thymidine incorporation by T cells harvested from diethylstilbestrol-exposed women was increased 3-fold over a range of concentrations in response to concanavalin A (P <.001), increased by 50% over a range of concentrations in response to phytohemagglutinin (P <.001), and increased 2-fold in response to the endogenous mitogen interleukin 2 (P <.05).
In vitro evidence suggests that women exposed to diethylstilbestrol have alterations in T-cell-mediated immunity. These changes require further attention with regard to their characterization, their role in the pathogenesis of cancer and autoimmunity, and their presence in normal women exposed to diethylstilbestrol in utero.
A model for the trans-generational effects of diethylstibesterol on uterine development and cancer
Hsp90 is a chaperone for over 100 ‘client proteins’ in the cell, most of which are involved in signaling pathways. For example, Hsp90 maintains several nuclear hormone receptors, such as the estrogen receptor (ER), as agonist-receptive monomers in the cytoplasm.
In the presence of agonist, Hsp90 dissociates and the receptors dimerize, enter the nucleus and ultimately activate transcription of the target genes. Increasing evidence suggests that Hsp90 also has a role in modifying the chromatin conformation of many genes. For example, Hsp90 has recently been shown to increase the activity of the histone H3 lysine-4 methyltransferase SMYD3, which activates the chromatin of target genes. Further evidence for chromatin-remodeling functions is that Hsp90 acts as a capacitor for morphological evolution by masking epigenetic variation. Release of the capacitor function of Hsp90, such as by environmental stress or by drugs that inhibit the ATP-binding activity of Hsp90, exposes previously hidden morphological phenotypes in the next generation and for several generations thereafter.
The chromatin-modifying phenotypes of Hsp90 have striking similarities to the trans-generational effects of the ER agonist diethylstilbesterol (DES). Prenatal and perinatal exposure to DES increases the predisposition to uterine developmental abnormalities and cancer in the daughters and granddaughters of exposed pregnant mice.
In this review, we propose that trans-generational epigenetic phenomena involving Hsp90 and DES are related and that chromatin-mediated WNT signaling modifications are required. This model suggests that inhibitors of Hsp90, WNT signaling and chromatin-remodeling enzymes might function as anticancer agents by interfering with epigenetic reprogramming and canalization in cancer stem cells.
Role of Epigenetic Regulation
Currently, there is a remarkable lack of knowledge regarding the involvement of chromatin assembly in the process by which adverse environmental exposures increase the overall risk of UF development. The precise mechanism underlying EDC-dependent effects on myometrial cell physiology are not adequately understood.
Uterine Fibroids [UF(s), AKA: leiomyoma] are the most important benign neoplastic threat to women’s health. They are the most common cause of hysterectomy imposing untold personal consequences and 100s of billions of healthcare dollars, worldwide. Currently, there is no long term effective FDA-approved medical treatment available, and surgery is the mainstay.
The etiology of UFs is not fully understood. In this regard, we and others have recently reported that somatic mutations in the gene encoding the transcriptional mediator subunit Med12 are found to occur at a high frequency (∼85%) in UFs. UFs likely originate when a Med12 mutation occurs in a myometrial stem cell converting it into a tumor-forming stem cell leading to a clonal fibroid lesion.
Although the molecular attributes underlying the mechanistic formation of UFs is largely unknown, a growing body of literature implicates unfavorable early life environmental exposures as potentially important contributors. Early life exposure to EDCs during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life. Neonatal exposure to the EDCs such as diethylstilbestrol (DES) and genistein during reproductive tract development has been shown to increase the incidence, multiplicity and overall size of UFs in the Eker rat model, concomitantly reprogramming estrogen-responsive gene expression.
Importantly, EDC exposure represses enhancer of zeste 2 (EZH2) and reduces levels of histone 3 lysine 27 trimethylation (H3K27me3) repressive mark through Estrogen receptor/Phosphatidylinositide 3-kinases/Protein kinase B non-genomic signaling in the developing uterus. Considering the fact that distinct Mediator Complex Subunit 12 (Med12) mutations are detected in different fibroid lesions in the same uterus, the emergence of each Med12 mutation is likely an independent event in an altered myometrial stem cell. It is therefore possible that a chronic reduction in DNA repair capacity eventually causes the emergence of mutations such as Med12 in myometrial stem cells converting them into fibroid tumor-forming stem cells, and thereby leads to the development of UFs.
Advancing our understanding of the mechanistic role epigenetic regulation of stem cells plays in mediating risk and tumorigenesis will help in pointing the way toward the development of novel therapeutic options.
Diethylstilbestrol alters positive and negative selection of T cells in the thymus and modulates T-cell repertoire in the periphery
In the current study, we investigated the effects of DES on T-cell differentiation in the thymus using the HY-TCR transgenic (Tg) mouse model in which the female mice exhibit positive selection of T cells bearing the Tg TCR, while the male mice show negative selection of such T cells.
In female HY-TCR-Tg mice, exposure to DES showed more pronounced decrease in thymic cellularity when compared to male mice. Additionally, female mice also showed a significant decrease in the proportion of double-positive (DP) T cells in the thymus and HY-TCR-specific CD8+ T cells in the periphery. Male mice exhibiting negative selection also showed decreased thymic cellularity following DES exposure. Moreover, the male mice showed increased proportion of double-negative (DN) T cells in the thymus and decreased proportion of CD8+ T cells. The density of expression of HY-TCR on CD8+ cells was increased following DES exposure in both females and males. Finally, the proliferative response of thymocytes to mitogens and peripheral lymph node T cells to male H-Y antigen was significantly altered in female and male mice following DES treatment.
Taken together, these data suggest that DES alters T-cell differentiation in the thymus by interfering with positive and negative selection processes, which in turn modulates the T-cell repertoire in the periphery.