Dicer is required for female reproductive tract development and fertility in the mouse.

Dicer encodes a riboendonuclease required for microRNA biosynthesis. Dicer was inactivated in Müllerian duct mesenchyme-derived tissues of the reproductive tract of the mouse, using an Amhr2-Cre allele. Although Amhr2-Cre; Dicer conditional mutant males appeared normal and were fertile, mutant females were infertile. In adult mutant females, there was a reduction in the size of the oviducts and uterine horns. The oviducts were less coiled compared to controls and cysts formed at the isthmus near the uterotubal junction. Unfertilized, degenerate oocytes were commonly found within these cysts, indicating a defect in embryo transit. Beads transferred into the mutant oviduct failed to migrate into the uterus. In addition, blastocysts transferred directly into the mutant uterus did not result in pregnancy. Histological analysis demonstrated that the mutant uterus contained less glandular tissue and often the few glands that remained were found within the myometrium, an abnormal condition known as adenomyosis. In adult mutants, there was ectopic expression of Wnt4 and Wnt5a in the luminal epithelium (LE) and glandular epithelium (GE) of the uterus, and Wnt11 was ectopically expressed in GE. These results demonstrate that Dicer is necessary for postnatal differentiation of Müllerian duct mesenchyme-derived tissues of the female reproductive tract, suggesting that microRNAs are important regulators of female reproductive tract development and fertility.

Life and death of the embryonic female reproductive tract

Regardless of genetic sex, amniotes develop two sets of genital ducts, the Wolffian and Müllerian ducts. Normal sexual development requires the differentiation of one duct and the regression of the other. I show that cells in the rostral most region of the coelomic epithelium (CE) are specified to a Müllerian duct fate beginning at Tail Somite Stage 19 (TS19). The Müllerian duct (MD) invaginates from the CE where it extends caudally to and reaches the Wolffian duct (WD) by TS22. Upon contact, the MD elongates to the urogenital sinus separating the WD from the CE and its formation is complete by TS34. During its elongation, the MD is associated with and dependent upon the WD and I have identified the mechanism for MD elongation. Using the Rosa26 reporter to fate map the WD, I show that the WD does not contribute cells to the MD. Using an in vitro recombinant explant culture assay I show that the entire length of the MD is derived from the CE. Furthermore, I analyzed cell proliferation and developed an in vitro assay to show that a small population of cells at the caudal tip proliferates, laying the foundation for the formation of the MD. I also show that during its formation, the MD has a distinctive mesoepithelial character. The MD in males regresses under the influence of Anti-Müllerian Hormone (AMH). Through tissue-specific gene inactivation I have identified that Acvr1 and Bmpr1a and Smad1, Smad5 and Smad8 function redundantly in transducing the AMH signal. In females the MD differentiates into an epithelial tube and eventually the female reproductive tract. However, the exact tissue into which the MD differentiates has not been determined. I therefore generated a MD specific Cre allele that will allow for the fate mapping of the MD in both females males. The MD utilizes a unique form of tubulogenesis during development and to my knowledge is the only tubule that relies upon a signal from and the presence of another distinct epithelial tube for its formation.^

Regulation of survivin gene expression in the human endometrium and endometrial cancer

In the United States, endometrial cancer is the leading cancer of the female reproductive tract. There are 40,100 new cases and 7,470 deaths from endometrial cancer estimated for 2008 (47). The average five year survival rate for endometrial cancer is 84% however, this figure is substantially lower in patients diagnosed with late stage, advanced disease and much higher for patients diagnosed in early stage disease (47). Endometrial cancer (EC) has been associated with several risk factors including obesity, diabetes, hypertension, previously documented occurrence of hereditary non-polyposis colorectal cancer (HNPCC), and heightened exposure to estrogen (25). As of yet, there has not been a dependable molecular predictor of endometrial cancer occurrence in women with these predisposing factors. The goal of our lab is to identify genes that are aberrantly expressed in EC and may serve as molecular biomarkers of EC progression. One candidate protein that we are exploring as a biomarker of EC progression is the cell survival protein survivin.

Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

Investigation of the molecular mechanisms of endometrial hyperplasia

While there is considerable information on the molecular aberrations associated with the development of endometrial cancer, very little is known of changes in gene expression associated with its antecedent premalignant condition, endometrial hyperplasia. In order to address this, we have compared the level of expression of components of the IGF-I signaling pathway in human endometrial hyperplasia to their level of expression in both the normal pre-menopausal endometrium and endometrial carcinoma. We have also characterized the molecular characteristics of endometrial hyperplasia as it occurs in a murine model of hormone-dependent tumorigenesis of the female reproductive tract. ^ There was a significant and selective increase in the expression of the IGF-I Receptor (IGF-IR) in both human hyperplasia and carcinoma as compared to the normal endometrium. The receptor was also activated, as judged by increased tyrosine phosphorylation. In addition, in hyperplasia and carcinoma there is activation of the downstream component Akt. The expression of the PTEN tumor suppressor is decreased in a subset of subjects with hyperplasia and in all of the carcinomas. The simultaneous loss of PTEN expression and increased IGF-IR activation in the hyperplastic endometrium was associated with an increased incidence of endometrial carcinoma elsewhere within the uterus. In the rodent hyperplasia, there was a significant increase in the expression and activation of Akt that appears to be attributable to a marked increase in the expression of IGF-II. ^ Our studies have demonstrated the pathologic proliferation of both the human and rodent endometrium is linked to a marked activation of the Akt pathway. However the cause of this dysregulation is different in the human disease and the animal model. In rodents, hyperplasia is linked to increased expression of one of the ligands of the IGF-IR, IGF-II. In humans the IGF-I receptor itself is upregulated and activated. Additional activation of the Akt pathway via the suppression of PTEN activity, results in conditions that are associated with the marked increase in the probability of developing endometrial cancer. Our data suggests that increased activity of the IGF-I pathway plays the key role in the hyperproliferative state characteristic of endometrial hyperplasia and cancer.^

Estrogen-induced gene expression and patient survival in high-grade serous carcinoma of the ovary

Background. Assessment of estrogen receptor (ER) expression has inconsistent utility as a prognostic marker in epithelial ovarian carcinoma. In breast and endometrial cancers, the use of estrogen-induced gene panels, rather than ER expression alone, has shown improved prognostic capability. Specifically, over-expression of estrogen-induced genes in these tumors is associated with a better prognosis and signifies estrogen sensitivity that can be exploited with hormone antagonizing agents. It was therefore hypothesized that estrogen-induced gene expression in ovarian carcinoma would successfully predict outcomes and differentiate between tumors of varying estrogen sensitivities. Methods. Two hundred nineteen (219) patients with ovarian cancer who underwent surgery at M. D. Anderson between 2004 and 2007 were identified. Of these, eighty-three (83) patients were selected for inclusion because they had advanced stage, high-grade serous carcinoma of the ovary or peritoneum, had not received neoadjuvant chemotherapy, and had readily available frozen tissue for study. All patients had also received adjuvant treatment with platinum and taxane agents. The expression of seven genes known to be induced by estrogen in the female reproductive tract (EIG121, sFRP1, sFRP4, RALDH2, PR, IGF-1, and ER) was measured using qRT-PCR. Unsupervised cluster analyses of multiple gene permutations were used to categorize patients as high or low estrogen-induced gene expressors. QPCR gene expression results were then compared to ER and PR immunohistochemical (IHC) expression. Cox proportional hazards models were used to evaluate the effects of both individual genes and selected gene clusters on patient survival. Results. Median follow-up time was 38.7 months (range 1-68 months). In a multivariate model, overall survival was predicted by sFRP1 expression (HR 1.10 [1.02-1.19], p=0.01) and EIG121 expression (HR 1.28 [1.10-1.49], p<0.01). A cluster defined by EIG121 and ER was further examined because that combination appeared to reasonably segregate tumors into distinct groups of high and low estrogen-induced gene expressors. Shorter overall survival was associated with high estrogen-induced gene expressors (HR 2.84 [1.11-7.30], p=0.03), even after adjustment for race, age, body mass index, and residual disease at debulking. No difference in IHC ER or PR expression was noted between gene clusters. Conclusion. In sharp contrast to breast and endometrial cancers, high estrogen-induced gene expression predicts shorter overall survival in patients with high-grade serous ovarian carcinoma. An estrogen-induced gene biomarker panel may have utility as prognostic indicator and may be useful to guide management with estrogen antagonists in this population.^

ROLE OF SOX9 IN UTERINE GLAND DEVELOPMENT AND DISEASE INITIATION

The female reproductive tract (FRT) develops midway through embryogenesis, and consists of oviducts, uterine horns, cervix and upper part of the vagina. The uterine horns are composed of an epithelial layer, luminal (LE) and glandular epithelium (GE), surrounded by a mesenchymal layer, the stroma and myometrium. Interestingly, in most mammals the GE forms after birth and it only becomes fully differentiated as the female reaches sexual maturity. Uterine glands (UG) are made up of GE and are present in all mammals. They secrete nutrients, cytokines and several other proteins, termed histotroph, that are necessary for embryo implantation and development. Experiments in ewes and mice have revealed that females who lack UGs are infertile mainly due to impaired implantation and early pregnancy loss, suggesting that UGs are essential for fertility. Fortunately for us, UGs develop after birth allowing us to peer into the genetic mechanism of tubulogenesis and branching morphogenesis; two processes that are disrupted in various adenocarcinomas (cancer derived from glands). We created 3D replicas of the epithelium lining the FRT using optical projection tomography and characterized UG development in mice using lineagetracing experiments. Our findings indicate that mouse UGs develop as simple tubular structures and later grow multiple secretory units that stem from the main duct. The main aim of this project was to study the role of SOX9 in the UGs. Preliminary studies revealed that Sox9 is mostly found in the nucleus of the GE. vii This observation led to the hypothesis that Sox9 plays a role in the formation and/or differentiation of the GE. To study the role of Sox9 in UGs differentiation, we conditionally knocked out and overexpressed Sox9 in both the LE and GE using the progesterone receptor (Pgr) promoter. Overexpressing Sox9 in the uterine epithelium, parts of the stroma, and myometrium led to formation of multiple cystic structures inside the endometrium. Histological analysis revealed that these structures appeared morphologically similar to structures present in histological tissue sections obtained from patients with endometrial polyps. We have accounted for the presence of simple and complex hyperplasia with atypia, metaplasia, thick-walled blood vessels, and stromal fibrosis; all “hallmarks” that indicate overexpressing Sox9 leads to development of a polyp-like morphology. Therefore, we can propose the use of Sox9-cOE mice to study development of endometrial cystic lesions and disease progression into hyperplastic lesions.

The relationship between rheumatoid arthritis and reproductive function

There is growing support for the theory that an interaction between the immune and reproductive/endocrine systems underlies the pathogenesis of autoimmune rheumatic diseases. Most of the recent evidence derives from studies of sex hormones and pregnancy in women with systemic lupus. Other than an ameliorative effect of pregnancy, little is known about reproductive factors in relation to rheumatoid arthritis. To elucidate the relationship, a population-based retrospective study was undertaken. Included were 378 female residents of Olmsted County, Minnesota diagnosed with rheumatoid arthritis between 1950 and 1982 (cases) and 325 arthritis-free, married female controls matched to the 324 married cases on birth-year, age at first marriage, and duration of Olmsted County residency. Information of reproductive factors was extracted from the medical records system maintained by the Mayo Clinic.^ Cases had lower fertility rates compared with the female population of Minnesota (rate ratio = 0.86, 95% confidence interval (CI)= 0.80-0.92). Fertility was significantly reduced even prior to the onset of rheumatoid factor positive arthritis. Restricting the comparison to married Olmsted County residents did not alter the results. Further adjustments for time not at risk of conception using survival analysis and proportional hazards modeling only intensified the fertility reduction in the married cases compared with controls. Nulligravidity was more common among cases than controls (odds ratio = 3.16, CI = 1.61-6.20). Independent of fertility, pregnancy had a protective effect against rheumatoid arthritis (odds ratio = 0.31, CI = 0.11-0.89), which was dramatically reversed in the 12 months postpartum (odds ratio = 4.67, CI = 1.50-14.47). Cases were younger at menopause than controls (p $<$ 0.01).^ Small but statistically insignificant associations were observed between rheumatoid arthritis and the following factors: increased frequency of complaints to a physician of infertility; increased frequency of spontaneous abortion, premature birth, and congenital malformations following arthritis onset; and increased prevalence of menopause at arthritis onset. Cases did not differ from controls on age at menarche, duration of pregnancy, or birth weight.^ The findings provide further support for the involvement of the reproductive/endocrine systems in the pathogenesis of autoimmune rheumatic disease. The search for biological mechanisms should be intensified. ^

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

Barriers to adolescent and young men's access of reproductive health care

Purpose. The purpose of this study was to identify the health needs and barriers that young men face in accessing health care and family planning services and to identify what health centers can do to attract young men to the clinic. A focus group format was used to elicit ideas from participants. ^ Methods. Forty-eight young men participated in nine focus groups. The young men were asked about the health issues they have, the barriers they face in accessing reproductive health care, and what clinics can do to attract young men to the clinic. Thematic analysis principles were used to identify the main themes that emerged in the focus groups. ^ Results. Sexually transmitted infections (STIs), mental health problems, and drug use were the major health issues that were mentioned in the majority of the focus groups. The main barriers discussed in the focus groups were attitudinal factors such as young men thinking it is unmanly to seek help, emotional factors such as young men not seeking help because of their ego or pride, and institutional factors such as the location of the clinic. The main suggestions for improvements in the health clinic included decreasing waiting times, emphasizing the fact that the clinics are free for males, having more female nurses, and encouraging clinic staff to treat the young men with respect. Young men suggested advertising and promoting the clinic in schools, in the community, and through the media. Focus group participants also provided their input about the design and format of the clinic flyer. ^ Conclusions. Many studies focus on the reproductive health care needs of adolescent and young females. This study has helped to show that young men also have health care needs and face barriers to accessing reproductive health care services.^

Incidence of breast cancer in first-degree female relatives of men with newly diagnosed prostate cancer

Prostate cancer is the most common incident cancer and the second leading cause of death in men in the United States. Although numerous attempts have been made to identify risk factors associated with prostate cancer, the results have been inconsistent and conflicting. The only established risk factors are age and ethnicity. A positive family history of prostate cancer has also been shown to increase the risk two- to three-fold among close relatives.^ There are several similarities between breast and prostate cancer that make the relationship between the two of interest. (1) Histologically, both cancers are predominantly adenocarcinomas, (2) both organs have a sexual and/or reproductive role, (3) both cancers occur in hormone-responsive tissue, (4) therapy often consists of hormonal manipulation, (5) worldwide distribution patterns of prostate and breast cancer are positively correlated.^ A family history study was conducted to evaluate the aggregation of prostate cancer and co-aggregation of breast cancer in 149 patients referred to The University of Texas, M.D. Anderson Cancer Center with newly diagnosed prostate cancer. All patients were white, less than 75 years of age at diagnosis and permanent residents of the United States. Through a personal interview with the proband, family histories were collected on 1,128 first-degree relatives. Cancer diagnoses were verified through medical records or death certificate. Standardized incidence ratios were calculated using a computer program by Monson incorporating data from Connecticut Tumor Registry.^ In this study, familial aggregation of prostate cancer was verified only among the brothers, not among fathers. Although a statistically significant excess of breast cancer was not found, the increased point estimates in mothers, sisters and daughters are consistent with a co-aggregation hypothesis. Rather surprising was the finding of a seven-fold increased risk of prostate cancer and a three-fold increased risk of breast cancer among siblings in the presence of a maternal history of any cancer. Larger family history studies including high risk (African-Americans) and lower-risk groups (Hispanics) and incorporating molecular genetic evaluations should be conducted to determine if genetic differences play a role in the differential incidence rates across ethnic groups. ^