Correlation of umbilical cord blood hormones and growth factors with stem cell potential: implications for the prenatal origin of breast cancer hypothesis.

INTRODUCTION: Prenatal levels of mitogens may influence the lifetime breast cancer risk by driving stem cell proliferation and increasing the number of target cells, and thereby increasing the chance of mutation events that initiate oncogenesis. We examined in umbilical cord blood the correlation of potential breast epithelial mitogens, including hormones and growth factors, with hematopoietic stem cell concentrations serving as surrogates of overall stem cell potential. METHODS: We analyzed cord blood samples from 289 deliveries. Levels of hormones and growth factors were correlated with concentrations of stem cell and progenitor populations (CD34+ cells, CD34+CD38- cells, CD34+c-kit+ cells, and granulocyte-macrophage colony-forming units). Changes in stem cell concentration associated with each standard deviation change in mitogens and the associated 95% confidence intervals were calculated from multiple regression analysis. RESULTS: Cord blood plasma levels of insulin-like growth factor-1 (IGF-1) were strongly correlated with all the hematopoietic stem and progenitor concentrations examined (one standard-deviation increase in IGF-1 being associated with a 15-19% increase in stem/progenitor concentrations, all P < 0.02). Estriol and insulin-like growth factor binding protein-3 levels were positively and significantly correlated with some of these cell populations. Sex hormone-binding globulin levels were negatively correlated with these stem/progenitor pools. These relationships were stronger in Caucasians and Hispanics and were weaker or not present in Asian-Americans and African-Americans. CONCLUSION: Our data support the concept that in utero mitogens may drive the expansion of stem cell populations. The correlations with IGF-1 and estrogen are noteworthy, as both are crucial for mammary gland development.

Cellular and molecular regulation of the primate endometrium: a perspective.

This contribution will trace some of the many seminal studies on the female uterus (endometrium) over the centuries and conclude with a description of some current research initiatives in our laboratory. Numerous contributions from many investigators over the years have contributed to our current understanding of endometrial function. The historical section of this chapter is intended to be a brief overall description of some of these efforts and not exhaustive. Additional information can be found in the review articles and books cited herein.

Reproducibility of assays for steroid hormones, prolactin and insulin-like growth factor-1 in umbilical cord blood.

We assessed the reproducibility of measurements of plasma hormone and binding protein levels in umbilical cord blood collected from 30 male and female babies. They were delivered as singleton births from full-term pregnancies (gestational age >or= 37 weeks) in a cord blood donation programme. We assayed three plasma replicates from each cord blood sample at two points in time. Plasma oestradiol, unconjugated oestriol, testosterone, progesterone, prolactin, sex-hormone binding globulin (SHBG), insulin-like growth factor-1 (IGF-1), and IGF binding protein-3 (IGFBP-3) levels were measured in duplicates in the same batch (batch 1). In addition, another set of assays was conducted for each cord blood 1 year apart in a different batch (batch 2). Means and standard deviations for each hormone and binding protein were similar in replicates assayed in batch 1 and 2. Pearson's correlation coefficients were 0.9 or higher in duplicates assayed in batch 1. The correlation coefficients were between 0.77 and 0.96 for between-batch assays. Intra-class correlation coefficients (ICC) were higher than 0.9 for assay of SHBG [95% CI 0.92, 1.0] and progesterone [95% CI 0.87, 0.97] and between 0.8 and 0.9 for assay of oestradiol, unconjugated oestriol, prolactin, IGF-1, and IGFBP-3. The lowest ICC value was found for testosterone (ICC = 0.74; [95% CI 0.56, 1.0]). These data indicate a high reproducibility of cord blood hormone measurements; minimal differences were observed between the calibrated and the original regression coefficients for the association of hormones/binding proteins with percent of CD34+ cells in mononuclear cells.

The 5' flanking region of the Rhesus monkey H3 (DMBT1) gene contains putative progesterone response elements.

DMBT1 (deleted in malignant brain tumors) encodes a large scavenger receptor cysteine rich (SRCR) protein with proposed tumor suppressor properties due to its frequent deletion or lack of expression in a variety of different tumors including endometrial cancers. The gene is alternatively spliced to produce a number of related proteins with suspected functions in mucosal inflammation and epithelial regeneration. Expression of DMBT1 has been demonstrated in a wide variety of cell types, mostly of epithelial origin, including tissues of the respiratory system, the alimentary system, brain, and reproductive system. We have previously identified a Rhesus monkey cDNA clone H3 (homologous to human DMBT1) as a progesterone-induced gene in Rhesus monkey endometrium during the secretory phase of the menstrual cycle. As an initial step in understanding the molecular mechanisms of H3 (DMBT1) regulation we have cloned and sequenced 1.5 kb of the 5'-flanking region expected to contain promoter sequences of the Rhesus monkey gene and identified six putative progesterone receptor binding sites in the 5'-upstream region.

Association of fetal hormone levels with stem cell potential: evidence for early life roots of human cancer.

Intrauterine and perinatal factors have been linked to risk of childhood leukemia, testicular cancer, and breast cancer in the offspring. The pool of stem cells in target tissue has been suggested as a critical factor linking early life exposures to cancer. We examined the relation between intrauterine hormone levels and measurements of stem cell potential in umbilical cord blood. Cord blood donors were 40 women, ages >/=18 years, who delivered, from August 2002 to June 2003, a singleton birth after a gestation of at least 37 weeks. We assayed plasma concentrations of estradiol, unconjugated estriol, testosterone, progesterone, prolactin, sex hormone binding globulin, insulin-like growth factor-I (IGF-I), and IGF binding protein-3. For stem cell potential, we measured concentrations of CD34(+) and CD34(+)CD38(-) cells and granulocyte-macrophage colony-forming unit (CFU-GM). We applied linear regression analysis and controlled for maternal and neonatal characteristics. We found strong positive associations between IGF-I and stem cell measures, 1 SD increase in IGF-I being associated with a 41% increase in CD34(+) (P = 0.008), a 109% increase in CD34(+)CD38(-) (P = 0.005), and a 94% increase in CFU-GM (P = 0.01). Similar associations were observed for IGF binding protein-3. Among steroid hormones, estriol and testosterone were significantly positively associated with CD34(+) and CFU-GM. These findings indicate that levels of growth factors and hormones are strongly associated with stem cell potential in human umbilical cord blood and point to a potential mechanism that may mediate the relationship between in utero exposure to hormones and cancer risk in the offspring.

Microarray profiling of progesterone-regulated endometrial genes during the rhesus monkey secretory phase.

BACKGROUND: In the endometrium the steroid hormone progesterone (P), acting through its nuclear receptors, regulates the expression of specific target genes and gene networks required for endometrial maturation. Proper endometrial maturation is considered a requirement for embryo implantation. Endometrial receptivity is a complex process that is spatially and temporally restricted and the identity of genes that regulate receptivity has been pursued by a number of investigators. METHODS: In this study we have used high density oligonucleotide microarrays to screen for changes in mRNA transcript levels between normal proliferative and adequate secretory phases in Rhesus monkey artificial menstrual cycles. Biotinylated cRNA was prepared from day 13 and days 21-23 of the reproductive cycle and transcript levels were compared by hybridization to Affymetrix HG-U95A arrays. RESULTS: Of approximately 12,000 genes profiled, we identified 108 genes that were significantly regulated during the shift from a proliferative to an adequate secretory endometrium. Of these genes, 39 were up-regulated at days 21-23 versus day 13, and 69 were down-regulated. Genes up-regulated in P-dominant tissue included: secretoglobin (uteroglobin), histone 2A, polo-like kinase (PLK), spermidine/spermine acetyltransferase 2 (SAT2), secretory leukocyte protease inhibitor (SLPI) and metallothionein 1G (MT1G), all of which have been previously documented as elevated in the Rhesus monkey or human endometrium during the secretory phase. Genes down-regulated included: transforming growth factor beta-induced (TGFBI or BIGH3), matrix metalloproteinase 11 (stromelysin 3), proenkephalin (PENK), cysteine/glycine-rich protein 2 (CSRP2), collagen type VII alpha 1 (COL7A1), secreted frizzled-related protein 4 (SFRP4), progesterone receptor membrane component 1 (PGRMC1), chemokine (C-X-C) ligand 12 (CXCL12) and biglycan (BGN). In addition, many novel/unknown genes were also identified. Validation of array data was performed by semi-quantitative RT-PCR of two selected up-regulated genes using temporal (cycle day specific) endometrial cDNA populations. This approach confirmed up-regulation of WAP four-disulfide core domain 2 (WFDC2) and SLPI during the expected window of receptivity. CONCLUSION: The identification of P-regulated genes and gene pathways in the primate endometrium is expected to be an important first step in elucidating the cellular processes necessary for the development of a receptive environment for implantation.

Role of progesterone in structural and biochemical remodeling of endometrium.

The endometrial response to the varying levels of ovarian steroids is exhibited as alterations in its form and function. These changes in endometrial morphology and physiology, especially those observed during the implantation window are prerequisites to support embryo attachment and invasion. However the state of endometrial receptivity to embryo results from an operative network of several molecular events triggered by estrogen, progesterone and probably some other factors, yet to be discovered. It is well established that estrogen and progesterone are the critical endocrine determinants of endometrial functions. However the precise delineation of hormone driven events and their interaction is yet to be ascertained. Several attempts have been made to understand these cascades, however most of these studies have been conducted in vitro using one or the other component of endometrial tissues. We have attempted to investigate in vivo morphological and biochemical/molecular changes in endometrium in response to neutralization of progesterone synthesis/ function in two primate animal models. In one of the models, ovariectomized rhesus monkeys, artificial menstrual cycles were simulated and subsequent effects on the _expression of various genes were investigated in presence and absence of sufficient progesterone levels. The results coincided with those observed in the endometrium of the other model, bonnet monkeys presenting normal hypothalamus-ovarian-pituitary function but displaying retarded endometrial growth due to blocked progesterone receptor. A significant decline was observed in the expression of transforming growth factor beta, transforming growth factor beta receptor, leukaemia inhibitory factor, whereas no remarkable changes were observed in the expression of estrogen receptor and progesterone receptors in response to neutralization of progesterone synthesis/function in these two animal models. Taking support from the inferences drawn from previously published in vitro studies and our data from in vivo studies conducted in these two models, we propose a hypothesis supporting a potential link between the expressions of transforming growth factor beta, leukaemia inhibitory factor, cyclooxygenases and integrins.

Temporal regulation of gene expression during the expected window of receptivity in the rhesus monkey endometrium.

Progesterone has been shown to regulate a number of genes and gene networks in the primate endometrium. This action of progesterone is essential to provide an appropriate milieu for embryo-endometrial communication that can lead to implantation and the successful initiation of pregnancy. A temporal regulation of endometrial genes is most likely required to achieve an appropriate state of receptivity in the primate endometrium. Using simulated menstrual cycles in the rhesus monkey, endometrial tissue was harvested at days that encompass the expected window of receptivity (4-10 days after the estradiol surge) and subsequently converted to cycle day-specific cDNA populations. Using differential display reverse transcriptase-polymerase chain reaction, 12 cDNA fragments were isolated and sequenced whose mRNA levels were elevated during this time frame. The temporal expression patterns of these mRNAs were confirmed by semiquantitative polymerase chain reaction. Two of these fragments exhibited high homology to previously characterized human genes: 1) secretory leukocyte protease inhibitor, also known as antileukoprotease, an endometrial neutrophil elastase inhibitor with antibacterial and anti-inflammatory properties; and 2) syncytin, also known as endogenous retrovirus W envelope protein, a highly fusogenic membrane glycoprotein that induces formation of giant syncytia and is believed to be important in decidual and placental development. The temporal regulation of these genes by progesterone supports their likely role in the orchestration of molecular and cellular events that are required to achieve a state of receptivity in the primate endometrium.

Gene expression in the rhesus monkey endometrium: differential display and laser capture microdissection.

The primate endometrium is a complex heterogeneous tissue that requires proper maturation to achieve a hospitable environment for implantation. Endometrial differentiation and maturation is primarily controlled through the action of progesterone during the secretory phase. Many of the genes and gene networks that are involved in this process are likely to be induced or inhibited in a temporal, spatial, and cell-type specific context within the endometrium. We have used several approaches to address these latter issues in the rhesus monkey endometrium. The use of differential display with hormonally distinct endometrial cDNA populations prepared from artificially controlled menstrual cycles has allowed us to identify different P-dependent mRNA regulatory patterns during simulated secretory phases. In addition, we have coupled differential display with laser capture microdissection to further study region and cell-type specific changes in the primate endometrium.

Assessment and application of laser microdissection for analysis of gene expression in the rhesus monkey endometrium.

We investigated the use of laser capture microdissection (LCM) to identify differences in gene expression between cell types or regions within the rhesus monkey endometrium. Different cell types were harvested from the two major regions of the endometrium during midsecretory phases (Days 21-23) of adequate artificial menstrual cycles: glandular epithelia (G) or stroma (S) from the functionalis (F) or the basalis (B). Amplification of the cDNA populations (primer-specific adaptors) was used to increase the amount of nucleic acid for further analysis. This single amplification step allowed us to detect the housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA) and the cDNA smears in the samples. Using differential display reverse transcription polymerase chain reaction (DDRT-PCR), six fragments were selected, cloned, and sequenced based on their regional and cell type localization. Primer-specific PCR analysis subsequently confirmed the localization of three fragments: F1, highly expressed in the functionalis but not the basalis, was homologous (93% identical) to the human leukotriene B4 receptor BLT2; FS-1, highly expressed only in the stroma of the functionalis, had a 94% homology with an as yet uncharacterized gene (FLJ124360); and BG-1, primarily expressed only in the glandular epithelia of the basalis, showed a 98% homology with an uncharacterized bacterial artificial chromosome clone sequence. These LCM-generated cDNA populations coupled with DDRT-PCR can provide an important avenue for the identification of new or novel gene fragments that display cell type- or region-specific gene expression in the rhesus monkey endometrium.