RESUMO
CONTEXT: Most in vitro fertilization (IVF) programs employ embryo cryopreservation to enhance pregnancies from a single ovarian stimulation. More embryos are created, some of which are not transferred to the uterus immediately, generating a need for improved cryopreservation protocols. One protocol may involve growing embryos to a further stage of development, allowing only embryos with proven developmental capabilities to be cryopreserved. Here we examined thaw survival, implantation and live birth rates of embryos cryopreserved at different stages. AIMS: We examined thaw survival, implantation and live birth rates of embryos cryopreserved at the zygote, day 3 (D3) embryos or blastocyst stage. SETTINGS AND DESIGN: This is a retrospective study from a single academic IVF program. PATIENTS AND METHODS: A retrospective study of all patients who had frozen embryos transferred to their uteri from year 2002 to 2008 at a single academic IVF program was conducted. STATISTICAL ANALYSIS USED: Analysis of variance followed by Fisher's Exact Test was performed to compare the survival after thaw, implantation and live birth rates between the three groups. RESULTS: One thousand nine hundred and ninety-one zygotes, 2880 D3 embryos and 503 blastocysts were frozen using a slow freeze technique, thawed and transferred. Significantly more D3 embryos and blastocysts survived the thawing process compared to zygotes and significantly higher implantation rate per number of thawed blastocysts was achieved than that for zygotes. Live birth rates were similar between the three groups. CONCLUSIONS: Growing embryos to blastocyst stage prior to cryopreservation is associated with fewer frozen embryos but does not appear compromise patients' chance of achieving pregnancy.
RESUMO
Uterine leiomyoma is the most common tumor of the female genital tract and the leading cause of hysterectomy. Although progesterone stimulates the proliferation of uterine leiomyoma cells, the mechanism of progesterone action is not well understood. We used chromatin immunoprecipitation (ChIP)-cloning approach to identify progesterone receptor (PR) target genes in primary uterine leiomyoma smooth muscle cells. We identified 18 novel PR-binding sites, one of which was located 20.5 kb upstream of the transcriptional start site of the Krüppel-like transcription factor 11 (KLF11) gene. KLF11 mRNA levels were minimally downregulated by progesterone but robustly upregulated by the progesterone antagonist RU486. Luciferase reporter assays showed significant baseline and RU486-inducible promoter activity in the KLF11 basal promoter or distal PR-binding region, both of which contained multiple Sp1-binding sequences but lacked classic progesterone response elements. RU486 stimulated recruitment of Sp1, RNA polymerase II, PR, and the coactivators SRC-1 and SRC-2 to the distal region and basal promoter. siRNA knockdown of PR increased KLF11 expression, whereas knockdown of KLF11 increased leiomyoma cell proliferation and abolished the antiproliferative effect of RU486. In vivo, KLF11 expression was significantly lower in leiomyoma tissues compared with adjacent myometrial tissues. Taken together, using a ChIP-cloning approach, we uncovered KLF11 as an integrator of PR signaling and proliferation in uterine leiomyoma cells.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proliferação de Células , Leiomioma/genética , Receptores de Progesterona/fisiologia , Proteínas Repressoras/fisiologia , Neoplasias Uterinas/genética , Proteínas Reguladoras de Apoptose , Proteínas de Ciclo Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antagonistas de Hormônios/farmacologia , Humanos , Leiomioma/patologia , Mifepristona/farmacologia , Progesterona/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Carga Tumoral/genética , Células Tumorais Cultivadas , Neoplasias Uterinas/patologiaRESUMO
BACKGROUND: Aromatase, the key enzyme in estrogen biosynthesis, is encoded by the Cyp19a1 gene. Thus far, 3 unique untranslated first exons associated with distinct promoters in the mouse Cyp19a1 gene have been described (brain, ovary, and testis-specific). It remains unknown whether aromatase is expressed in other mouse tissues via novel and tissue-specific promoters. METHODS: Real-time PCR was used to examine the aromatase expression levels in various C57BL/6 mouse tissues. 5'-rapid amplification of cDNA ends (5'-RACE) was used to determine the transcriptional start sites of Cyp19a1 transcripts. Promoter activity was measured using serial deletion mutants of DNA fused to the luciferase reporter gene. Primary mouse adipose fibroblasts were isolated and cultured from 16-week-old mouse gonadal fat pads. RESULTS: We systematically analyzed Cyp19a1 expression in a large number of mouse tissues, and demonstrated for the first time that aromatase was expressed in the male but not female gonadal fat pad. Subcutaneous and brown adipose tissue did not contain detectable Cyp19a1 mRNA. We used 5'-RACE to clone a novel gonadal fat-specific untranslated first exon, which is spliced onto a common junction 15 bp upstream of the translation start site. This adipose-specific first exon was mapped to approximately 75 kb upstream of the translation start site. Transfection of luciferase reporter gene plasmids containing the promoter region upstream of the adipose-specific first exon into murine 3T3-L1 adipose fibroblasts demonstrated significant basal promoter activity conferred primarily by the sequence located at -343/-1 bp. Dexamethasone significantly induced activity of this adipose-specific promoter region. Adipose-specific Cyp19a1 mRNA was expressed in primary mouse adipose fibroblasts and significantly induced by dexamethasone alone or serum plus dexamethasone. CONCLUSION: Taken together, this research identified a novel, adipose-specific first exon of Cyp19a1 and its hormonally regulated promoter region in male murine gonadal fat. These results expand the known 5'-regulatory region of the murine Cyp19a1 gene to 75 kb upstream of the translation start site. Cyp19a1 expression in mouse adipose tissue may play an important role in reproductive biology and lipid metabolism.
Assuntos
Tecido Adiposo/enzimologia , Aromatase/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Tecido Adiposo/citologia , Animais , Células Cultivadas , Dexametasona/farmacologia , Éxons/genética , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Gônadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: Regulation of fetoplacental blood flow is likely mediated by factors such as prostanoids. Estrogen and its receptors affect prostanoid biosynthesis. Previously, we demonstrated that villous endothelial cells express estrogen receptor-beta (ESR2), and we sought to determine its role in the mediation of fetoplacental vascular function. STUDY DESIGN: Villous endothelial cells from uncomplicated pregnancies were isolated, cultured, and treated with estrogen. RNA interference, real-time polymerase chain reaction, Western blotting, and enzyme immunoassays were performed. RESULTS: Cyclooxygenase-2 (COX-2) expression levels were not altered consistently by estrogen. RNA interference of ESR2 led to a concomitant decrease in COX-2 messenger RNA (P < .0001) and protein (P < .05) in the presence and absence of estradiol. ESR2 knock-down also led to diminished prostacyclin and thromboxane concentrations in the absence of estradiol (P < .005). CONCLUSION: ESR2 mediates COX-2 expression levels and both prostacyclin and thromboxane concentrations in the basal state, which suggests the possibility of ligand-independent regulation of COX-2 activity and prostaglandin H2 substrate availability. Further investigation regarding ESR2 regulation of prostanoid biosynthesis and its effects on the fetoplacental vasculature is warranted.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Células Endoteliais/metabolismo , Receptor beta de Estrogênio/fisiologia , Placenta/citologia , Prostaglandinas/biossíntese , Células Cultivadas , Epoprostenol/análise , Epoprostenol/biossíntese , Feminino , Humanos , Placenta/irrigação sanguínea , Prostaglandinas/análise , Tromboxanos/análise , Tromboxanos/biossínteseRESUMO
To date, 10 promoters were reported to regulate the expression of the human aromatase (CYP19) gene, giving rise to transcripts with an identical coding region but tissue-specific first exons comprising unique 5'-untranslated regions. We describe the identification and characterization of a new CYP19 exon I, designated exon I.8, in a 5'-rapid amplification of complementary DNA ends-generated library of human THP-1 monocytic cells. A construct containing exon I.8 and its 5'-flanking sequence was sufficient to drive transcription in THP-1 cells. This novel promoter was located approximately 2-kb upstream of promoter I.4 and approximately 75-kb upstream of the common splice junction. We detected several I.8-containing splice variants, 2 of which also contained a sequence from exon I.4. Analysis of human tissues revealed a unique pattern of promoter I.8 usage. The placenta contained the highest level of I.8-specific transcripts. This work underscores the complexity of the mechanisms that regulate normal and pathologic aromatase expression.
Assuntos
Aromatase/genética , Regiões Promotoras Genéticas/genética , Aromatase/biossíntese , Aromatase/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Éxons/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leucemia Mieloide/enzimologia , Leucemia Mieloide/genética , Dados de Sequência MolecularRESUMO
Aromatase is the key enzyme for estrogen biosynthesis. A distal promoter, PI.4, maintains baseline levels of aromatase in normal breast adipose tissue. In contrast, malignant breast epithelial cells secrete prostaglandin E(2) (PGE(2)), which stimulates aromatase expression via proximal promoters PI.3/PII in a cyclic AMP (cAMP)- and protein kinase C (PKC)-dependent manner in adjacent breast adipose fibroblasts (BAF), leading to increased local concentrations of estrogen. Although an effective treatment for breast cancer, aromatase inhibitors indiscriminately abolish estrogen synthesis in all tissues, causing major side effects. To identify drug targets to selectively block aromatase and estrogen production in breast cancer, we investigated PGE(2)-stimulated signaling pathways essential for aromatase induction downstream of cAMP and PKC in human BAFs. Here, we show that PGE(2) or its surrogate hormonal mixture dibutyryl cAMP (Bt(2)cAMP) + phorbol diacetate (PDA) stimulated the p38, c-jun NH(2)-terminal kinase (JNK)-1, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathways. Inhibition or small interfering RNA-mediated knockdown of p38 or JNK1, but not ERK, inhibited PGE(2)- or Bt(2)cAMP + PDA-induced aromatase activity and expression via PI.3/PII. Conversely, overexpression of wild-type p38alpha or JNK1 enhanced PGE(2)-stimulated aromatase expression via PII. PGE(2) or Bt(2)cAMP + PDA stimulated c-Jun and activating transcription factor-2 (ATF2) phosphorylation and binding to the PI.3/PII region. Specific activation of protein kinase A (PKA) or EPAC with cAMP analogues stimulated p38 and JNK1; however, only PKA-activating cAMP analogues induced aromatase expression. The PKC activator PDA effectively stimulated p38 and JNK1 phosphorylation but not aromatase expression. Taken together, PGE(2) activation of p38 and JNK1 via PKA and PKC is necessary for aromatase induction in BAFs, and p38 and JNK1 are potential new drug targets for tissue-specific ablation of aromatase expression in breast cancer.
Assuntos
Tecido Adiposo/enzimologia , Aromatase/biossíntese , Neoplasias da Mama/enzimologia , Dinoprostona/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Aromatase/genética , Neoplasias da Mama/genética , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacologia , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
CONTEXT: Uterine leiomyomas are smooth muscle cell tumors that cause irregular uterine bleeding and pregnancy loss in many reproductive-age women. Progesterone stimulates their growth, whereas treatment with progesterone receptor (PR) antagonists or selective progesterone receptor modulators shrinks these tumors. Molecular mechanisms underlying these observations are unknown. OBJECTIVE: Bcl-2 is a key protein that inhibits apoptosis. It was proposed that growth enhancement of leiomyoma cells by progesterone was mediated via bcl-2 induction. Here we test the hypothesis that PR regulates the bcl-2 gene by directly binding to its promoter. RESULTS: The pure progesterone agonist R5020 increased the total number of viable primary human leiomyoma smooth muscle (LSM) cells in culture. Progesterone or R5020 (10(-6) m) significantly increased bcl-2 mRNA levels after 2 and 4 h by 9.2- and 3.4-fold, respectively, in LSM cells. Transient transfection with deletion mutants of bcl-2 promoter showed that the -1281/-258-bp region conferred responsiveness to progesterone induction in the presence of PR-A. We identified a palindromic progesterone response element (PRE) at -553/-539 bp. EMSA showed that PR in nuclear extracts from LSM cells bound specifically to this PRE. Chromatin immunoprecipitation-PCR confirmed in situ recruitment of PR to the -629/-388-bp region bearing the PRE. In vivo, bcl-2 mRNA levels correlated significantly with total PR mRNA levels in leiomyoma tissues. CONCLUSION: Taken together, progesterone via PR interacts with the bcl-2 promoter to induce its expression in leiomyoma tissue. This may explain, in part, the progesterone-dependent enhancement of growth in uterine leiomyoma.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genes bcl-2/genética , Leiomioma/genética , Regiões Promotoras Genéticas/genética , Receptores de Progesterona/fisiologia , Neoplasias Uterinas/genética , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Leiomioma/metabolismo , Luciferases/genética , Progesterona/farmacologia , Promegestona/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Progesterona/agonistas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Neoplasias Uterinas/metabolismoRESUMO
Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (-197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (-197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.
Assuntos
Metilação de DNA , Endometriose/genética , Endométrio/metabolismo , Receptor beta de Estrogênio/genética , Regulação da Expressão Gênica/genética , Adulto , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Células Cultivadas , Ilhas de CpG , Decitabina , Endométrio/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores de Progesterona/genética , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
Pathogenesis and growth of three common women's cancers (breast, endometrium and ovary) are linked to estrogen. A single gene encodes the key enzyme for estrogen biosynthesis named aromatase, inhibition of which effectively eliminates estrogen production in the entire body. Aromatase inhibitors successfully treat breast cancer, whereas their roles in endometrial and ovarian cancers are less clear. Ovary, testis, adipose tissue, skin, hypothalamus and placenta express aromatase normally, whereas breast, endometrial and ovarian cancers overexpress aromatase and produce local estrogen exerting paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kb regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. In cancers of breast, endometrium and ovary, aromatase expression is primarly regulated by increased activity of the proximally located promoter I.3/II region. Promoters I.3 and II lie 215 bp from each other and are coordinately stimulated by PGE(2) via a cAMP-PKA-dependent pathway. In breast adipose fibroblasts exposed to PGE(2) secreted by malignant epithelial cells, PKC is also activated, and this potentiates cAMP-PKA-dependent induction of aromatase. Thus, inflammatory substances such as PGE(2) may play important roles in inducing local production of estrogen that promotes tumor growth.
Assuntos
Aromatase/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias do Endométrio/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/enzimologia , Animais , Aromatase/genética , Neoplasias da Mama/genética , Neoplasias do Endométrio/genética , Feminino , Humanos , Neoplasias Ovarianas/genéticaRESUMO
17-beta hydroxysteroid dehydrogenase type 2 (HSD17B2) oxidizes estradiol to estrone, testosterone to androstenedione, and 20 alpha-dihydroprogesterone to progesterone. HSD17B2 is highly expressed in human placental tissue where it is localized to placental endothelial cells lining the fetal compartment. The aim of this study was to investigate the effects of potential regulatory factors including progesterone, estradiol, and retinoic acid (RA) onHSD17B2 expression in primary human placental endothelial cells in culture.HSD17B2 mRNA expression was not regulated by progesterone, the progesterone agonist R5020, or estradiol treatment. RA significantly induced HSD17B2 mRNA levels and enzyme activity in a dose- and time-dependent manner. Maximal stimulation occurred at Hour 48 at an RA concentration of 10(-6) M. Both retinoic acid receptor alpha (RARA) and retinoid X receptor alpha (RXRA) were readily detected by immunoblotting in isolated placental endothelial cells. RNA interference directed against RARA or RXRA led to reduced basal levels of HSD17B2 mRNA levels and significantly abolished RA-stimulated HSD17B2 expression. Together, these data indicate that regulation of HSD17B2 mRNA levels and enzymatic activity by RA in the placenta is mediated by RARA and RXRA.
Assuntos
17-Hidroxiesteroide Desidrogenases/biossíntese , Estradiol/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Placenta/enzimologia , Progesterona/farmacologia , Promegestona/farmacologia , 17-Hidroxiesteroide Desidrogenases/genética , Células Endoteliais/enzimologia , Feminino , Imunofluorescência , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Immunoblotting , Placenta/citologia , Congêneres da Progesterona/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores do Ácido Retinoico/biossíntese , Receptores do Ácido Retinoico/genética , Receptor alfa de Ácido Retinoico , Receptor X Retinoide alfa/biossíntese , Receptor X Retinoide alfa/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Endometriosis is the most common cause of pelvic pain and affects an estimated 5 million women in the US. The biologically active estrogen estradiol (E2) is the best-defined mitogen for the growth and inflammation processes in the ectopic endometriotic tissue that commonly resides on the pelvic organs. Progesterone and progestins may relieve pain by limiting growth and inflammation in endometriosis but a portion of patients with endometriosis and pelvic pain do not respond to treatment with progestins. Moreover, progesterone-induced molecular changes in the eutopic (intrauterine) endometrial tissue of women with endometriosis are either blunted or undetectable. These in vivo observations are indicative of resistance to progesterone action in endometriosis. The molecular basis of progesterone resistance in endometriosis may be related to an overall reduction in the levels of progesterone receptors (PRs) and the lack of the PR isoform named progesterone receptor B (PR-B). In normal endometrium, progesterone acts on stromal cells to induce secretion of paracrine factor(s). These unknown factor(s) act on neighboring epithelial cells to induce the expression of the enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD-2), which metabolizes the biologically active estrogen E2 to estrone (E1). In endometriotic tissue, progesterone does not induce epithelial 17beta-HSD-2 expression due to a defect in stromal cells. The inability of endometriotic stromal cells to produce progesterone-induced paracrine factors that stimulate 17beta-HSD-2 may be due to the lack of PR-B and very low levels of progesterone receptor A (PR-A) observed in vivo in endometriotic tissue. The end result is deficient metabolism of E2 in endometriosis giving rise to high local concentrations of this local mitogen. The cellular and molecular mechanisms underlying progesterone resistance and failure to metabolize E2 in endometriosis are reviewed.