transforming-growth-factor-beta and Infertility--Female

Polycystic ovarian syndrome (PCOS) is a common endocrine disorder in reproductive age affecting 5 to 7% of women. It is characterized by anovulatory infertility, hyperandrogenism, and polycystic ovaries. Angiogenesis in the ovary is critical for follicular growth, ovulation, and the subsequent development and regression of the corpus luteum. Accumulating evidence suggests that multiple angiogenic factors are dysregulated in PCOS, including vascular endothelial growth factor, angiopoietins, platelet-derived growth factor, transforming growth factor-β, and basic fibroblast growth factor. This angiogenic factor imbalance likely underlies the increased stromal vascularity observed in PCOS. Angiogenic factor dysregulation may play an important role in the pathophysiology of PCOS and may contribute to ovulatory dysfunction, subfertility, and ovarian hyperstimulation syndrome, which are commonly seen in women with PCOS. Further experimental studies are needed to gain a better understanding of the growth factors that are involved in normal and pathological ovarian angiogenesis, and to assess the potential of angiogenesis-based treatment strategies in PCOS.

Topics: Angiopoietins; Female; Fibroblast Growth Factors; Humans; Infertility, Female; Neovascularization, Pathologic; Ovarian Hyperstimulation Syndrome; Ovary; Platelet-Derived Growth Factor; Polycystic Ovary Syndrome; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by chronic oligoanovulation and hyperandrogenism and associated with insulin resistance, type 2 diabetes, and cardiovascular risk. In recent years, genetic studies have linked PCOS to a dinucleotide marker D19S884 in the fibrillin 3 gene. Fibrillins make up the major component of microfibrils in the extracellular matrix (ECM) and interact with molecules in the ECM to regulate transforming growth factor β (TGF-β) signaling. Therefore, variations in fibrillin 3 and subsequent dysregulation of TGF-β may contribute to the pathogenesis of PCOS. Here, we review the evidence from genetic studies supporting the role of TGF-β in PCOS and describe how TGF-β dysregulation may contribute to (1) the fetal origins of PCOS, (2) reproductive abnormalities in PCOS, and (3) cardiovascular and metabolic abnormalities in PCOS.

Topics: Animals; Female; Fertility; Fibrillins; Genetic Predisposition to Disease; Genetic Variation; Humans; Infertility, Female; Microfilament Proteins; Ovary; Phenotype; Polycystic Ovary Syndrome; Signal Transduction; Transforming Growth Factor beta

Premature ovarian failure (POF) is an ovarian defect characterized by the premature depletion of ovarian follicles; POF affects approximately 1-2% of women under the age of 40 yr, thus representing one major cause of female infertility. POF relevance is continuously growing because women tend to conceive always more frequently beyond 30 yr. Frequently, POF is the end-stage of an occult process [primary ovarian insufficiency (POI)]. POI is a heterogeneous disease caused by a variety of mechanisms. Though the underlying cause remains unexplained in the majority of cases, several data indicate that POI has a strong genetic component. These data include the existence of several causal genetic defects in human, experimental, and natural models, as well as the frequent familiarity. The candidate genes are numerous, but POF remains unexplained in most of the cases. Several recent evidences have driven the attention of researchers on the possible involvement of various elements belonging to the transforming growth factor β family, which includes bone morphogenetic proteins, growth/differentiation factors, and inhibins. These peptides are produced by either the oocyte or granulosa cells to constitute a complex paracrine network within the ovarian follicle. Here, we review the studies reporting the genetic alterations of these factors in human and animal defects of ovarian folliculogenesis which support the fundamental roles played by these signals in ovarian morphogenesis and function.

Topics: Animals; Bone Morphogenetic Protein 15; Bone Morphogenetic Protein Receptors, Type I; Female; Growth Differentiation Factor 9; Humans; Infertility, Female; Inhibins; Ovarian Follicle; Primary Ovarian Insufficiency; Transforming Growth Factor beta

Topics: Collagen; Connective Tissue Growth Factor; Embryo Implantation; Extracellular Matrix; Female; Humans; Immediate-Early Proteins; Infertility, Female; Intercellular Signaling Peptides and Proteins; Left-Right Determination Factors; Matrix Metalloproteinases; Progesterone; Transforming Growth Factor beta; Uterine Hemorrhage

This study was designed to identify and quantify concentrations of growth factors/cytokines released by Vero cells during the co-culture interval. The factors screened for in this preliminary investigation, namely platelet-derived growth factor (PDGF), transforming growth factor beta (TGFbeta), interleukin-6 (IL-6), leukaemia inhibitory factor (LIF) and epidermal growth factor (EGF) have each been identified to impact on early embryo development or are secreted by embryos themselves, suggesting an autocrine regulatory role. Vero cell culture supernatants were collected at 2, 3, 4, 5 and 6 days after seeding. Samples were assessed by enzyme-linked immunoassay for growth factor/cytokine secretion at each designated time interval. Conditioned medium from all days contained IL-6, PDGF and LIF. The concentration of IL-6 increased from 294 pg/well on day 2 to almost 1600 pg/well on day 6. PDGF also accumulated rapidly in co-culture wells, rising from 19-40 pg/well early in the culture period to around 500 pg/ well by day 6. In the second half of this study, medium supernatants from patients enrolled in our co-culture programme were analysed. Retrospective evaluation of medium supernatants collected at the time of transfer from co-cultures from 11 randomly selected patients showed considerable patient-to-patient variation in concentrations of secreted growth factors and cytokines. These findings indicate that during the co-culture interval embryos are exposed to a dynamic environment, with increasing concentrations of growth factors and cytokines. The positive effects of co-culture on embryo quality and in-vitro blastulation need to be balanced against the variation that this technique can potentially introduce into the embryo culture system.

Topics: Animals; Blastomeres; Cell Communication; Chlorocebus aethiops; Coculture Techniques; Culture Media, Conditioned; Embryo Transfer; Epidermal Growth Factor; Female; Fertilization in Vitro; Humans; Infertility, Female; Interleukin-6; Platelet-Derived Growth Factor; Pregnancy; Transforming Growth Factor beta; Vero Cells

Intrauterine adhesion (IUA) is one of the main causes of female infertility. This study reveals that endoplasmic reticulum stress activation upregulates the TGF-β/SMAD pathway to induce epithelial-mesenchymal transition and promote endometrial fibrosis in an IUA model.. IUA is a common gynecological disease and is a leading cause of female infertility. Mechanical or infectious damage to the endometrial basal layer can lead to endometrial fibrosis, which is the most common cause of IUA. Endoplasmic reticulum stress (ERS), the transforming growth factor beta signaling pathway (TGF-β/SMAD) and epithelial-mesenchymal transition (EMT) are important factors promoting endometrial fibrosis. The purpose of this study was to determine the up- and downstream regulatory relationships of the above three in the process of endometrial fibrosis. The rat IUA model was induced by double injury method and prophylactic injection of the ERS inhibitor 4-phenylbutyric acid (4-PBA) was given in vivo. The ERS activator tunicamycin and the TGF-β/SMAD pathway inhibitor A 83-01 were used in human endometrial epithelial cells (HEECs) in vitro. Masson's trichrome, Sirius red staining, immunohistochemistry, immunofluorescence and Western blot analyses were used to determine ERS, TGF-β/SMAD pathway, EMT and fibrosis markers in the uterine tissue and HEECs of the different treatment groups. In animal experiments, ERS and the TGF-β/SMAD pathway had been activated and EMT occurred in an in vivo model of IUA but was suppressed in animals treated with prophylactic 4-PBA. In in vitro experiments, tunicamycin-treated HEECs had increased the activation of ERS, the abundance of TGF-β/SMAD pathway and fibrosis markers while EMT occurred, but the TGF-β/SMAD pathway and EMT were significantly inhibited in the tunicamycin+A 83-01 group. Our data suggest that increased ERS can induce EMT and promote endometrial fibrosis through the TGF-β/SMAD pathway.

Topics: Animals; Endoplasmic Reticulum Stress; Epithelial-Mesenchymal Transition; Female; Fibrosis; Humans; Infertility, Female; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tunicamycin; Uterine Diseases

Inhibins and their co-receptor betaglycan are members of the transforming growth factor β superfamily, a group of signaling molecules that control the differentiation of human endometrium in the secretory phase of the menstrual cycle.. Since endometriosis is associated with endometrial dysfunction and infertility, this study aimed at evaluating the expression of α-inhibin and betaglycan mRNA and proteins in endometrial samples of infertile women with and without endometriosis.. This was a cross-sectional study. Participants/Materials: Endometrial samples of women with (n = 17) and without (n = 22) endometriosis were subdivided according to the menstrual cycle phase into proliferative and secretory.. University hospital.. We used real-time RT-PCR to quantify mRNA levels and immunohistochemistry to localize the proteins.. α-inhibin mRNA levels were significantly increased in the secretory phase (p < 0.01 vs. proliferative phase) only among women with endometriosis. Conversely, betaglycan mRNA levels were downregulated in the secretory endometrium of controls (p < 0.01 vs. proliferative) but failed to change between cycle phases of patients with endometriosis. Both proteins were present in the glandular epithelium and stroma in the endometrium of women with and without endometriosis. Immunostaining analysis showed that while α-inhibin protein expression did not vary significantly, the intensity of betaglycan immunostaining decreased in the secretory phase in the control group (p = 0.038 vs. proliferative phase) but not in the endometriosis group.. We cannot determine whether endometriosis causes the abnormal expression of α-inhibin and betaglycan in the eutopic endometrium or if this alteration already existed before the establishment of endometriotic lesions.. Our findings suggest an abnormally increased expression of α-inhibin mRNA (not protein) and betaglycan (mRNA and protein) in the secretory-phase endometrium of women with endometriosis.

Topics: Cross-Sectional Studies; Endometriosis; Endometrium; Female; Humans; Infertility, Female; Inhibins; Proteoglycans; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

Oocyte competence and quality depend on communication between the oocyte and the cumulus and theca cells. In the preantral phase, the members of the transforming growth factor β (TGF-β) superfamily are responsible for this communication and play an important role in folliculogenesis. Members of the TGF-β superfamily are related to endometriosis (overexpression in the ectopic endometrium); however, few studies have explored these proteins as influencing fertility in endometriosis. Considering endometriosis-related infertility and to better understand the role of the TGF-β superfamily members in the antral phase in women with endometriosis, this research investigated the gene expression of the genes for ligands AMH, BMP-6, GDF-9, INHA, INHBB, and TGFβ3; receptors AMHR2, BMPR2, and TGFβR3; and intracellular signalling: SMAD3 and SMAD4.. The gene expression of AMH, BMP-6, GDF-9, INHA, INHBB, TGFβ3, AMHR2, BMPR2, TGFβR3, SMAD3, and SMAD4 in cumulus cells was investigated through quantitative real-time PCR in a case-control study including infertile women with and without peritoneal endometriosis undergoing in vitro fertilization.. Age and outcomes of assisted reproduction were similar between the groups (P > .05). However, women with endometriosis showed reduced expression of BMP-6 and SMAD4 (P < .05) in cumulus cells compared with the control group, other genes did not present altered gene expression in women with endometriosis (P > .05).. The reduced expression of BMP-6 and SMAD4 in women with peritoneal endometriosis compared with the control group indicates that granulosa (cumulus) cell function could be altered in these women.

Topics: Adult; Anti-Mullerian Hormone; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein Receptors, Type II; Case-Control Studies; Cumulus Cells; Endometriosis; Female; Gene Expression; Growth Differentiation Factor 9; Humans; Infertility, Female; Inhibin-beta Subunits; Inhibins; Intracellular Signaling Peptides and Proteins; Ligands; Proteoglycans; Real-Time Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Peptide; Receptors, Transforming Growth Factor beta; Smad3 Protein; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta3

Endometriosis (EMS) is a gynecologic disorder associated with infertility and characterized by the endometrial-type mucosa outside the uterine cavity. Currently available treatment modalities are limited to undesirable effects. Thus, in the present study, we sought to study the pathogenesis mechanism of EMS. For this purpose, the ectopic and eutopic endometrial tissues were resected from 86 patients with EMS and 54 infertile patients without EMS, respectively. The regulatory mechanism among HES family bHLH transcription factor 5 (HES5), transforming growth factor-beta (TGF-β)-induced factor 1 (TGIF1), F-box, and WD repeat domain containing 7 (FBXW7) was studied by performing co-immunoprecipitation, dual-luciferase reporter gene assay, and chromatin immunoprecipitation, respectively. A mouse model of EMS was established to verify the aforementioned regulatory mechanism in vivo. Upregulation of HES5 and TGIF1, as well as downregulation of FBXW7, was observed in EMS endometrial tissues and human endometrial stromal cells (hESCs), respectively. The overexpression of HES5 was found to suppress the FBXW7 transcription and TGIF1 degradation, resulting in the inactivation of the TGF-β signaling pathway, as well as inhibition of hESC proliferation and invasion, thereby enhancing apoptosis. Results from a mouse model of EMS showed that the presence of HES5 contributed to the alleviation of EMS. Collectively, we attempted to provide a mechanistic insight into the unrecognized roles of the HES5/FBXW7 in EMS progression.

Topics: Adult; Animals; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Disease Progression; Endometriosis; Endometrium; F-Box-WD Repeat-Containing Protein 7; Female; Humans; Infertility, Female; Mice; Mice, Inbred BALB C; Middle Aged; Repressor Proteins; Signal Transduction; Stromal Cells; Transfection; Transforming Growth Factor beta

Uterine gland development, also known as adenogenesis, is a key uterine morphogenic process indispensable for normal uterine function and fertility. Our earlier studies have reported that overactivation of TGFB receptor 1 (TGFBR1) in the mouse uterus using progesterone receptor (Pgr)-Cre recombinase causes female infertility, defective decidualization, and reduced uterine gland formation, a developmental milestone of postnatal uterus. To understand mechanisms that underpin the disrupted uterine gland formation in mice with sustained activation of TGFBR1, we raised the question of whether early postnatal adenogenesis was compromised in these mice. Experiments were designed using mice with constitutive activation of TGFBR1 driven by Pgr-Cre to determine the timing of adenogenic defects and potential mechanisms associated with dysregulation of adenogenic genes, luminal epithelial cell proliferation and endometrial fibrotic changes. Uterine tissues from mice with constitutive activation of TGFBR1 were collected during the critical time window of adenogenesis and analyzed together with age-matched controls. Multiple approaches including immunohistochemistry, immunofluorescence, Trichrome staining, quantitative real-time PCR, western blot, conditional knockout and human endometrial cell culture were utilized. TGFBR1 activation in the mouse uterus suppressed adenogenesis during postnatal uterine development, concomitant with the aberrant differentiation of uterine stromal cells. Analysis of transcript expression of WNT pathway components revealed dysregulation of adenogenesis-associated genes. Notably, the adenogenic defects occurred in spite of the increased proliferation of uterine luminal epithelial cells, accompanied by increased expression of genes associated with fibrotic changes. Moreover, the adenogenic defects were alleviated in mice where TGFBR1 was activated in presumably half of the complement of uterine cells. Our results suggest that altered differentiation of endometrial stromal cells and formation of stromal compartment promote adenogenic defects.

Topics: Animals; Cell Differentiation; Cell Line; Epithelial Cells; Female; Humans; Infertility, Female; Mice; Mice, Transgenic; Organogenesis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Progesterone; Signal Transduction; Stromal Cells; Transforming Growth Factor beta; Uterus

Human chorionic gonadotropin (hCG) and regulatory T cells (Tregs) have been suggested to play important roles during the initial stage of pregnancy. However, the clinical relevance and mechanism of the effects of hCG on Treg functions in women with recurrent implantation failure (RIF) remain to be elucidated.. The results of this study provide novel evidence supporting a role of hCG in regulating the differentiation of peripheral FOXP3

Topics: Adolescent; Adult; Cell Movement; Cells, Cultured; Chorionic Gonadotropin; Embryo Implantation; Endometrium; Female; Forkhead Transcription Factors; Humans; Infertility, Female; Pregnancy; Pregnancy Outcome; Receptors, CCR4; Receptors, LH; Receptors, Lymphocyte Homing; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Young Adult

Calnexin (CANX) and calreticulin (CALR) chaperones mediate nascent glycoprotein folding in the endoplasmic reticulum. Here we report that these chaperones have distinct roles in male and female fertility. Canx null mice are growth retarded but fertile. Calr null mice die during embryonic development, rendering indeterminate any effect on reproduction. Therefore, we conditionally ablated Calr in male and female germ cells using Stra8 (mcKO) and Zp3 (fcKO) promoter-driven Cre recombinase, respectively. Calr mcKO male mice were fertile, but fcKO female mice were sterile despite normal mating behavior. Strikingly, we found that Calr fcKO female mice had impaired folliculogenesis and decreased ovulatory rates due to defective proliferation of cuboidal granulosa cells. Oocyte-derived, TGF-beta family proteins play a major role in follicular development and molecular analysis revealed that the normal processing of GDF9 and BMP15 was defective in Calr fcKO oocytes. These findings highlight the importance of CALR in female reproduction and demonstrate that compromised CALR function leads to ovarian insufficiency and female infertility.

Topics: Animals; Bone Morphogenetic Protein 15; Calnexin; Calreticulin; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cumulus Cells; Endoplasmic Reticulum; Female; Fertility; Growth Differentiation Factor 9; Infertility, Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oocytes; Organ Culture Techniques; Ovarian Follicle; Ovulation; Primary Ovarian Insufficiency; Protein Folding; Transforming Growth Factor beta

Follicle-stimulating hormone (FSH) is an essential regulator of gonadal function and fertility. Loss-of-function mutations in the FSHB/Fshb gene cause hypogonadotropic hypogonadism in humans and mice. Both gonadotropin-releasing hormone (GnRH) and activins, members of the transforming growth factor β (TGFβ) superfamily, stimulate FSH synthesis; yet, their relative roles and mechanisms of action in vivo are unknown. Here, using conditional gene-targeting, we show that the canonical mediator of TGFβ superfamily signaling, SMAD4, is absolutely required for normal FSH synthesis in both male and female mice. Moreover, when the Smad4 gene is ablated in combination with its DNA binding cofactor Foxl2 in gonadotrope cells, mice make essentially no FSH and females are sterile. Indeed, the phenotype of these animals is remarkably similar to that of Fshb-knockout mice. Not only do these results establish SMAD4 and FOXL2 as essential master regulators of Fshb transcription in vivo, they also suggest that activins, or related ligands, could play more important roles in FSH synthesis than GnRH.

Topics: Animals; Cells, Cultured; Crosses, Genetic; Female; Fertility; Follicle Stimulating Hormone; Forkhead Box Protein L2; Forkhead Transcription Factors; Gonadotrophs; Hypogonadism; Infertility, Female; Infertility, Male; Luteinizing Hormone; Male; Mice; Mice, Knockout; Ovary; Phenotype; Sexual Maturation; Smad4 Protein; Sperm Count; Testis; Transforming Growth Factor beta

The TGF-β signaling pathway is involved with multiple processes in the mammalian ovary, including primordial follicle formation, granulosa cell (GC) proliferation, follicle atresia, ovulation, and feedback regulation between the pituitary and ovary. The transcriptional factor SMAD4 (Sma- and Mad-related protein 4) is the central component of the canonical TGF-β signaling pathway. Smad4 knockout (KO) using Amhr2-Cre, which is expressed in GCs of immature developing follicles, causes premature luteinization. In this study, we specifically depleted Smad4 in GCs of preovulatory follicles using Cyp19-Cre mice. As different from results with Smad4(fl/fl);Amhr2-Cre mice, Smad4 depletion in preovulatory follicles did not cause premature luteinization or suppress GC proliferation; rather, it increased follicle atresia. In addition, Nppc and Npr2 expressions were reduced by Smad4 depletion; thus, their effect of maintaining oocyte meiotic arrest was weakened in Smad4 conditional KO mice. Smad4(fl/fl);Cyp19-Cre female mice were subfertile and had irregular estrous cycles and ovulation defects. Smad4 KO also blocked LH-induced cumulus expansion and follicle rupture, but not oocyte meiotic resumption. Our results also indicated that SMAD4 was required for LH-stimulated activation of ERK1/2 and the expressions of ovulation-related genes. The defects arising from SMAD4 depletion could not be rescued by intraovarian mediators of LH actions, such as epidermal growth factor-like factors and prostaglandin E2. Furthermore, corpus lutea did not form in Smad4(fl/fl);Cyp19-Cre female mice, indicating that SMAD4 was crucial for GCs terminal differentiation. Thus, by characterizing the ovarian phenotypes of preovulatory follicle-specific Smad4 KO mice, we identified the developmental stage-specific functions of the canonical TGF-β signaling pathway in ovulation and luteinization.

Topics: Amphiregulin; Animals; Cell Cycle Checkpoints; Cell Differentiation; Cells, Cultured; Dinoprostone; EGF Family of Proteins; Female; Follicular Atresia; Glycoproteins; Granulosa Cells; Infertility, Female; Intercellular Signaling Peptides and Proteins; Luteinization; Meiosis; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovarian Follicle; Ovulation; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-B) plays an important role in embryo implantation; however, TGF-B requires liberation from its inactive latent forms (i.e., large latent TGF-B complex [LLC] and small latent TGF-B complex [SLC]) to its biologically active (i.e., monomer or dimer) forms in order to act on its receptors (TGF-BRs), which in turn activate SMAD2/3. Activation of TGF-B1 from its latent complexes in the uterus is not yet deciphered. We investigated uterine latent TGF-B1 complex and its biologically active form during implantation, decidualization, and delayed implantation. Our study, utilizing nonreducing SDS-PAGE followed by Western blotting and immunoblotting with TGF-B1, LTBP1, and latency-associated peptide, showed the presence of LLC and SLC in the uterine extracellular matrix and plasma membranous protein fraction during stages of the implantation period. A biologically active form of TGF-B1 (~17-kDa monomer) was highly elevated in the uterine plasma membranous compartment at the peri-implantation stage (implantation and nonimplantation sites). Administration of hydroxychloroquine (an inhibitor of pro-TGF-B processing) at the preimplantation stage was able to block the liberation of biologically active TGF-B1 from its latent complex at the postimplantation stage; as a consequence, the number of implantation sites was reduced at Day 5 (1000 h), as was the number of fetuses at Day 13. The inhibition of TGF-B1 showed reduced levels of phosphorylated SMAD3. Further, the delayed-implantation mouse model showed progesterone and estradiol coordination to release the active TGF-B1 form from its latent complex in the receptive endometrium. This study demonstrates the importance of liberation of biologically active TGF-B1 during the implantation period and its regulation by estradiol.

Topics: Animals; Decidua; Disease Models, Animal; Embryo Implantation; Embryo Implantation, Delayed; Endometrium; Estradiol; Female; Infertility, Female; Latent TGF-beta Binding Proteins; Mice; Peptides; Phosphorylation; Placentation; Pregnancy; Progesterone; Protein Precursors; Protein Processing, Post-Translational; Protein Sorting Signals; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

This study aimed to determine the immunohistochemical characteristics of intramural leiomyomata that enlarged during controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF).. For this retrospective case-control clinical study and immunohistochemical analysis, 5 patients with enlarged intramural leiomyomata during COH for IVF, who had undergone myomectomy immediately after a failed IVF cycle, were recruited retrospectively. Fifteen consecutive patients who had had myomectomy for intramural leiomyomata <5 cm, but had never undergone any infertility treatment, served as the control group. Histological examinations and immunohistochemical staining with proliferating cell nuclear antigen (PCNA), transforming growth factor-beta (TGF-beta) and fibronectin were performed on all specimens taken from the study and control groups. The main outcome measures were defined as histological and immunohistochemical scores.. Hematoxylin and eosin as well as PCNA staining showed increased mitotic index, cellularity and proliferation index in these growing leiomyomata (study group). There were neither increased mitoses nor cellularity in the leiomyomata of the control group, while PCNA, TGF-beta and fibronectin scores in the study group were significantly higher than those in the control group (p < 0.001).. This study is the first to report increased TGF-beta, PCNA, cellularity and mitosis in leiomyomata enlarging during COH for IVF. Further studies with larger sample size are needed to determine the role of TGF-beta, PCNA and fibronectin in the growth of leiomyomata during COH.

Topics: Adult; Case-Control Studies; Female; Fertilization in Vitro; Fibronectins; Follicle Stimulating Hormone; Gene Expression; Hormones; Humans; Immunohistochemistry; Infertility, Female; Leiomyoma; Mitosis; Ovary; Ovulation Induction; Proliferating Cell Nuclear Antigen; Retrospective Studies; Transforming Growth Factor beta; Uterine Neoplasms

The process of implantation, necessary for all viviparous birth, consists of tightly regulated events, including apposition of the blastocyst, attachment to the uterine lumen, and differentiation of the uterine stroma. In rodents and primates the uterine stroma undergoes a process called decidualization. Decidualization, the process by which the uterine endometrial stroma proliferates and differentiates into large epithelioid decidual cells, is critical to the establishment of fetal-maternal communication and the progression of implantation. The role of bone morphogenetic protein 2 (Bmp2) in regulating the transformation of the uterine stroma during embryo implantation in the mouse was investigated by the conditional ablation of Bmp2 in the uterus using the (PR-cre) mouse. Bmp2 gene ablation was confirmed by real-time PCR analysis in the PR-cre; Bmp2fl/fl (termed Bmp2d/d) uterus. While littermate controls average 0.9 litter of 6.2+/-0.7 pups per month, Bmp2d/d females are completely infertile. Analysis of the infertility indicates that whereas embryo attachment is normal in the Bmp2d/d as in control mice, the uterine stroma is incapable of undergoing the decidual reaction to support further embryonic development. Recombinant human BMP2 can partially rescue the decidual response, suggesting that the observed phenotypes are not due to a developmental consequence of Bmp2 ablation. Microarray analysis demonstrates that ablation of Bmp2 leads to specific gene changes, including disruption of the Wnt signaling pathway, Progesterone receptor (PR) signaling, and the induction of prostaglandin synthase 2 (Ptgs2). Taken together, these data demonstrate that Bmp2 is a critical regulator of gene expression and function in the murine uterus.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Proliferation; Cyclooxygenase 2; Decidua; Embryo Implantation; Female; Gene Deletion; Gene Expression Regulation; Humans; Infertility, Female; Mice; Microarray Analysis; Models, Genetic; Neovascularization, Physiologic; Ovary; Signal Transduction; Tacrolimus Binding Proteins; Transforming Growth Factor beta; Wnt Proteins

TGFbeta1 is implicated in regulation of ovarian function and the events of early pregnancy. We have investigated the effect of null mutation in the Tgfbeta1 gene on reproductive function in female mice. The reproductive capacity of TGFbeta1 null mutant females was severely impaired, leading to almost complete infertility. Onset of sexual maturity was delayed, after which ovarian function was disrupted, with extended ovarian cycles, irregular ovulation, and a 40% reduction in oocytes ovulated. Serum FSH and estrogen content were normal, but TGFbeta1 null mutant mice failed to display the characteristic proestrus surge in circulating LH. Ovarian hyperstimulation with exogenous gonadotropins elicited normal ovulation rates in TGFbeta1 null mutant mice. After mating with wild-type stud males, serum progesterone content was reduced by 75% associated with altered ovarian expression of mRNAs encoding steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase-1 and P450 17 alpha-hydroxylase/C17-20-lyase. Embryos recovered from TGFbeta1 null mutant females were developmentally arrested in the morula stage and rarely progressed to blastocysts. Attempts to rescue embryos by exogenous progesterone administration and in vitro culture were unsuccessful, and in vitro fertilization and culture experiments demonstrated that impaired development is unlikely to result from lack of maternal tract TGFbeta1. We conclude that embryo arrest is due to developmental incompetence in oocytes developed in a TGFbeta1-deficient follicular environment. This study demonstrates that TGFbeta1 is a critical determinant of normal ovarian function, operating through regulation of LH activity and generation of oocytes competent for embryonic development and successful initiation of pregnancy.

Topics: Animals; Embryonic Development; Estrous Cycle; Female; Infertility, Female; Mice; Mice, Knockout; Mice, SCID; Mutation; Ovary; Ovulation; Ovum; Pregnancy; Pregnancy, Animal; Progesterone; Sexual Maturation; Transforming Growth Factor beta; Transforming Growth Factor beta1

A receptive endometrial environment requires adequate immunological tolerance to protect the implanting embryo from maternal immune rejection. Studies in mice implicate CD4+CD25+ T-regulatory (Treg) cells as essential mediators of immune tolerance in pregnancy. The aim of this study was to evaluate the link between Treg cells and fertility in women. Expression of Foxp3, a master regulator of Treg cell differentiation, was quantified in endometrial tissue from women experiencing primary unexplained infertility and normal fertile women. Endometrial biopsies were collected during the mid-secretory phase of the menstrual cycle from women meeting rigorously defined criteria for unexplained infertility after experiencing repeated failed cycles of IVF treatment (infertile, n = 10), or women classified as proven fertile (control, n = 12). Expression of Foxp3 mRNA was reduced approximately two-fold in the tissue of infertile women. In contrast, mRNAs encoding T cell transcription factors T-bet and GATA3, associated with differentiation of Th1 and Th2 CD4+ T cells respectively, were unchanged. Treg cell differentiation is controlled by TGFbeta, but the relative abundance in endometrial tissue of TGFbeta1, TGFbeta2, TGFbeta3 mRNAs was not changed in infertile women. Cytokines influencing Th1 and Th2 cell differentiation, including IFNgamma, IL-2, IL-4, IL-5, IL-10 and IL-12p40, as well as dendritic cell-regulating cytokines IL-1alpha, IL-1beta, IL-6, LIF, GM-CSF and TNFalpha were also expressed similarly regardless of fertility status. The finding of reduced endometrial Foxp3 implicates impaired differentiation of uterine T cells into the Treg phenotype as a key determinant of fertility in women. The factors underpinning this aberration in the immune response remain to be identified.

Topics: Adult; CD4-Positive T-Lymphocytes; Cytokines; Endometrium; Female; Forkhead Transcription Factors; GATA3 Transcription Factor; Gene Expression; Humans; Infertility, Female; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Box Domain Proteins; Th1 Cells; Th2 Cells; Transcription Factors; Transforming Growth Factor beta

To quantify the expression of transforming growth factor beta1 in nerve fibers in endometriotic lesions and to correlate it with dysmenorrhea and appearance of endometriotic implants.. Prospective comparative study.. University hospital.. Peritoneal endometriotic specimens obtained from 35 patients diagnosed with endometriosis were compared with biopsies of normal peritoneum from 10 patients without endometriosis.. Endometriosis-associated dysmenorrhea for each patient was evaluated before surgery using a 10-point visual analog scale, which was followed by a laparoscopic staging of the patient's endometriosis.. Immunohistochemical analysis of the peritoneal endometriotic specimens evaluated the maximal intensity of staining (INTMMAX) of TGFbeta1, defined as higher staining intensity found within a selected structure.. When the nerve fibers of endometriotic lesions were compared with those of normal peritoneum, statistically significant differences were found in the INTMMAX of TGFbeta1. Greater TGFbeta1 INTMMAX was found in red lesions and deep endometriotic foci than in black lesions and normal peritoneum. A statistically significant relationship was found between the TGFbeta1 INTMMAX score and dysmenorrhea; a relationship also was found to the color of the lesions.. The physical appearance of endometriotic implants and the severity of dysmenorrhea appear to be related to the expression of TGFbeta1 in nerve fibers.

Topics: Adult; Biopsy; Color; Dysmenorrhea; Endometriosis; Endometrium; Female; Humans; Hysteroscopy; Immunohistochemistry; Infertility, Female; Multivariate Analysis; Nerve Fibers; Pelvic Pain; Peritoneum; Prospective Studies; ROC Curve; Severity of Illness Index; Staining and Labeling; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Abortion, Habitual; Animals; Apoptosis; Complement System Proteins; Corticotropin-Releasing Hormone; Female; Fertilization in Vitro; Fetus; Histocompatibility Antigens Class I; HLA Antigens; HLA-G Antigens; Humans; Immune System; Immune Tolerance; Infertility, Female; Killer Cells, Natural; Male; Mice; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Reproduction; Semen; T-Lymphocytes; Transforming Growth Factor beta; Trophoblasts; Tryptophan Oxygenase; Uterus

To determine the effect of ovarian stimulation on TH1, TH2 and natural killer (NK) lymphocytes, plasma cytokines, leptin and nitrite levels.. Women with reproductive failure were studied during the implantation window, at baseline (n = 18) and under ovarian stimulation (gonadotropins + progesterone, n = 6).. eight fertile women. Lymphocyte subpopulations and NK function were determined by flow cytometry. Interleukin-2 (IL-2), IL-4, IL-10, IFN-gamma, TNF-alpha, TGF-beta1 and leptin were measured by enzyme immunoassay (EIA); nitrite by the Griess reaction.. At baseline, patients had higher values of NK effectors, NK activity and plasma IFN-gamma and IL-2 than controls. Conversely, TGF-beta1 values were lower. Hormones induced leukocytosis. Under stimulation, THI CD4+ cells, NK effectors and function and plasma IFN-gamma and IL-2 decreased, while transforming growth factor (TGF)-beta1 increased. Other variables did not change.. The abnormal distribution of leukocytes, high TH1 cytokines and a low TGF-beta1 associated with reproductive failure, respond to ovarian stimulation, achieving total or partial normalization.

Topics: Adult; Chorionic Gonadotropin; Cytokines; Cytotoxicity, Immunologic; Female; Humans; Immunophenotyping; Infertility, Female; Interferon-gamma; Interleukin-10; Interleukin-2; Interleukin-4; Killer Cells, Natural; Leptin; Leuprolide; Lymphocyte Count; Nitrites; Ovulation Induction; Progesterone; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

In a preliminary study the hypothesis was tested that cytokine profiles in peripheral blood were higher in women with deep infiltrating endometriosis and cytokine profiles in peritoneal fluid were higher in women with superficial endometriosis. Thirteen women of reproductive age having laparoscopy for infertility (n=9), pain (n=3) or combined pain and infertility (n=1). Peripheral blood and peritoneal fluid were obtained and analyzed for Interleukin-6 (IL-6), Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin-10 (IL-10), Transforming Growth Factor-betal (TGFbeta1), and Interferon-gamma (IFN-gamma). No significant cytokine differences were observed in either peritoneal fluid or peripheral blood between IL-6, TGFbeta1, IFNgamma, TNF-alpha and IL-10 of women with superficial endometriosis (n=7) and women with deeply infiltrating endometriosis (n=6). The results of this preliminary study do not show significant differences in peripheral blood and peritoneal fluid cytokine levels between women with deep infiltrating endometriosis compared to women with superficial disease. Future studies with increased sample size are required to either confirm or refute these preliminary findings.

Topics: Adult; Ascitic Fluid; Cytokines; Endometriosis; Female; Humans; Infertility, Female; Interferon-gamma; Interleukin-10; Interleukin-6; Laparoscopy; Pain; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The expression profiles of leukemia inhibitory factor (LIF), transforming growth factor beta2 (TGFbeta2), and transforming growth factor beta2 receptor (TGFbeta2R) were analyzed during the peri-implantation period in regularly menstruating, fertile bonnet monkeys and in animals in which endometrial nonreceptivity was induced by administering an antiprogestin, onapristone. Based on our previous experiences, a dose of 2.5 or 5 mg of onapristone was administered s.c. every third day during the menstrual cycle, because these dosages impair endometrial development without upsetting the normal gonadal endocrine profiles. Endometrial biopsy specimens were collected during the proliferative phase (estradiol levels about 200 pg/ml, n = 5) and peri-implantation period (Day 8 after midcycle peak in estradiol levels, n = 5) from normal ovulatory animals and during the peri-implantation period from onapristone-treated animals (n = 10). The biopsy specimens were processed to determine the expression patterns of LIF, TGFbeta2, and TGFbeta2R by immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) methods. Levels of both protein and mRNA for LIF, TGFbeta2, and TGFbeta2R (analyzed by immunohistochemistry and RT-PCR, respectively) were greater in the endometrial samples collected during the peri-implantation period compared to samples collected during the proliferative phase in control animals. Treatment with either of the two doses (2.5 or 5 mg) of onapristone caused a significant (P < 0.05) down-regulation in the expression of LIF in the peri-implantation endometria. The endometrial expressions of TGFbeta2 and TGFbeta2R mRNAs were reduced significantly in animals treated with 5 mg of onapristone, but not in those treated with the lower dose. However, immunoreactive TGFbeta2 and TGFbeta2R proteins were significantly (P < 0.05) down-regulated in the endometrial samples from both the 2.5- and 5-mg-treated groups. The alterations observed in the expression patterns of LIF, TGFbeta2, and TGFbeta2R were specific, because the expression levels of epidermal growth factor receptor remained unaffected in the endometria from the treated groups. The present study demonstrates derangement in the expression profiles of LIF, TGFbeta2, and TGFbeta2R during the peri-implantation period in infertile bonnet monkeys. It may be hypothesized that TGFbeta2 function is one of the early steps in the regulation of the progesterone-driven cascade of events leading to end

Topics: Animals; Endometrium; Estradiol; Female; Fertility Agents, Female; Gonanes; Growth Inhibitors; Immunohistochemistry; Infertility, Female; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines; Macaca radiata; Progesterone; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

To determine if exposure of human gametes to macrophage secretory products reduces sperm binding to the zona pellucida, and to determine which cytokine(s) may be responsible for this effect.. A human macrophage cell line was cultured and either activated with lipopolysaccharide for 2 hours and then washed or left unactivated. Culture-conditioned media from activated or unactivated cells was used in hemizona assay. Hemizonae were incubated with sperm suspended in culture medium from either unactivated macrophages or activated macrophages, with the matching hemizona incubated with sperm suspended in control medium. Matching hemizonae were incubated with sperm suspended in unactivated macrophage medium paired with sperm suspended in activated macrophage culture medium. Conditioned medium from activated macrophages was found to have elevated levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, and transforming growth factor-beta, therefore, gametes were also exposed to these cytokines followed by the hemizona assay. After each incubation, the number of sperm tightly bound to the outer surface of each hemizona was determined.. Exposure of gametes to activated and unactivated macrophage culture-conditioned media significantly decreases sperm binding to the zona pellucida, with medium from activated macrophages inducing the greatest effect (P < .05). Exposure of sperm to TNF-alpha significantly impaired sperm binding (P < .05), whereas other cytokines tested had no effect.. These results suggest that macrophage secretory products in the basal and activated state may be a factor in endometriosis-associated infertility through the interference of sperm binding to the zona pellucida, and that TNF-alpha is a key cytokine responsible for this effect.

Topics: Culture Media, Conditioned; Dose-Response Relationship, Drug; Endometriosis; Female; Humans; Infertility, Female; Interleukin-1; Lipopolysaccharides; Macrophages; Male; Spermatozoa; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Zona Pellucida

We recently described the expression of ebaf, a novel member of the transforming growth factor-beta superfamily in human endometrium. ebaf messenger ribonucleic acid was expressed in late secretory and menstrual endometria. Here, we show that ebaf is secreted as 42-, 34-, 28-, and 14-kDa proteins into the conditioned medium of transfected cells, endometrial fluid, and serum. The amount of secreted proteins was markedly reduced during the implantation window in the endometria and sera of normal fertile subjects. The expression of ebaf was dysregulated in the endometria of a subset of women with infertility during the receptive phase of the menstrual cycle. Abundant secreted protein was present in the endometria of these women during the implantation window. During the critical period of endometrial receptivity, ebaf protein was more abundant in patients with endometriosis who did not conceive than in patients who became pregnant. These findings show that ebaf is a secreted product and is released into body fluids. Some types of infertility are associated with dysregulated expression of ebaf in human endometrium, suggesting that a molecular defect in uterine receptivity may be identified using such a marker protein.

Topics: Adult; Amino Acid Sequence; Blotting, Northern; Blotting, Western; Endometrium; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Infertility, Female; Left-Right Determination Factors; Menstrual Cycle; Molecular Sequence Data; Plasmids; RNA; Transfection; Transforming Growth Factor beta

We have analysed the content of the growth and differentiation regulating peptide, transforming growth factor beta1 (TGFbeta1), in follicular fluid from patients undergoing in-vitro fertilization (IVF), and correlated concentrations of TGFbeta1 with the outcome of the IVF treatment and the concentrations of 17beta oestradiol in serum at ovum retrieval. A total of 88 women with infertility of >3 years duration and age <38 years participated in the study. During IVF treatment, follicular fluid and matched serum samples were collected at ovum retrieval and analysed for TGFbeta1, oestradiol, progesterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) using radioimmunoassay and enzyme-linked immunosorbent assay. We found that the TGFbeta1 content in the follicular fluid at the time of oocyte retrieval correlated positively with subsequent pregnancy. In 29 women who became pregnant following IVF, follicular fluid TGFbeta1 values were significantly higher (P=0.005) than in 59 women where IVF was unsuccessful. In the pregnant group, TGFbeta1 values correlated positively with oestradiol at ovum retrieval. TGFbeta1 also correlated positively with the number of fertilized oocytes. TGFbeta1 may thus be important for successful human pre-embryo development, contribute to successful embryo implantation and development and may be necessary for the establishment of pregnancy.

Topics: Adult; Embryo Transfer; Estradiol; Female; Fertilization in Vitro; Follicle Stimulating Hormone; Follicular Fluid; Humans; Infertility, Female; Luteinizing Hormone; Ovulation Induction; Pregnancy; Progesterone; Transforming Growth Factor beta

The purpose of this study was to clarify the relationship between NK activity and TG-beta in the immune system in endometriosis. We investigated (1) the changes in the NK activity and concentration of TGF-beta in human peritoneal fluid (HPF), and (2) the effects of HPF and TGF-beta on the development of early mice embryos. In a rat model of experimental endometriosis, we observed the effects of tissue culture supernatants of peritoneum on NK activity in rat spleen cells, and obtained the following results. (1) NK activity of peripheral lymphocytes in healthy women was significantly suppressed in the presence of HPF of endometriosis. (2) The concentrations of TGF-beta was significantly higher in HPF of endometriosis than in HPF of healthy women. (3) Both HPF of endometriosis and TGF-beta significantly inhibited the development of early mice embryos. (4) The supernatants prepared from the intact peritoneum of the rat model showed marked inhibition of NK activity compared to control rats, although the peritoneum was obtained from a region distant from the implanted endometrium. These results suggest that ectopic endometrial tissues may cause a change in the cell-mediated immune system and subsequently exert an adverse effect on human reproduction.

Topics: Animals; Ascitic Fluid; Cells, Cultured; Embryonic and Fetal Development; Endometriosis; Endometrium; Female; Humans; In Vitro Techniques; Infertility, Female; Killer Cells, Natural; Mice; Mice, Inbred ICR; Rats; Rats, Wistar; Transforming Growth Factor beta

To investigate the presence of transforming growth factor-beta in peritoneal fluid of women with and without endometriosis.. Fifty-two peritoneal fluid samples, obtained during laparoscopies performed for tubal ligation (n = 10), infertility (n = 38), or pain (n = 4), were examined for the presence of transforming growth factor-beta using the Mv1Lu cell growth inhibition assay. At laparoscopy, 26 women had endometriosis. The other 26 women had no endometriosis; 16 of them had infertility, and ten who had no pelvic pathology at tubal sterilization served as fertile controls.. The concentration of transforming growth factor-beta was increased in the peritoneal fluid from women with endometriosis (11.4 +/- 3.3 ng/mL) compared to both the fertile control group without endometriosis (1.1 +/- 0.29 ng/mL) and the infertile control group without endometriosis (3.6 +/- 1.4 ng/mL). Twenty-five of the 52 women (48%) demonstrated levels of transforming growth factor-beta higher than 2 ng/mL. Patients with endometriosis were significantly more likely to have elevated concentrations of transforming growth factor-beta than were women without endometriosis (16 of 26, 61.5%, versus nine of 26, 34.6%).. These findings demonstrate the presence of transforming growth factor-beta in peritoneal fluid. Elevated levels in women with endometriosis could be important in the pathophysiology of this disease.

Topics: Ascitic Fluid; Endometriosis; Female; Humans; Infertility, Female; Killer Cells, Natural; Laparoscopy; Sterilization, Tubal; Transforming Growth Factor beta