RESUMO
BACKGROUND: Extravillous trophoblast cell (EVT) differentiation and its communication with maternal decidua especially the leading immune cell type natural killer (NK) cell are critical events for placentation. However, appropriate in vitro modelling system and regulatory programs of these two events are still lacking. Recent trophoblast organoid (TO) has advanced the molecular and mechanistic research in placentation. Here, we firstly generated the self-renewing TO from human placental villous and differentiated it into EVTs (EVT-TO) for investigating the differentiation events. We then co-cultured EVT-TO with freshly isolated decidual NKs for further study of cell communication. TO modelling of EVT differentiation as well as EVT interaction with dNK might cast new aspect for placentation research. RESULTS: Single-cell RNA sequencing (scRNA-seq) was applied for comprehensive characterization and molecular exploration of TOs modelling of EVT differentiation and interaction with dNKs. Multiple distinct trophoblast states and dNK subpopulations were identified, representing CTB, STB, EVT, dNK1/2/3 and dNKp. Lineage trajectory and Seurat mapping analysis identified the close resemblance of TO and EVT-TO with the human placenta characteristic. Transcription factors regulatory network analysis revealed the cell-type specific essential TFs for controlling EVT differentiation. CellphoneDB analysis predicted the ligand-receptor complexes in dNK-EVT-TO co-cultures, which relate to cytokines, immunomodulation and angiogenesis. EVT was known to affect the immune properties of dNK. Our study found out that on the other way around, dNKs could exert effects on EVT causing expression changes which are functionally important. CONCLUSION: Our study documented a single-cell atlas for TO and its applications on EVT differentiation and communications with dNKs, and thus provide methodology and novel research cues for future study of human placentation.
Assuntos
Placenta , Trofoblastos , Gravidez , Feminino , Humanos , Trofoblastos/metabolismo , Decídua/metabolismo , Diferenciação Celular , Organoides , Células Matadoras Naturais/metabolismo , Movimento CelularRESUMO
BACKGROUND: Preimplantation genetic testing for aneuploidy (PGT-A) was demonstrated to be superior to conventional IVF in reducing the incidence of miscarriage and abnormal offspring after the first embryo transfer (ET). PGT-A requires several embryo trophectoderm cells, but its negative impacts on embryo development and long-term influence on the health conditions of conceived children have always been a concern. As an alternative, noninvasive PGT-A (niPGT-A) approaches using spent blastocyst culture medium (SBCM) achieved comparable accuracy with PGT-A in several pilot studies. The main objective of this study is to determine whether noninvasive embryo viability testing (niEVT) results in better clinical outcomes than conventional IVF after the first embryo transfer. Furthermore, we further investigated whether niEVT results in higher the live birth rate between women with advanced maternal age (AMA, > 35 years old) and young women or among patients for whom different fertilization protocols are adopted. METHODS: This study will be a double-blind, multicenter, randomized controlled trial (RCT) studying patients of different ages (20-43 years) undergoing different fertilization protocols (in vitro fertilization [IVF] or intracytoplasmic sperm injection [ICSI]). We will enroll 1140 patients at eight reproductive medical centers over 24 months. Eligible patients should have at least two good-quality blastocysts (better than grade 4 CB). The primary outcome will be the live birth rate of the first embryo transfer (ET). Secondary outcomes will include the clinical pregnancy rate, ongoing pregnancy rate, miscarriage rate, cumulative live birth rate, ectopic pregnancy rate, and time to pregnancy. DISCUSSION: In this study, patients who undergo noninvasive embryo viability testing (niEVT) will be compared to women treated by conventional IVF. We will determine the effects on the pregnancy rate, miscarriage rate, and live birth rate and adverse events. We will also investigate whether there is any difference in clinical outcomes among patients with different ages and fertilization protocols (IVF/ICSI). This trial will provide clinical evidence of the effect of noninvasive embryo viability testing on the clinical outcomes of the first embryo transfer. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR) Identifier: ChiCTR2100051408. 9 September 2021.
Assuntos
Aborto Espontâneo , Coeficiente de Natalidade , Criança , Feminino , Gravidez , Humanos , Adulto , Aborto Espontâneo/epidemiologia , Aborto Espontâneo/etiologia , Injeções de Esperma Intracitoplásmicas , Taxa de Gravidez , Aneuploidia , Fertilização in vitro , Ensaios Clínicos Controlados Aleatórios como Assunto , Estudos Multicêntricos como AssuntoRESUMO
Connective tissue growth factor (also known as CTGF or CCN2) is a secreted matricellular protein that belongs to the CCN family. With wide-ranging biological activities and tissue expression patterns, CTGF plays a critical role in regulating various cellular functions. In the female reproductive system, CTGF is highly expressed in granulosa cells in growing ovarian follicles and is involved in the regulation of follicular development, ovulation, and luteal function. In the mammalian ovary, bone morphogenetic protein 6 (BMP6) is an important intraovarian modulator of follicular development. In this study, we demonstrated that BMP6 treatment significantly increased the expression of CTGF in both primary and immortalized human granulosa cells. Using both pharmacological inhibitors and Small interfering RNA-mediated knockdown approaches, we showed that ALK2 and ALK3 type I receptors are required for BMP6-induced cellular activities. Furthermore, this effect is most likely mediated by a Sma- and Mad-related protein (SMAD)-dependent pathway. Our studies provide novel insight into the molecular mechanisms by which an intraovarian growth factor affects the production of another factor via a paracrine effect in human granulosa cells.
Assuntos
Proteína Morfogenética Óssea 6/farmacologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células da Granulosa/metabolismo , Proteínas Smad/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Células da Granulosa/efeitos dos fármacos , Humanos , Luteinização , Hormônio Luteinizante , Transdução de Sinais , Proteínas Smad/genéticaRESUMO
BACKGROUND: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. METHODS: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. RESULTS: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. CONCLUSIONS: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.
Assuntos
Antígenos HLA-G/metabolismo , Tolerância Imunológica/fisiologia , Células-Tronco Neurais/metabolismo , Diferenciação Celular , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Antígenos HLA-G/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Lentivirus/genética , Células-Tronco Neurais/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Teratoma/patologia , TransfecçãoRESUMO
The human leukocyte antigen G (HLA-G) is expressed on the fetal-maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell-cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (ß-hCG) were elevated. HLA-G up-regulates ß-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in ß-hCG compared with control cells. The defect in ß-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating ß-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.
Assuntos
Fusão Celular , Coriocarcinoma/imunologia , Gonadotropina Coriônica/biossíntese , Antígenos HLA-G/imunologia , Sistema de Sinalização das MAP Quinases , Trofoblastos/citologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Primers do DNA , Imunofluorescência , Técnicas de Silenciamento de Genes , Antígenos HLA-G/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Regulação para CimaRESUMO
OBJECTIVE: To investigate whether embryonic soluble human leukocyte antigen-G (sHLA-G) could be a noninvasive marker for embryo competency in assisted reproductive technology (ART), which is still controversial due to the different detection assays used and the different culture conditions in laboratories. STUDY DESIGN: Based on the standardization of IVF procedures and the embryo culture condition, a total of 205 embryo culture supernatants (ESs) from 92 ART cycles were evaluated for sHLA-G contents by chemiluminescent ELISA assay. RESULTS: sHLA-G presence could be detected in 30.7% of the ESs tested. In the cycles where at least one of the embryos transferred was positive for sHLA-G, 51.9% of patients (27/52) achieved a clinical pregnancy. In cycles where none of the embryos transferred secreted detectable amounts of sHLA-G, the pregnancy rate was only 30.0% (12/40, p < 0.05). The implantation rate in sHLA-G-positive cycles was also significantly higher (31.5%) than that in sHLA-G-negative cycles (14.9%, p < 0.05). CONCLUSION: The results suggested sHLA-G in ESs as a potential marker of embryo competency in ART programs for the Chinese population.
Assuntos
Biomarcadores/análise , Embrião de Mamíferos/química , Embrião de Mamíferos/fisiologia , Antígenos HLA-G/análise , Técnicas de Reprodução Assistida , China , Meios de Cultivo Condicionados/química , Técnicas de Cultura Embrionária , Implantação do Embrião , Embrião de Mamíferos/imunologia , Feminino , Fertilização in vitro , Humanos , Gravidez , Taxa de Gravidez , SolubilidadeRESUMO
OBJECTIVE: To evaluate the role and efficacy of preventing bone mineral loss in patients with endometriosis treated by gonadotrophin-releasing hormone analogues (GnRH-a) combined with add-back therapy. METHODS: Prospective, randomized controlled studies of the use of GnRHa with add-back therapy in treatment of endometriosis were enrolled in this study from Medline, Embase, Cochrane library, China National Knowledge Internet (CNKI), Chinese Biological Medicine Disk (CBM) and Data Base of Wanfang.After quality assessment and data extraction, meta-analysis were conducted in the change of BMD, reproductive hormone (E2) and visual pain score(VAS) by Stata 11.0 software. RESULTS: A total of 785 patients from 13 randomized controlled trail (RCT) studies enrolled in this study after exclude no following up, poor quality and repeat published studies.377 patients were in group of GnRH-a with add-back treatment and 408 patients were in group of GnRna alone.The findinds were showed in meta-analysis: (1) there was a significant difference in percentage change of bone mineral density (BMD) between two groups, the add-back therapy was more effective in prevention of bone loss which was (SMD = 0.223, 95%CI:0.003 to 0.443, P = 0.047). (2) There was no significant difference in the level of reproductive hormone between two groups (SMD = -0.053, 95% CI:-0.479 to 0.373, P = 0.807). (3) There was also no significant difference in the visual pain score between the two groups (SMD = -0.157, 95% CI: -0.474 to 0.160, P = 0.332). CONCLUSIONS: GnRH-a with add-back therapy have been shown to be more effective in preventing loss of BMD than GnRH-a treatment alone.However, the long term effect of preventing BMD should be studied.
Assuntos
Densidade Óssea/efeitos dos fármacos , Endometriose/tratamento farmacológico , Estrogênios/administração & dosagem , Hormônio Liberador de Gonadotropina/análogos & derivados , Esquema de Medicação , Quimioterapia Combinada , Antagonistas de Estrogênios/administração & dosagem , Antagonistas de Estrogênios/uso terapêutico , Estrogênios/uso terapêutico , Feminino , Hormônio Liberador de Gonadotropina/administração & dosagem , Humanos , Leuprolida/administração & dosagem , Leuprolida/uso terapêutico , Vértebras Lombares , Medição da Dor , Dor Pélvica/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
PROBLEM: The phenotypes and functions of B and CD4+ T-helper cell subsets during chronic inflammation of the endometria remain largely unexplored. This study aimed to investigate the characteristics and functions of follicular helper T (Tfh) cells to understand the pathological mechanisms of chronic endometritis (CE). METHOD OF STUDY: Eighty patients who underwent hysteroscopic and histopathological examinations for CE were divided into three groups-those with positive results for hysteroscopy and CD138 staining (DP), negative results for hysteroscopy but positive CD138 staining (SP), and negative results for hysteroscopy and CD138 staining (DN). The phenotypes of B cells and CD4+ T-cell subsets were analyzed using flow cytometry. RESULTS: CD38+ and CD138+ cells were mainly expressed in the non-leukocyte population of the endometria, and the endometrial CD19+ CD138+ B cells were fewer than the CD3+ CD138+ T cells. The percentage of Tfh cells increased with chronic inflammation in the endometria. Additionally, the elevated percentage of Tfh cells correlated with the number of miscarriages. CONCLUSIONS: CD4+ T cells, particularly Tfh cells, may be critical in chronic endometrial inflammation and affect its microenvironment, thereby regulating endometrial receptivity, compared to B cells.
Assuntos
Resultado da Gravidez , Linfócitos T Auxiliares-Indutores , Humanos , Gravidez , Feminino , Linfócitos B , Endométrio , InflamaçãoRESUMO
Maternal ageing is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age-related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aged oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. We cultivated endometrial mesenchymal stem cells (EnMSCs) from aged mice and extracted mitochondria from EnMSCs. To improve the quality of oocytes, GV oocytes were supplemented with mitochondria via microinjection. And MII oocytes from aged mice were fertilized by intracytoplasmic sperm injection (ICSI), combining EnMSCs' mitochondrial microinjection. In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aged oocytes. Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aged mice after ICSI. We also found that the birth rate of mitochondria-injected ageing oocytes was significantly increased after embryo transplantation. Our study demonstrates that supplementation with EnMSC-derived mitochondria can improve the quality of oocytes and promote embryo development in ageing mice, which might provide a prospective strategy for clinical treatment.
Assuntos
Oócitos , Sêmen , Masculino , Feminino , Animais , Camundongos , Oócitos/metabolismo , Mitocôndrias , Fertilização , Suplementos NutricionaisRESUMO
The human placenta is a unique temporary organ with a mysterious immune tolerance. The formation of trophoblast organoids has advanced the study of placental development. HLA-G is uniquely expressed in the extravillous trophoblast (EVT) and has been linked to placental disorders. With older experimental methodologies, the role of HLA-G in trophoblast function beyond immunomodulation is still contested, as is its role during trophoblast differentiation. Organoid models incorporating CRISPR/Cas9 technology were used to examine the role of HLA-G in trophoblast function and differentiation. JEG-3 trophoblast organoids (JEG-3-ORGs) were established that highly expressed trophoblast representative markers and had the capacity to differentiate into EVT. CRISPR/Cas9 based on HLA-G knockout (KO) significantly altered the trophoblast immunomodulatory effect on the cytotoxicity of natural killer cells, as well as the trophoblast regulatory effect on HUVEC angiogenesis, but had no effect on the proliferation and invasion of JEG-3 cells and the formation of TB-ORGs. RNA-sequencing analysis further demonstrated that JEG-3 KO cells followed similar biological pathways as their wild-type counterparts during the formation of TB-ORGs. In addition, neither HLA-G KO nor the exogenous addition of HLA-G protein during EVT differentiation from JEG-3-ORGs altered the temporal expression of the known EVT marker genes. Based on the JEG-3 KO (disruption of exons 2 and 3) cell line and the TB-ORGs model, it was determined that HLA-G has a negligible effect on trophoblast invasion and differentiation. Despite this, JEG-3-ORG remains a valuable model for studying trophoblast differentiation.
Assuntos
Placenta , Trofoblastos , Gravidez , Feminino , Humanos , Trofoblastos/metabolismo , Placenta/metabolismo , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Linhagem Celular Tumoral , OrganoidesRESUMO
[This corrects the article DOI: 10.3389/fgene.2022.919301.].
RESUMO
Purpose: Recurrent implantation failure (RIF) is an enormous challenge for in vitro fertilization (IVF) clinicians. An understanding of the molecular mechanisms of RIF helps to predict prognosis and develop new therapeutic strategies. The study is designed to identify diagnostic biomarkers for RIF as well as the potential mechanisms underlying RIF by utilizing public databases together with experimental validation. Methods: Two microarray datasets of RIF patients and the healthy control endometrium were downloaded from the Gene Expression Omnibus (GEO) database. First, differentially expressed microRNAs (miRNAs) (DEMs) were identified and their target genes were predicted. Then, we identified differentially expressed genes (DEGs) and selected hub genes through protein-protein interaction (PPI) analyses. Functional enrichment analyses of DEGs and DEMs were conducted. Furthermore, the key DEMs which targeted these hub genes were selected to obtain the key miRNA-target gene network. The key genes in the miRNA-target gene network were validated by a single-cell RNA-sequencing dataset of endometrium from GEO. Finally, we selected two miRNA-target gene pairs for further experimental validation using dual-luciferase assay and quantitative polymerase chain reaction (qPCR). Results: We identified 49 DEMs between RIF patients and the fertile group and found 136,678 target genes. Then, 325 DEGs were totally used to construct the PPI network, and 33 hub genes were selected. Also, 25 DEMs targeted 16 key DEGs were obtained to establish a key miRNA-target gene network, and 16 key DEGs were validated by a single-cell RNA-sequencing dataset. Finally, the target relationship of hsa-miR-199a-5p-PDPN and hsa-miR-4306-PAX2 was verified by dual-luciferase assay, and there were significant differences in the expression of those genes between the RIF and fertile group by PCR (p < 0.05). Conclusion: We constructed miRNA-target gene regulatory networks associated with RIF which provide new insights regarding the underlying pathogenesis of RIF; hsa-miR-199a-5p-PDPN and hsa-miR-4306-PAX2 could be further explored as potential biomarkers for RIF, and their detection in the endometrium could be applied in clinics to estimate the probability of successful embryo transfer.
RESUMO
Reprogramming human somatic cells to pluripotency represents a valuable resource for research aiming at the development of in vitro models for human diseases and regenerative medicines to produce patient-specific induced pluripotent stem (iPS) cells. Seeking appropriate cell resources for higher efficiency and reducing the risk of viral transgene activation, especially oncogene activation, are of significance for iPS cell research. In this study, we tested whether human amnion-derived cells (hADCs) could be rapidly and efficiently reprogrammed into iPS cells by the defined factors: OCT4/SOX2/NANOG. hADCs from normal placenta were isolated and cultured. The 3rd passage cells were infected with the lentiviral vectors for the delivery of OCT4, SOX2, and NANOG. Afterwards, the generated iPSCs were identified by morphology, pluripotency markers, global gene expression profiles, and epigenetic status both in vitro and in vivo. The results showed that we were able to reprogram hADCs by the defined factors (OCT4/SOX2/NANOG). The efficiency was significantly high (about 0.1%), and the typical colonies appeared on the 9th day after infection. They were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and the epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into various cell types of all three germ layers both in vitro and in vivo. These results demonstrate that hADCs were an ideal somatic cell resource for the rapid and efficient generation of iPS cells by OCT4/SOX2/NANOG.
Assuntos
Âmnio/citologia , Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/fisiologia , Fator 3 de Transcrição de Octâmero/fisiologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição SOXB1/fisiologia , Fosfatase Alcalina/metabolismo , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Imunofluorescência , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Fatores de Transcrição SOXB1/genéticaRESUMO
OBJECTIVE: The purpose of this study was to gain a further understanding of the relationship between miR-152 and human leukocyte antigen (HLA)-G in human trophoblast cell line (JEG-3). STUDY DESIGN: The JEG-3 cells were transfected with pre-miR-152. The effect of the overexpressed miR-152 on HLA-G expression, trophoblast invasion, and natural killer (NK) cell-mediated cytolysis were assessed by reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, transwell invasion assay, and NK cell cytotoxicity assay, respectively. RESULTS: The miR-152 repressed HLA-G expression but exerted no effect on JEG-3 cell invasion, and overexpression of miR-152 led to increased NK cell-mediated cytolysis in JEG-3 cells. CONCLUSION: The data indicate that miR-152 may function as an immune system enhancer through up-regulating NK cell-mediated cytolysis of host cells.
Assuntos
Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/metabolismo , Trofoblastos/metabolismo , Análise de Variância , Western Blotting , Linhagem Celular , Células Cultivadas , Citotoxicidade Imunológica/genética , Citotoxicidade Imunológica/imunologia , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos HLA-G , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/imunologia , MicroRNAs/genética , MicroRNAs/imunologia , MicroRNAs/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trofoblastos/citologia , Trofoblastos/imunologia , Regiões não Traduzidas/genética , Regiões não Traduzidas/imunologiaRESUMO
OBJECTIVE: The purpose of this study was to perform a comprehensive analysis of the microRNA expression profile in placentas from preeclamptic pregnancies vs normal placentas. STUDY DESIGN: Placentas were obtained from patients with (1) mild preeclampsia (n = 8) and (2) severe preeclampsia (n = 15) and (3) in a normal control group (n = 11) with elective cesarean delivery. The microRNA expression profile was assessed by microRNA microarray and real-time reverse transcriptase-polymerase chain reaction analysis. RESULTS: Thirty-four microRNAs were expressed differentially in preeclamptic placentas, compared with normal placentas. Of these, 11 microRNAs were overexpressed, and 23 microRNAs were underexpressed in preeclamptic pregnancies. Notably, several microRNA clusters on human chromosome 19q13.42, 13q31.3, Xq26.2, Xq26.3, and 14q32.31 (a human imprinted region) were expressed differentially in preeclamptic placentas. These results were confirmed with the use of real-time polymerase chain reaction for selected microRNAs (miR-210, -152, -411, and so on). CONCLUSION: The results show that 34 microRNAs are deregulated in preeclamptic pregnancies, which suggests the involvement of these microRNAs in the pathogenesis of preeclampsia.
Assuntos
MicroRNAs/genética , Placenta , Pré-Eclâmpsia/genética , Adulto , Feminino , Expressão Gênica , Humanos , Gravidez , Índice de Gravidade de DoençaRESUMO
OBJECTIVE: To evaluate the feasibility of laparoscopically robotic assisted radical hysterectomy and pelvic lymphadenectomy in treatment of cervical cancer. METHODS: From Dec. 2008 to Aug. 2009, 5 cervical cancer patients at stage Ib1 to IIa underwent laparoscopically robotic assisted radical hysterectomy and pelvic lymphadenectomy. The following clinical parameters were recorded and compared, including operative time, blood loss, intraoperative and postoperative complications, the changes of hemoglobin before and after surgery, postoperative temperature, the time of postoperative anus exhaust and urination, hospitalization, pathologic exam, and the number of lymph nodes. RESULTS: Laparoscopically robotic assisted radical hysterectomy and pelvic lymphadenectomy were performed successfully on those 5 patients without the conversion to laparotomy. No intraoperative and postoperative complications were observed. The operative time were 305, 365, 275, 240 and 245 minutes, respectively, with a mean value of 286 minutes. Estimated blood loss was 200, 400, 650, 300 and 400 ml, respectively. The mean blood loss was 390 ml. Temperatures of all patients were not higher than 37.5 degrees C and anus exhaust was recovered at 36 hours after surgery. Those five patients were hospitalized for 11, 13, 9, 12 and 12 days respectively. Squamous carcinoma of cervix were diagnosed by the pathologic examination. The resected margin of vagina and parametrium was clear. The numbers of pelvic lymph nodes were 14, 22, 16, 21 and 18, respectively. No evidence of lymph nodes metastasis was found. CONCLUSION: Laparoscopically robotic assisted radical hysterectomy and pelvic lymphadenectomy is feasible as a novel approach in the treatment of cervical cancer.
Assuntos
Robótica , Neoplasias do Colo do Útero , Feminino , Humanos , Histerectomia , Excisão de Linfonodo , LinfonodosRESUMO
OBJECTIVE: To investigate the effect of embryotrophic factors (ETF) from human oviductal cells on gene expression of mouse early developmental embryos and discuss the role of fallopian tube in early development of embryos. METHODS: ETF was isolated from conditioned medium of human oviductal cell line by sequential liquid chromatographic systems. Mouse embryos were treated by ETF in vitro. Using differential display RT-PCR, the gene expression of embryos treated by ETF was compared with embryos without ETF treatment. The differentially expressed genes were separated, re-amplified, cloned and sequenced. RESULTS: Gene expression profiles of embryos with ETF treatment was different from embryos without this treatment. Eight differentially expressed genes were cloned and sequenced. These genes functioned in RNA degradation, synthesis, splicing, protein trafficking, cellular differentiation and embryo development. CONCLUSION: Embryotrophic factors from human oviductal cells affect gene expression of early developmental embryos. The human oviductal cells play wide roles in early developmental stages of embryos.
Assuntos
Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Alquil e Aril Transferases/genética , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/genética , Tubas Uterinas/citologia , Feminino , Glicoproteínas/genética , Humanos , Camundongos , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Oocyte maturation, the important process to produce female haploid gamete, accompanies with polarity establishment and highly asymmetric cell division to emit minor polar body within little cytoplasm. Microfilaments play central roles in polarity establishment and asymmetric cell division. Several actin regulators like WASP protein family as well as small GTPases function in microfilament dynamics, involving the process. Rac1, one member of RhoGTPases, has been reported to regulate the polarity and asymmetric cell division in mouse oocytes in vitro. The physiological role of Rac1 in mouse oocyte remains unknown. By conditional knockout technology, we specifically deleted Rac1 gene in mouse oocyte, and found that Rac1 deletion exerted little effect on mouse oocyte maturation including polarity establishment and asymmetric division, and the mutant mice showed normal fertility.
Assuntos
Fertilidade/genética , Neuropeptídeos/metabolismo , Oócitos/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Feminino , Técnicas de Silenciamento de Genes , Meiose , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Neuropeptídeos/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Elder women suffer from low or loss of fertility because of decreasing oocyte quality as maternal aging. As energy resource, mitochondria play pivotal roles in oocyte development, determining oocyte quality. With advanced maternal age, increased dysfunctions emerge in oocyte mitochondria, which decrease oocyte quality and its developmental potential. Mitochondria supplement as a possible strategy for improving egg quality has been in debate due to ethnic problems. Heterogeneity is an intractable problem even transfer of germinal vesicle, spindle, pronuclei or polar body is employed. We proposed that the autologous adipose tissue-derived stem cell (ADSC) mitochondria could improve the fertility in aged mice. We found that autologous ADSC mitochondria could promote oocyte quality, embryo development and fertility in aged mice, which may provide a promising strategy for treatment of low fertility or infertility in elder women.
Assuntos
Envelhecimento , Infertilidade Feminina , Células-Tronco Mesenquimais , Mitocôndrias/transplante , Oócitos , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Camundongos , GravidezRESUMO
OBJECTIVE: To investigate the expression of fms-like tyrosine kinase receptor 1 (Flt-1) in placentas of pre-eclampsia. METHODS: The expression of Flt-1 mRNA in the placentas from 20 pre-eclampsia patients and 20 pregnant women with normal blood pressure was detected by semi-quantitative reverse transcription-polymerase chain reaction. The protein expression of Flt-1 was analyzed using western blot in 18 pre-eclampsia patients and 18 normotensive pregnant women. RESULTS: Placental Flt-1 mRNA level in pre-eclampsia was 2.25 +/- 0.19 (intensity ratios of Flt-1 mRNA to beta-actin mRNA), significantly higher than in normotensive pregnant women 1.23 +/- 0.29 (P < 0.05). Western blot showed that Flt-1 protein level in pre-eclamptic placenta was 2.67 +/- 1.19 [western blot signal intensity ratios of Flt-1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], significantly higher than in pregnant women with normal blood pressure 0.94 +/- 0.51 (P < 0.05). CONCLUSION: Increased Flt-1 expression in pre-eclamptic placenta may be involved in pathogenesis of pre-eclampsia.