RESUMO
The birthweight after frozen embryo transfer (FET) was significantly higher compared with fresh embryo transfer (fresh ET), while the mechanism remains unclear. In this study, we transferred vitrified-warmed or fresh mice blastocysts into pseudopregnant recipients (n = 11 each group) produced by natural mating to avoid the influence of superovulation. The fetal weight, placental weight, placental efficiency and placental architecture were studied at E18.5. Placental RNA-Seq analysis was used to identify candidate different lncRNAs and mRNAs between the FET group and the fresh ET group. We found that the fetal weight was increased in the FET group, with increased placental efficiency and the proportion of placental function related labyrinth zone area. 554 lncRNAs and 1012 mRNAs were differentially expressed. KEGG and GO enrichment analyses showed these differentially expressed lncRNAs and their targeted mRNAs might be related to placental morphogenesis. Furthermore, the most differentially expressed 15 lncRNAs and 15 mRNAs were validated by qRT-PCR, we found the LncRNA embryonic stem cells expressed 1 (Lncenc1) was significantly decreased, and Gjb5, which played an important role in labyrinth zone development, was increased. Gjb5 protein increase was further confirmed by Western blot. Lncenc1 and Gjb5 had 48 predicted co-targeted miRNAs, while the correlation analysis of Lncenc1 and Gjb5 mRNA showed a significant inverse correlation. The results showed that FET treatment might enhance the placental function to increase mouse fetal weight via the network diagram of Lncenc1-miRNA-Gjb5.
Assuntos
MicroRNAs , RNA Longo não Codificante , Animais , Blastocisto , Criopreservação , Transferência Embrionária/métodos , Feminino , Peso Fetal , Camundongos , MicroRNAs/genética , Placenta/metabolismo , Gravidez , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Aumento de PesoRESUMO
OBJECTIVE: To minimize twin birth rate by establishing an elective single cleavage embryo transfer strategy based on a twin live birth prediction model from fresh double cleavage embryos transfer (cleavage DET) patients. METHODS: A total of 2478 patients underwent fresh cleavage DET in Nanjing Tower Hospital were enrolled to establish the twin live birth prediction model by logistic regression analysis and the cutoff value was calculated by ROC curve. Another 300 fresh cleavage DET patients and 550 cleavage single-embryo transfer (SET) patients were selected to testify the sensitivity, specificity and usefulness of this model. RESULTS: The twin live birth probability (TLBP) = eX/(eX + 1), e is a natural logarithm, X = -1.763 - (0.319 × female age) + (0.329 × endometrial thickness) + (0.282 × the number of transferred top embryos) - (0.314 × previous transfer times), and the cutoff value of TLBP was 24.2%. The sensitivity of this model for predicting twin live birth was 75.6%, while the specificity was 52.5% in the external validation of 300 DET patients. Furthermore, the validation of 550 SET patients showed that the live birth rate of TLBP value positive patients was significantly higher than that in negative patients (54.3% vs. 35.5%, p < .001). When adopted an elective single cleavage embryo transfer strategy, the patients with a positive TLBP value choose SET, while still undergo DET who with a negative TLBP value, the live birth rate would maintain as 56.7%; however, the twin birth rate would significantly decline to 7.4%. CONCLUSION: Female age, endometrial thickness, the number of transferred top embryos and previous embryo transfer times were critical variables for the twin live birth prediction model. An elective single cleavage embryo transfer strategy according to this model can maintain the relatively high live birth rate, meanwhile get the acceptable low twin birth rate.
Assuntos
Coeficiente de Natalidade , Fertilização in vitro , Transferência Embrionária , Feminino , Humanos , Nascido Vivo , Gravidez , Taxa de Gravidez , Gravidez de Gêmeos , Transferência de Embrião ÚnicoRESUMO
PURPOSE: To investigate the clinical factors that could be used predict the number of transferable blastocysts in preimplantation genetic testing (PGT) cycles based on next-generation sequencing (NGS) and formed form a mathematical model to predict the chance likelihood of obtaining one transferable blastocyst, which is helpful for genetic counseling. METHODS: This retrospective study enrolled couples undergoing PGT cycles for chromosomal structural rearrangement (PGT-SR, n = 363, 202 with reciprocal translocation carriers, 131 with Robertsonian translocation carriers, 30 with inversion carriers), monogenic diseases (PGT-M, n = 47), and for Aneuploidies (PGT-A, n = 132) from January 2015 to October 2018. Stepwise multiple linear regression analysis was used to identify the factors relevant for obtaining at least one transferable blastocyst. The factors that predict the number of biopsied blastocysts were further analyzed. RESULTS: The transferable blastocyst rates were 29.94, 41.99, 49.09, 41.42, and 44.37% in the reciprocal translocation carrier, Robertsonian translocation carrier, inversion carrier, PGT-M, and PGT-A cycles, respectively. The number of transferable blastocysts in these cycles were 0.3004 × the number of biopsied blastocysts (NBB) - 0.0031, 0.4063 × NBB + 0.0460, 0.5762 × NBB - 0.3128, 0.3611 × NBB + 0.1910, and 0.4831 × NBB - 0.0970, respectively. Furthermore, the number of MII oocytes and female age were clinical predictors of NBB in reciprocal translocation and PGT-A couples, while the number of MII oocytes was the only clinical predictor in Robertsonian translocation carriers, inversion carriers, and PGT-M couples. CONCLUSIONS: The number of biopsied blastocysts was the only clinical predictor of the ability to obtain a transferable blastocyst in PGT cycles; therefore, for clinical practice, theoretically the minimum numbers of biopsied blastocysts is 4 in reciprocal translocation carrier and 3 in couples undergoing PGT for other reasons. The number of MII oocytes and female age were clinical predictors of the number of biopsied blastocysts. With the mathematical models in our study as a reference, in clinical practice, clinicians will be able to conduct a more targeted genetic consultation for different kinds of PGT patients.
Assuntos
Diagnóstico Pré-Implantação , Aneuploidia , Blastocisto , Feminino , Fertilização in vitro , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Teóricos , Gravidez , Estudos RetrospectivosRESUMO
Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against mitochondrial injury in the granulosa cells of PCOS remain unclear. In this study, decreased mitochondrial membrane potential and mtDNA content, increased number of autophagosomes were found in the granulosa cells of PCOS patients and the dihydrotestosterone (DHT)-treated KGN cells, with decreased protein level of the autophagy substrate p62 and increased levels of the cellular autophagy markers Beclin 1 and LC3B-II, while the protein levels of PTEN-induced kinase-1 (PINK1) and Parkin were increased and the level of sirtuin 1 (SIRT1) was decreased. DHT-induced PCOS-like mice also showed enhanced mitophagy and decreased SIRT1 mRNA expression. Melatonin treatment significantly increased the protein level of SIRT1 and decreased the levels of PINK1/Parkin, whereas it ameliorated the mitochondrial dysfunction and PCOS phenotype in vitro and in vivo. However, when the KGN cells were treated with SIRT1 siRNA to knock down SIRT1 expression, melatonin treatment failed to repress the excessive mitophagy. In conclusion, melatonin protects against mitochondrial injury in granulosa cells of PCOS by enhancing SIRT1 expression to inhibit excessive PINK1/Parkin-mediated mitophagy.
Assuntos
Antioxidantes/farmacologia , Células da Granulosa/efeitos dos fármacos , Melatonina/farmacologia , Mitofagia/efeitos dos fármacos , Síndrome do Ovário Policístico/metabolismo , Proteínas Quinases/efeitos dos fármacos , Sirtuína 1/efeitos dos fármacos , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Adulto , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Proteína Beclina-1/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Estudos de Casos e Controles , Linhagem Celular , DNA Mitocondrial/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Di-Hidrotestosterona/farmacologia , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/ultraestrutura , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia/fisiologia , Síndrome do Ovário Policístico/fisiopatologia , Proteínas Quinases/metabolismo , Sirtuína 1/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Human chorionic gonadotropin (hCG) can attract regulatory T cells (Tregs) into the fetal-maternal interface regulating maternal immune tolerance in pregnancy. The objective of this study was to investigate whether hCG recruits the Tregs into endometrium by inducing chemokines. The number of Tregs in the endometrium was analyzed by immunohistochemistry. The expression of CCL2 was analyzed in vivo and in vitro with hCG stimulation. CD4+CD25+ Tregs were isolated from peripheral blood for Tregs migration assay with hCG, CCL2 siRNA and CCR2 antagonist stimulation. Our results showed that the number of endometrial Tregs in RIF patients was significantly decreased (9.4⯱â¯5.3 vs. 23.1⯱â¯3.1, Pâ¯<â¯0.01), while intrauterine infusion of 2000 IU hCG increased the endometrial Tregs (18.6⯱â¯9.8 vs. 9.4⯱â¯5.3, Pâ¯<â¯0.05) and CCL2 expression (0.21⯱â¯0.01 vs. 0.17⯱â¯0.01, Pâ¯<â¯0.01). Horn injecting with 10 IU hCG also increased the endometrial Tregs in pseudopregnant mice (46⯱â¯16.8 vs. 7⯱â¯4.3, Pâ¯<â¯0.01). Furthermore, the CCL2 protein and mRNA levels were significantly increased in human endometrial stromal cells (hESCs) with the stimulation of hCG. Migration assays showed that hESCs with hCG stimulation promoted Tregs migration (2597⯱â¯833.2 vs. 1115⯱â¯670.7, Pâ¯<â¯0.05), while the number of migrated Tregs significantly decreased with CCL2 siRNA (1061⯱â¯105.4 vs. 2598⯱â¯294.7, Pâ¯<â¯0.05) or CCR2 antagonist (356.7⯱â¯138.8 vs. 2597⯱â¯833.2, Pâ¯<â¯0.05) treatment. In conclusion, intrauterine perfusion of hCG might promote the recruitment of Tregs into endometrium by inducing chemokine CCL2.
Assuntos
Quimiocina CCL2/metabolismo , Gonadotropina Coriônica/administração & dosagem , Implantação do Embrião/imunologia , Transferência Embrionária/métodos , Linfócitos T Reguladores/imunologia , Adulto , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Implantação do Embrião/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Endométrio/imunologia , Endométrio/metabolismo , Feminino , Humanos , Tolerância Imunológica/efeitos dos fármacos , Infertilidade/terapia , Masculino , Camundongos , Modelos Animais , Perfusão/métodos , Gravidez , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Células Estromais , Falha de TratamentoRESUMO
RESEARCH QUESTION: Can next-generation sequencing (NGS) based on copy number variation sequencing (CNV-Seq) identify normal/balanced embryos in balanced reciprocal translocation carriers and what are their reproductive outcomes? DESIGN: One hundred couples with balanced reciprocal translocation who underwent a total of 134 preimplantation genetic testing (PGT) cycles between January 2015 and October 2017 were evaluated. Trophectoderm cells of blastocysts were biopsied for CNV-Seq-based NGS. All the balanced/normal blastocysts were vitrified and cryopreserved. Single balanced/normal blastocysts were warmed and transferred in the subsequent frozen embryo transfer (FET) cycle. RESULTS: During the study period, 400 blastocysts were analysed by NGS-PGT, of which 109 (27.25%) were balanced and euploid. A total of 52 blastocysts were transferred in the FET cycle. Clinical pregnancy was confirmed in 34 women (65.38%), with a miscarriage rate of 2.94%; 26 healthy term babies were born, including 24 singletons and one set of twins, while eight couples had ongoing pregnancies. Amniocentesis revealed a fetal chromosome status that was consistent with the NGS-PGT results. Female carriers had a significantly higher blastocyst rate than did the male carriers (37.01% versus 31.27%, Pâ¯=â¯0.04). The transferable blastocyst rate was higher in couples treated with gonadotrophin-releasing hormone (GnRH) antagonist than in those treated with GnRH agonist (38.20% versus 24.37%, P = 0.01). However, neither carrier sex nor ovarian stimulation protocol influenced the clinical pregnancy rate. CONCLUSIONS: CNV-Seq-based NGS is an efficient and reliable PGT method for balanced reciprocal translocation.