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RESEARCH QUESTION: Are TUBB8 gene variations present in Iranian infertile women with oocyte maturation arrest or embryo cleavage arrest? DESIGN: TUBB8 gene variations were investigated by polymerase chain reaction sequencing on blood samples from 16 women with oocyte maturation arrest and 12 women with cleavage arrest, collectively referred to as the experimental cohort, as well as 56 fertile women as the control group. The Exome Sequencing Project and dbSNP databases and the Genome Aggregation Database were used to search the frequency of corresponding variants. PolyPhen and SIFT were used to conduct in-silico analysis of gene variations and Align-GVGD was used to predict the effect of missense variants on proteins. The homology modelling and structure evaluation of variations was also checked. RESULTS: Two likely pathogenic variants [c.713C>T (p.Thr238Met), c.1054G>T (p.Ala352Ser)] were identified in patients with oocyte maturation arrest and one likely pathogenic variant [c.G763A, (p.Val255Met)] was identified in a patient with cleavage arrest. These changes were absent in controls. CONCLUSIONS: Three deleterious variants in TUBB8 related to oocyte maturation arrest or cleavage arrest and infertility were identified. TUBB8 variant screening for patients with oocyte maturation and cleavage arrest is recommended.
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Infertilidad Femenina , Humanos , Femenino , Infertilidad Femenina/genética , Irán , Oocitos , Fertilidad , Fase de Segmentación del Huevo , Tubulina (Proteína)/genéticaRESUMEN
Vitrification affects fertilization ability and developmental competence of mammalian oocytes. This effect may be more closely associated with an intracellular calcium rise induced by cryoprotectants. The present study aimed to assess whether addition of Ethylene Glycol Tetraacetic acid (EGTA) to vitrification solution could improve quality and developmental competence of in vitro matured ovine oocytes. Vitrified groups were designed according to the presence or absence of EGTA and/or calcium in base media, including: mPB1+ (modified PBS with Ca2+), mPB1- (modified PBS without Ca2+), mPB1+/EGTA (mPB1+ containing EGTA), mPB1-/EGTA (mPB1- containing EGTA). In vitro development, numerical chromosome abnormalities, hardening of zona pellucida, mitochondrial distribution and function of viable oocytes were evaluated and compared between groups. Quality of blastocysts was assessed by differential and TUNEL staining. Also, mRNA expression levels of six candidate genes (KIF11, KIF2C, CENP-E, KIF20A, KIF4A and KIF2A), were quantitatively evaluated by RT-PCR. Our results showed that calcium-free vitrification and EGTA supplementation can significantly increase the percentage of normal haploid oocytes and maintain normal distribution and function of mitochondria in vitrified ovine oocytes, consequently improving developmental rate after in vitro fertilization. qRT-PCR analysis showed no significant difference in mRNA expression levels of kinesin genes between vitrified and fresh oocytes. Also, the presence of calcium in vitrification solution significantly increased zona hardening. In conclusion, we have shown for the first time that supplementation of vitrification solution with EGTA, as a calcium chelator, improved the ability of vitrified ovine oocytes to preserve mitochondrial distribution and function, as well as normal chromosome segregation.
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Criopreservación/métodos , Crioprotectores/farmacología , Ácido Egtácico/farmacología , Vitrificación , Animales , Quelantes del Calcio/farmacología , Femenino , Fertilización In Vitro , Técnicas de Maduración In Vitro de los Oocitos , Oocitos/efectos de los fármacos , Ovinos , Oveja DomésticaRESUMEN
This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.
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Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oveja Doméstica/embriología , Trehalosa/administración & dosificación , Vitrificación/efectos de los fármacos , Animales , Blastocisto/fisiología , Femenino , Oocitos/fisiología , Zona Pelúcida/fisiologíaRESUMEN
Vitrification of embryos is a routine procedure in many IVF laboratories. The effect of vitrification on gene expression and some modifications of H3 histone in H19 and MEST imprinted genes in blastocysts produced in vitro from non-vitrified and vitrified two-cell embryos was investigated. The expression level of the chosen imprinted genes increased significantly (P < 0.05) in experimental groups compared with in-vivo blastocysts (control group). H3K9me2 decreased, whereas H3K9ac increased in the experimental group compared with the control group. The increases in the expression levels of the imprinted genes, and the attendant changes in histone and chromatin status associated with in-vitro culture of embryos from the two-cell stage, are unaffected by prior vitrification and warming. In the present study, it was shown that such changes are solely caused by the effect of in-vitro culture, irrespective of vitrification. Although these genes are sensitive to environmental changes, vitrification seems to have no additional effect on these genes and on the histone marks, and can threfore be considered to be a process with minimum damage for embryo cryopreservation in assisted reproductive technology applications.
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Blastocisto/citología , Criopreservación/métodos , Técnicas de Cultivo de Embriones/métodos , Histonas/metabolismo , Proteínas/metabolismo , ARN Largo no Codificante/metabolismo , Animales , Blastocisto/metabolismo , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , VitrificaciónRESUMEN
OBJECTIVE: Transforming growth factor-beta (TGF-ß) superfamily and its members that include bone morphogenetic protein 15 (BMP15), anti-Mullerian hormone (AMH), growth /differentiation factor-9 (GDF9), and their respective receptors: BMPR1A, BMPR1B, and BMPR2 have been implicated as key regulators in various aspects of ovarian function. The abnormal function of the ovaries is one of the main contributing factors to polycystic ovarian syndrome (PCOS), so this study aimed to investigate the mRNA expression proï¬le of these factors in granulosa (GCs) and cumulus cells (CCs) of those patients. MATERIALS AND METHODS: The case-control research was conducted on 30 women (15 infertile PCOS and 15 normo-ovulatory patients, 22≤age ≤38 years old) who underwent ovarian stimulation for in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycle. GCs/CCs were obtained during ovarian puncture. The expression analysis of the aforementioned genes was quantified using real-time polymerase chain reaction (PCR). RESULTS: AMH and BMPR1A expression levels were significantly increased in GCs of PCOS compared to the control group. In contrast, GDF9, BMP15, BMPR1B, and BMPR2 expressions were decreased. PCOS' CC showed the same expression patterns. GDF9 and AMH were effectively expressed in normal CCs, and BMP15 and BMPR1B in normal GCs (P<0.05). CONCLUSION: Differential gene expression levels of AMH and its regulatory factors and their primary receptors were detected in granulosa and cumulus cells in PCOS women. Since the same antagonist protocol for ovarian stimulation was used in both PCOS and control groups, the results were independent of the protocols. This diversity in gene expression pattern may contribute to downstream pathways alteration of these genes, which are involved in oocyte competence and maturation.
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OBJECTIVE: Genomic imprinting is an epigenetic phenomenon that plays a critical role in normal development of embryo. Using exogenous hormones during assisted reproductive technology (ART) can change an organism hormonal profile and subsequently affect epigenetic events. Ovarian stimulation changes gene expression and epigenetic pattern of imprinted genes in the organs of mouse fetus. MATERIALS AND METHODS: For this experimental study, expression of three imprinted genes H19, Igf2 (Insulin-like growth factor 2) and Cdkn1c (Cyclin-dependent kinase inhibitor 1C), which have important roles in development of placenta and embryo, and the epigenetic profile of their regulatory region in some tissues of 19-days-old female fetuses, from female mice subjected to ovarian stimulation, were evaluated by quantitative reverse-transcription PCR (qRT-PCR) and Chromatin immunoprecipitation (ChIP) methods. RESULTS: H19 gene was significantly lower in heart (P<0.05), liver (P<0.05), lung (P<0.01), placenta (P<0.01) and ovary (P<0.01). It was significantly higher in kidney of ovarian stimulation group compared to control fetuses (P<0.05). Igf2 expression was significantly higher in brain (P<0.05) and kidney (P<0.05), while it was significantly lower in lung of experimental group fetuses in comparison with control fetuses (P<0.05). Cdkn1c expression was significantly higher in lung (P<0.05). It was significantly decreased in placenta of experimental group fetuses rather than the control fetuses (P<0.05). Histone modification data and DNA methylation data were in accordance to the gene expression profiles. CONCLUSION: Results showed altered gene expressions in line with changes in epigenetic pattern of their promoters in the ovarian stimulation group, compared to normal cycle.
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The aim of this study was to determine if laser zona thinning could improve the rates of pregnancy and implantation for vitrified-warmed embryo transfer at the cleavage stage. A total of 400 vitrified-warmed embryo transfer cycles were randomly assigned to either the test group or the control group. The zona pellucida of vitrified-warmed embryos in the patients of the control group was untreated, whereas in the test group it was partially thinned by laser just before embryo transfer. In the test group, the clinical pregnancy and implantation rates were significantly lower as compared with that of the control group (28.5 versus 43.0, P=0.002, and 11.2 versus 16.7, P=0.004, respectively). Therefore the results of this investigation show that laser zona thinning may have an unexpected adverse effect on the rates of clinical pregnancy and implantation of vitrified-warmed embryos at the cleavage stage.
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Técnicas Reproductivas Asistidas , Zona Pelúcida/fisiología , Adulto , Criopreservación , Implantación del Embrión , Transferencia de Embrión , Embrión de Mamíferos , Femenino , Humanos , Masculino , Embarazo , Índice de Embarazo , Zona Pelúcida/ultraestructuraRESUMEN
PURPOSE: The objective of this retrospective study was to evaluate the efficacy of vitrification and slow freezing for the cryopreservation of human cleavage stage embryos in terms of post-warming survival rate, post-warming embryo morphology and clinical outcomes. METHODS: The embryos of 305 patients at cleavage stages were cryopreserved either with vitrification (153 patients) or slow-freezing (152 patients) methods. After warming; the survival rate, post-warmed embryo morphology, clinical pregnancy and implantation rates were evaluated and compared between the two groups. RESULT(S): In the vitrification group versus slow freezing group, the survival rate (96.9% vs. 82.8%) and the post-warmed excellent morphology with all blastomeres intact (91.8% vs. 56.2%) were higher with an odds ratio of 6.607 (95% confidence interval; 4.184-10.434) and 8.769 (95% confidence interval; 6.460-11.904), respectively. In this group, the clinical pregnancy rate (40.5% vs. 21.4%) and the implantation rate (16.6% vs. 6.8%) were also higher with an odds ratio of 2.427 (95%confidence interval; 1.461-4.033) and 2.726 (95% confidence interval; 1.837-4.046), respectively. CONCLUSION(S): Vitrification in contrast to slow freezing is an efficient method for cryopreservation of human cleavage stage embryos. Vitrification provides a higher survival rate, minimal deleterious effects on post-warming embryo morphology and it can improve clinical outcomes.
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Criopreservación/métodos , Embrión de Mamíferos , Congelación , Resultado del Embarazo , Adulto , Técnicas de Cultivo de Embriones , Implantación del Embrión , Transferencia de Embrión , Femenino , Humanos , Inducción de la Ovulación , Embarazo , Estudios RetrospectivosRESUMEN
BACKGROUND: Intraindividual copy number variation (CNV) origin is largely unknown. They might be due to aging and/or common genome instability at the preimplantation stage while contribution of preimplantation in human intraindividual CNVs occurrence is unknown. To address this question, we investigated mosaicism and its origin in the fetuses of natural conception. METHODS: We studied normal fetuses following therapeutic abortion due to maternal indications. We analyzed the genome of 22 tissues of each fetus by array comparative genomic hybridization for intraindividual CNVs. Each tissue was studied in 2 microarray experiments; the reciprocal aberrations larger than 40 Kb, identified by comparing tissues of each fetus, were subsequently validated using quantitative polymerase chain reaction. RESULTS: Through intraindividual comparison, frequency of reciprocal events varied from 2 to 9. According to the distribution pattern of the frequent CNV in derivatives of different germ layers, we found that its origin is early development including preimplantation, whereas CNVs with low frequency have occurred in later stages. Shared CNVs in both fetuses were belonged to thymus and related to the functional role of genes located in these CNVs. CONCLUSIONS: The origin of some of fetal CNVs is preimplantation stage. Each organ might inherit CNVs with an unpredictable pattern due to the extensive cell mixing/migration in embryonic development.
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Variaciones en el Número de Copia de ADN , Feto , Variación Genética , Mosaicismo , Hibridación Genómica Comparativa , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , EmbarazoRESUMEN
Bone marrow stromal cells (BMSCs) were reported to transdifferentiate into Schwann cells by a two-stage protocol, using beta-mercaptoethanol and retinoic acid (BME-RA) as preinducers (preinduction stage: PS) and platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), forskolin (FSK) and heregulin (HRG) as inducers (induction stage: IS). In this study, six groups were used, group one was used as control (PS: BME-RA; IS: PDGF, bFGF, FSK and HRG). In group 2, the preinducer was similar to group 1, and in the induction stage, progesterone replaced HRG. In groups 3 and 4, the preinducer was progesterone; and at the induction stage, the inducer was similar to groups 1 and 2. Accordingly, in groups 5 and 6, the preinducer was FSK. The immunohistochemical differentiation markers were S-100 and P0, and RT-PCR markers were OCT-4 and P0 at the preinduction stage, while at the induction stage P0 and NeuroD were used. The results of the study showed that S-100 was expressed in the groups after the induction stage, however, P0 was not expressed in any group. There was not any significant difference between the percentage of S100 positive cells in the 1st and 2nd groups. P0 was expressed at the mRNA level in the undifferentiated BMSCs and in the 3rd and 4th groups after the preinduction and the induction stages. The conclusion of this study is that progesterone can induce BMSCs into Schwann cell phenotype.
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Células de la Médula Ósea/citología , Diferenciación Celular/efectos de los fármacos , Progesterona/farmacología , Progestinas/farmacología , Células de Schwann/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Transdiferenciación Celular , Células Cultivadas , Colforsina/farmacología , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurregulina-1/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas S100/metabolismo , Células de Schwann/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
OBJECTIVES: Vitrification is increasingly used in assisted reproductive technology (ART) laboratories worldwide. In this study the effect of vitrification on the expression and modifications of H3 histones of Igf2 and Oct4 was investigated in blastocysts cultured from vitrified and non-vitrified two-cell embryos. MATERIALS AND METHODS: In this experimental study, two-cell embryos were cultured in KSOM medium to reach the blastocyst stage. Expression of Igf2 and Oct4 and modifications of H3 histones in regulatory regions of both genes were compared with in vivo blastocysts, which comprise the control group. To gene expression evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the ChIP assay method were carried out to assess expression and histone modifications of the two genes. RESULTS: The expression level of Igf2 was significantly higher in both experimental groups than the control group. In the regulatory region of Igf2, H3K9 methylation decreased whereas H3K9 acetylation increased in the experimental group compared with the control group. In contrast, the expression level of Oct4 was significantly lower in experimental groups. The Oct4 gene promoter showed a significant increase in H3K9 methylation and decrease in H3K9 acetylation (P<0.05). CONCLUSIONS: According to our results, both vitrification and cultivation conditions may lead to changes in expression level and modification of histones in Igf2 and Oct4. However, these effects were the same in vitrified and non-vitrified groups. Indeed, the embryo is most affected by culture environment and in vitro culture. Therefore, vitrification may be used as a low-risk technique for embryo cryopreservation in ART.
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BACKGROUND: Peripheral nerve injury is a common lesion in clinical practice and transplantation is one of the most common approaches to its treatment. While nerve graft is used for restoring the defected nerve using autologous or allogenic tissues, Schwann cells are considered as an alternative source. In this study, bone marrow stromal cells (BMSCs) were induced to transdifferentiate into Schwann-like cells (SLCs) using progesterone. METHODS: The BMSCs were collected from the long bones of rats and were transdifferentiated in vitro into SLCs by preinduction with ß-mercaptoethanol and retinoic acid, followed by induction with bFGF, PDGF, forskelin and progesterone. The SLCs were then transplanted in a rat model of sciatic nerve injury with 1-cm gaps. A sciatic function index (SFI), histological, immunohistochemical and ultrastructural studies were used in evaluating the improvement in the nerves regeneration. RESULTS: The results show significant differences in the SFI between the control and the treated groups (P<0.05). The transplant was immunoreactive to S100, and the electron microscopy showed myelination in the transplanted cells. CONCLUSIONS: There were functional and structural improvements in the progesterone-induced SLCs, which were not significantly different from the heregulin-treated ones (positive control) but still significantly different from negative controls.
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Transdiferenciación Celular/efectos de los fármacos , Traumatismos de los Nervios Periféricos , Progesterona/farmacología , Células de Schwann/trasplante , Animales , Axotomía , Modelos Animales de Enfermedad , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Células de Schwann/efectos de los fármacos , Nervio CiáticoRESUMEN
BACKGROUND: The aim of this study is to investigate the effect of ISM1 culture medium on embryo development, quality and outcomes of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles. This study compares culture medium commonly used in the laboratory setting for oocyte recovery and embryo development with a medium from MediCult. We have assessed the effects of these media on embryo development and newborn characteristics. MATERIALS AND METHODS: In this prospective randomized study, fertilized oocytes from patients were randomly assigned to culture in ISM1 (MediCult, cycles: n=293) or routine lab culture medium (G-1TM v5; Vitrolife, cycles: n=290) according to the daily media schedule for oocyte retrieval. IVF or ICSI and embryo transfer were performed with either MediCult media or routine lab media. Embryo quality on days 2/3, cleavage, pregnancy and implantation rates, baby take home rate (BTHR), in addition to the weight and length of newborns were compared between groups. RESULTS: There were similar cleavage rates for ISM1 (86%) vs. G-1TM v5 (88%). We observed a significantly higher percentage of excellent embryos in ISM1 (42.7%) compared to G-1TM v5 (39%, p<0.05). Babies born after culture in ISM1 had both higher birth weight (3.03 kg) and length (48.8 cm) compared to G-1TM v5 babies that had a birth weight of 2.66 kg and a length of 46.0 cm (p<0.001 for both). CONCLUSION: This study suggests that ISM1 is a more effective culture medium in generating higher quality embryos, which may be reflected in the characteristics of babies at birth.
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OBJECTIVE: The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. MATERIALS AND METHODS: In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student's t-test (one sample t-test). RESULTS: Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05). CONCLUSION: Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from the natural cycle. The results observed in this study have shown the possibility of an unfavorable effect on implantation and pregnancy rate.