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To assess the value of deep learning in selecting the optimal embryo for in vitro fertilization, a multicenter, randomized, double-blind, noninferiority parallel-group trial was conducted across 14 in vitro fertilization clinics in Australia and Europe. Women under 42 years of age with at least two early-stage blastocysts on day 5 were randomized to either the control arm, using standard morphological assessment, or the study arm, employing a deep learning algorithm, intelligent Data Analysis Score (iDAScore), for embryo selection. The primary endpoint was a clinical pregnancy rate with a noninferiority margin of 5%. The trial included 1,066 patients (533 in the iDAScore group and 533 in the morphology group). The iDAScore group exhibited a clinical pregnancy rate of 46.5% (248 of 533 patients), compared to 48.2% (257 of 533 patients) in the morphology arm (risk difference -1.7%; 95% confidence interval -7.7, 4.3; P = 0.62). This study was not able to demonstrate noninferiority of deep learning for clinical pregnancy rate when compared to standard morphology and a predefined prioritization scheme. Australian New Zealand Clinical Trials Registry (ANZCTR) registration: 379161 .
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RESEARCH QUESTION: Can predictive post-warm parameters that support the decision to transfer a warmed blastocyst or to warm another blastocyst be identified in women with multiple frozen-vitrified blastocysts? DESIGN: Retrospective single-centre observational cohort analysis. A total of 1092 single vitrified-warmed blastocyst transfers (SVBT) with known Gardner score, maternal age and live birth were used to develop live birth prediction models based on logistic regression, including post-warm re-expansion parameters. Time-lapse incubation was used for pre-vitrification and post-warm embryo culture. A dataset of 558 SVBT with the same inclusion criteria was used to validate the model, but with known clinical pregnancy outcome instead of live birth outcome. RESULTS: Three different logistic regression models were developed for predicting live birth based on post-warm blastocyst re-expansion. Different post-warm assessment times indicated that a 2-h post-warm culture period was optimal for live birth prediction (model 1). Adjusting for pre-vitrification Gardner score (model 2) and in combination with maternal age (model 3) further increased predictability (area under the curve [AUC]â¯=â¯0.623, 0.633, 0.666, respectively). Model validation gave an AUC of 0.617, 0.609 and 0.624, respectively. The false negative rate and true negative rate for model 3 were 2.0 and 10.1 in the development dataset and 3.5 and 8.0 in the validation dataset. CONCLUSIONS: Clinical application of a simple model based on 2 h of post-warm re-expansion data, pre-vitrification Gardner score and maternal age can support a standardized approach for deciding if warming another blastocyst may increase the likelihood of live birth in SVBT.
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Transferência Embrionária , Resultado da Gravidez , Gravidez , Feminino , Humanos , Estudos Retrospectivos , Vitrificação , Blastocisto , Taxa de Gravidez , Nascido Vivo , CriopreservaçãoRESUMO
Human IVF embryos that are not used for fresh transfer are cryopreserved by vitrification for later embryo transfers. This study evaluates pre-vitrification and post-warming embryo characteristics that are suitable to predict the chance of clinical pregnancy in single vitrified blastocyst transfer (SVBT) cycles. In a multicenter observational trial (IMBOS trial), embryos were cultured in a time-lapse system before and after vitrification. Associations between clinical pregnancy, morphokinetic parameters, blastocyst collapse, KIDScore D5, pre-vitrification and post-warming Gardner scores, post-warming blastocyst size and re-expansion rates before SVBT were analyzed in 182 SVBTs which resulted in 89 clinical pregnancies. No association was found between clinical pregnancy after SVBT and the number of collapses or the maximal collapse size before vitrification. The multifactorial analysis of pre-vitrification Gardner scores showed a significant association with clinical pregnancy for trophectoderm grading but not for expansion/hatching status and inner cell mass grading. A significant association with clinical pregnancy was found for the time to reach a blastocyst after pronuclear fading (tB-tPNf), KIDScore D5 and post-warming size but not the rate of expansion or maximal expansion size. The selection of blastocysts for SVBT could benefit from using pre-vitrification parameters like tB-tPNf, trophectoderm grading and post-warming blastocyst size.
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INTRODUCTION: Worldwide infertility is highly prevalent, and lifestyle factors, such as food intake, could have an essential role in the success of a fertility treatment. The literature is not consistent and adequate for recommendations to the increasing number of women and men of reproductive age who ask for lifestyle guidance. Therefore, the aims of the Food & Fertility Study will be to investigate the possible association between food intake and semen quality in men, and pregnancy and live birth rates in women undergoing intrauterine insemination or assisted reproductive technology treatment. METHODS AND ANALYSIS: The Food & Fertility Study is a multicentre prospective cohort study which plans to enrol a total of 4000 women and men between 2022 and 2024. Data collection will take place in four fertility clinics through a web-based Food Frequency Questionnaire. Data on sperm quality and pregnancy and live birth rates will be obtained from medical records and national registers. ETHICS AND DISSEMINATION: The study is registered with and approved by the Danish Data Protection Agency, the North Denmark Region (j.nr: 2019-055298). Further, a Statement of Work and a Master Collaboration Agreement have been submitted and approved by the regional legal departments (AGR-2019-731-9667). Dissemination of the results will be through national and international conferences, in scientific environments, in the form of lectures to the broader public, and by peer-reviewed publications in international scientific journals. TRIAL REGISTRATION NUMBER: NCT05454046.
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Infertilidade Feminina , Feminino , Humanos , Masculino , Gravidez , Dinamarca/epidemiologia , Ingestão de Alimentos , Infertilidade Feminina/terapia , Estudos Multicêntricos como Assunto , Estudos Prospectivos , Sêmen , Análise do SêmenRESUMO
PURPOSE: This article aims to assess how differences in maternal age distributions between IVF clinics affect the performance of an artificial intelligence model for embryo viability prediction and proposes a method to account for such differences. METHODS: Using retrospectively collected data from 4805 fresh and frozen single blastocyst transfers of embryos incubated for 5 to 6 days, the discriminative performance was assessed based on fetal heartbeat outcomes. The data was collected from 4 clinics, and the discrimination was measured in terms of the area under ROC curves (AUC) for each clinic. To account for the different age distributions between clinics, a method for age-standardizing the AUCs was developed in which the clinic-specific AUCs were standardized using weights for each embryo according to the relative frequency of the maternal age in the relevant clinic compared to the age distribution in a common reference population. RESULTS: There was substantial variation in the clinic-specific AUCs with estimates ranging from 0.58 to 0.69 before standardization. The age-standardization of the AUCs reduced the between-clinic variance by 16%. Most notably, three of the clinics had quite similar AUCs after standardization, while the last clinic had a markedly lower AUC both with and without standardization. CONCLUSION: The method of using age-standardization of the AUCs that is proposed in this article mitigates some of the variability between clinics. This enables a comparison of clinic-specific AUCs where the difference in age distributions is accounted for.
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Inteligência Artificial , Blastocisto , Humanos , Estudos Retrospectivos , Imagem com Lapso de Tempo , Aprendizado de Máquina , Fertilização in vitroRESUMO
In this study we investigated whether age of men undergoing assisted reproductive technology (ART) treatment was associated with day of transfer, stage, morphology, and initial hCG-rise of the competent blastocyst leading to a live birth? The design was a multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial hCG-rise) from men whose partner underwent single blastocyst transfer resulting in singleton pregnancy/birth. The ART treatments were carried out at sixteen private and university-based public fertility clinics. We included 7246 men and women, who between 2014 and 2018 underwent controlled ovarian stimulation (COS) or Frozen-thawed Embryo Transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. 4842 men with a partner giving birth were included, by linking data to the Danish Medical Birth Registry. We showed that the adjusted association between paternal age and transfer day in COS treatments was OR 1.06, 95% CI (1.00;1.13). Meaning that for every increase of one year, men had a 6% increased probability that the competent blastocyst was transferred on day 6 compared to day 5. Further we showed that the mean difference in hCG values when comparing paternal age group 30-34, 35-39 and 40-45 with the age group 25-29 in those receiving COS treatment, all showed significantly lower adjusted values for older men. In conclusion we hypothesize that the later transfer (day 6) in female partners of older men may be due to longer time spent by the oocyte to repair fragmented DNA of the sperm cells, which should be a focus of future research in men.
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Nascido Vivo , Idade Paterna , Blastocisto , Estudos de Coortes , Feminino , Humanos , Masculino , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , SêmenRESUMO
PURPOSE: The aim of this study was to compare the outcomes of three endometrial preparation methods prior to frozen embryo transfer (FET): Natural cycle (NC), modified natural cycle (mNC), and programmed/artificial cycle (AC) protocols. Primary outcomes investigated were clinical pregnancy rate (CPR) and live birth rate (LBR). METHODS: A retrospective study on 2080 FET cycles including patients ≤ 35 years with a BMI ≤ 30 who underwent FET with a single autologous blastocyst stage embryo at Aarhus University Hospital or Horsens Regional Hospital in the period 2013-2019. Only blastocysts frozen by vitrification were included. No luteal phase support (LPS) was used in natural cycles. RESULTS: In NC, mNC and AC, CPRs were 34.9%, 40.6% and 32.0%, while LBRs were 32.3%, 36.3% and 26.6%, respectively. There were no significant differences in main outcomes when comparing AC with NC [LBR: OR = 0.9 (0.6; 1.2), p = 0.4]. Compared to NC, mNC-FET displayed significantly higher positive hCG, implantation rate, CPR and LBR [LBR: OR = 1.4 (1.0; 1.9), p = 0.03]. An analysis with mNC as reference group demonstrated significantly better outcomes in the mNC group compared to AC [LBR: OR 0.6 (0.5; 0.8), p = < 0.01]. CONCLUSION: The present study overall demonstrated better outcomes including LBR with mNC protocol as compared to NC and AC protocol, while comparison of AC and NC showed both protocols to be equally effective. A programmed cycle may be necessary for women with anovulatory cycles; however, normo-ovulating women may be offered a natural cycle protocol. TRIAL REGISTRATION NUMBER: 3-3013-3047/1 and 31-1522-44. Date of registration: June 24, 2019 and April 23, 2020.
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Criopreservação , Transferência Embrionária , Criopreservação/métodos , Transferência Embrionária/métodos , Feminino , Humanos , Nascido Vivo , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , VitrificaçãoRESUMO
RESEARCH QUESTION: Is it possible to identify monochorionic twin pregnancies before blastocyst transfer based on maternal or embryo characteristics registered by time lapse? DESIGN: A retrospective cohort study including women who received fertility treatment (nâ¯=â¯6501) between 2010 and 2019 at two fertility clinics in central Denmark. The treatment resulted in 2239 pregnancies after single embryo transfer (SET) and, of those, 43 (1.92%) were monochorionic twins. Baseline characteristics, information on assisted reproductive technology and ultrasonic findings at gestational week 8 were collected on all women. Furthermore, a blinded time lapse annotation analysis was conducted by two independent laboratory technicians on a total of 85 embryos. A total of 22 embryos leading to monochorionic pregnancies were matched with 63 embryos leading to singleton pregnancies. RESULTS: A monochorionic twin rate of 1.92% was found. No significant difference was found in maternal age, the use of intracytoplasmic sperm injection compared with IVF, indication for treatment or other maternal characteristics. In the blinded annotation analysis, inner cell mass grade A was associated with an increased risk of twinning (Pâ¯=â¯0.04) in fresh embryos. The s3 division timing was found to be significantly shorter in fresh twin compared with singleton embryos (Pâ¯=â¯0.006). No other time lapse parameters were found to be characteristic of twin embryos. CONCLUSION: To the best of our knowledge, this is the first blinded annotation study to identify aspects in time lapse resulting in monochorionic twins after SET. Whether inner cell mass grade A and s3 reflect a biological background for monochorionic twinning merits further investigation.
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Gravidez de Gêmeos/estatística & dados numéricos , Transferência de Embrião Único/estatística & dados numéricos , Imagem com Lapso de Tempo , Adulto , Feminino , Humanos , Gravidez , Estudos RetrospectivosRESUMO
OBJECTIVE: To study if the age of women undergoing assisted reproductive technology treatment associates with stage, morphology, and implantation of the competent blastocyst. DESIGN: Multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial human chorionic gonadotrophin [hCG] rise) from women undergoing single blastocyst transfer resulting in singleton pregnancy/birth. SETTING: Sixteen private and university-based facilities. PATIENT(S): In this study, 7,246 women who, between 2014 and 2018, underwent controlled ovarian stimulation (COS) or frozen-thawed embryo transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. Linking data to the Danish Medical Birth Registry resulted in a total of 4,842 women with a live birth being included. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The competent blastocyst development stage (1-6), inner cell mass (A, B, C), trophectoderm (A, B, C), and initial serum hCG value. RESULT(S): Adjusted analysis of age and stage in COS treatments showed that for every 1-year increase in age there was a 5% reduced probability of the competent blastocyst assessed as being in a high stage at transfer. Comparison between hCG values in women 18-24 years and 25-29 years in both COS and FET showed significantly lower levels in the youngest women. CONCLUSION(S): The initial hCG rise was influenced by the age of the woman, with an identical pattern for hCG values in COS and FET treatments. In COS, the competent blastocyst had a reduced stage with increasing women's age.
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Implantação do Embrião/fisiologia , Transferência Embrionária/tendências , Desenvolvimento Embrionário/fisiologia , Idade Materna , Adolescente , Adulto , Blastocisto/fisiologia , Gonadotropina Coriônica/sangue , Estudos de Coortes , Dinamarca/epidemiologia , Feminino , Humanos , Pessoa de Meia-Idade , Gravidez , Taxa de Gravidez/tendências , Sistema de Registros , Técnicas de Reprodução Assistida/tendências , Adulto JovemRESUMO
Cartilage regeneration is a fast growing field that combines biotechnology and molecular techniques in creating new tissue mimicking the native microenvironment. Human embryonic stem cells (hESCs) are a highly potent cell source for cartilage regeneration owing to their infinite proliferation capacity and pluripotency. Thus, lineage-specific differentiation of hESCs often results in populations with cellular heterogeneity. Chondrogenesis was induced through high-density micromass culture of hESCs and by addition of chondrogenic medium; 1:100 ITS(+), 100 nM dexamethasone, 40 µg/ml l-proline, 50 µg/mL ascorbic acid-2-phosphate, 1:100 Knockout serum, and 10 ng/mL TGFß3. At day 14 micromasses were dissociated and chondrogenically committed cell separated in a fraction-based discontinuous density gradient. After fractionation the chondrogenically committed cells were analyzed with regard to embryonic- and chondrogenic gene expression and fraction F3 and F4 with histology. In general, we found that the chondrogenic condition compared with the control condition had a significant effect on the following gene expression levels: NANOG, OCT4, SOX5, SOX9, ACAN, and COL2A1 in all fractions. Furthermore, we found in the chondrogenic condition that NANOG, OCT4, and SOX9 were significantly higher in F4 compared with F3, whereas COL2A1 and the ratio COL2A1:COL1A1 were significantly lower. Additionally, toluidine blue pH 4 stains of pellet cultures of F3 and F4 revealed that cells from F3 were more homogenous in morphology than F4. In conclusion, we propose a simple strategy to obtain more homogenous population of chondrogenically committed cells from hESCs using micromass culture and discontinuous density gradient separation.
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The goal of embryo selection models is to select embryos with the highest reproductive potential, whilst minimizing the rejection of viable embryos. Ultimately, any embryo selection model must be tested on clinical outcome. We therefore retrospectively tested a published blastocyst prediction model on a large combined set of transferred embryos with known clinical outcome. The model was somewhat effective in that we found a relative increase of 30% for implantation in the model-selected group of embryos. There was, however, a concomitant large rejection of embryos from our test cohort, which actually resulted in pregnancy. This hypothetical experiment highlights the limitations of predicting blastulation only. Crucially, it illustrates that both sensitivity and specificity are important parameters when developing embryo selection models for prospective clinical use.
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Blastocisto , Modelos Biológicos , Humanos , Técnicas de Reprodução Assistida , Estudos Retrospectivos , Imagem com Lapso de TempoRESUMO
OBJECTIVE: To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium on ongoing implantation rate (OIR). DESIGN: Multicenter, randomized, placebo-controlled, double-blinded prospective design. SETTING: Fourteen Scandinavian fertility clinics. PATIENT(S): A total of 1,332 women with indication for in vitro fertilization or intracytoplasmic sperm injection; 1,149 received embryo transfer (GM-CSF: n = 564; control: n = 585). INTERVENTION(S): Oocytes were fertilized, and embryos cultured and transferred in control medium or test medium containing 2 ng/mL GM-CSF. MAIN OUTCOME MEASURE(S): OIR at gestational week 7, with follow-up at week 12 and birth. RESULT(S): At week 7, OIRs were 23.5% (GM-CSF), and 20.0% (control) (odds ratio [OR] 1.26, 95% confidence interval [CI] 0.91-1.75). At week 12, OIRs were 23.0% (GM-CSF) and 18.7% (control) (OR 1.35, 95% CI 1.06-1.72), and live birth rates were 28.9% and 24.1%, respectively (OR 1.35, 95% CI 1.03-1.78). The effect of GM-CSF was influenced by the human serum albumin concentration in the medium. Birth weight and abnormality incidence were similar in both groups. Exploratory analyses showed that GM-CSF increased OIR in women with previous miscarriage, especially in women with more than one miscarriage. CONCLUSION(S): Addition of GM-CSF to embryo culture medium elicits a significant increase in survival of transferred embryos to week 12 and live birth. Our results are consistent with a protective effect of GM-CSF on culture-induced embryo stress. GM-CSF may be particularly efficacious in women with previous miscarriage. CLINICAL TRIAL REGISTRATION NUMBER: NCT00565747.
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Técnicas de Cultura Embrionária/métodos , Fertilização in vitro/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Adulto , Estudos de Coortes , Método Duplo-Cego , Implantação do Embrião/fisiologia , Transferência Embrionária/métodos , Feminino , Seguimentos , Humanos , Recém-Nascido , Nascido Vivo/epidemiologia , Gravidez , Estudos Prospectivos , Resultado do TratamentoRESUMO
Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added to the media for 12 weeks. None of these genes are related to the germ lineage, whereas genes for neuronal cells (PAX6 and NR6A1) and endothelial cells (FLT-1 and PTF1A) were up-regulated. To induce and support the differentiation towards the germ lineage we stimulated hESCs with different concentrations of ATRA for 7 and 14 days. We observed no significant difference in gene expression on RNA level when combining all cell lines. Whereas, the overall outcome was negative, one of these cell lines demonstrated an up-regulation of DDX4 on RNA and protein level after 7 days of ATRA stimulation. In summary, our data showed that the lack of exogenous FGF2 results in up-regulation of genes crucial for neuronal and endothelial cell differentiation of hESCs, but not in the up-regulation of genes related to germ cell differentiation when cultured on hFFs. Additionally, we demonstrated that ATRA supplementation did not result in a general specific direction of hESCs towards the germ lineage.
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Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , RNA Helicases DEAD-box/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Técnicas In Vitro , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Proteínas de Ligação a RNA/genética , Tretinoína/farmacologiaRESUMO
Evidently, children born after intracytoplasmic sperm injection (ICSI) are at an increased risk of having sex chromosomal abnormalities. Here we evaluate the change in methods used for prenatal diagnostics in patients having ICSI with epididymal or testicular sperm from the introduction of the procedure in 1995 until December 2007. Four hundred and fifty pregnancies resulted in the birth of 553 children. Of the Danish subpopulation 115 (34.2%) received nuchal translucency examination (NT) and 43 (12.8%) received invasive prenatal diagnostics (IPD). IPD was carried out in 11 out of 23 couples (48%) during the period 1995-1998. Since 2002, less than 10% chose to receive IPD. Twenty-one (57%) of 37 Danish women 37-44 years of age underwent IPD compared to only 22 (7.4%) of the 299 women less than 37 years of age (p < 0.001). Conversely, since 1999 the use of NT has gradually increased to a frequency of 88.9% in 2007. The partners of vasectomized men had significantly more often NT performed compared to those of non-vasectomized men. IPD were not otherwise associated with the etiology of azoospermia. This study documents a shift in prenatal diagnostics from IPD to NT for testicular sperm aspiration/percutaneous epididymal sperm aspiration (TESA/PESA) couples.
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Medição da Translucência Nucal , Diagnóstico Pré-Natal , Recuperação Espermática , Dinamarca , Feminino , Humanos , Masculino , Gravidez , Inquéritos e QuestionáriosRESUMO
The maintenance of pluripotency of human embryonic stem cells (hESCs) requires a high efficiency of self-renewal. During in vitro propagation, however, spontaneous differentiation occurs frequently, and there is also a risk of chromosomal changes. In this study, we assessed the properties of hESCs after long-term culture at ambient air and 5% oxygen growth conditions. The hESC lines were grown for up to 42 and 18 mo in normoxic and hypoxic conditions, respectively, and their proliferation; expression of Oct4, SSEA1, Nanog, and Notch1; karyotype; telomerase activity; and differentiation potential in vitro were evaluated. In contrast to cultures at 20% oxygen, where the central zones of the colonies underwent spontaneous differentiation, during exposure to 5% oxygen, the hESC colonies maintained a homogenous and flat morphology that was consistent with the presence of Oct4-positive undifferentiated phenotype. Irrespective of oxygen concentration, the undifferentiated cells expressed high levels of Nanog and Oct4 transcripts, normal karyotype, and high telomerase activity. When assayed for differentiation potential, they yielded derivatives of all three embryonic germ layers. Our data thus indicate that hypoxic exposure has the capacity to sustain enhanced long-term self-renewal of hESCs. The hESC lines described in the current paper can be obtained for research purposes from the Laboratory for Stem Cell Research, Aalborg University.
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Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Oxigênio/farmacologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Masculino , Fatores de TempoRESUMO
In September 2003, legislation approved in Denmark legalized work on surplus human embryos from IVF for clinical purposes to establish human embryonic stem (ES) cell cultures. The aim of this study was to establish such stem cell lines. Fresh surplus embryos were donated after informed consent from the donors. Embryos were cultured into blastocysts and using the immunosurgery procedure, inner cell masses were isolated and cultured on irradiated human foreskin fibroblasts in KnockOut D-MEM supplemented with KnockOut Serum Replacement, bFGF, and LIF. Within a period of 12 months, 198 embryos were donated. Four isolated inner cell masses developed into putative ES cell lines, CLS1, CLS2, CLS3, CLS4, which have now been continuously cultured for eight months, corresponding to 30 passages. These cells expressed markers for undifferentiated human ES cells: stage-specific embryonic antigen-4, tumour-related antigen (TRA)-1-60, TRA-1-81, OCT4, NANOG, SOX2, and FGF4. The cells expressed high levels of telomerase activity, had a normal karyotype, and have been successfully cryopreserved and thawed. Finally, the cells displayed the potential to differentiate in vitro into cell types originating from all three germ layers. It is thought that the cell lines described in this study are the first human ES cells established in Denmark.
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Técnicas de Cultura de Células , Linhagem Celular , Criopreservação/métodos , Embrião de Mamíferos/citologia , Células-Tronco Totipotentes/citologia , Antígenos de Superfície/metabolismo , Diferenciação Celular/fisiologia , Meios de Cultura , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Dinamarca , Fator 4 de Crescimento de Fibroblastos/metabolismo , Glicoesfingolipídeos/metabolismo , Proteínas HMGB/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Cariotipagem , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , Proteoglicanas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1 , Antígenos Embrionários Estágio-Específicos , Telomerase/metabolismo , Células-Tronco Totipotentes/metabolismo , Células-Tronco Totipotentes/fisiologia , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: To assess the degree of apoptosis in the cumulus cells and the variation of the zona pellucida and the maturity and fertilization of the corresponding oocyte. DESIGN: Retrospective study. SETTING: Private fertility clinic. PATIENT(S): Fifty couples undergoing ICSI. INTERVENTION(S): ICSI. MAIN OUTCOME MEASURE(S): Correlation between apoptosis in the cumulus cells and the zona pellucida thickness variation, maturation stage, fertilization rate, and embryo score. RESULT(S): This study demonstrated no correlation between apoptosis in cumulus cells and the thickness and variation of the zona pellucida in oocytes and embryos. The incidence of apoptosis was significantly higher in cumulus cells from empty zona pellucidas and germinal vesicle stage and metaphase I oocytes compared with metaphase II oocytes. Non-fertilized metaphase II oocytes showed significantly higher incidence of apoptosis compared with fertilized metaphase II oocytes. There was a correlation between embryo score and the zona pellucida thickness variation. CONCLUSION(S): Apoptosis in cumulus cells had no impact on the zona pellucida thickness and variation in oocytes and embryos. The zona pellucida thickness variation was positively correlated to good embryo score. A higher degree of apoptosis was seen in cumulus cells from immature oocytes compared with mature oocytes. Furthermore, apoptosis in cumulus cells impaired the fertilization rate of metaphase II oocytes after ICSI.