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RESEARCH QUESTION: Do cell numbers and degree of fragmentation in cleavage-stage embryos, assessed manually, correlate with evaluations made by deep learning algorithm model iDAScore v2.0? DESIGN: Retrospective observational study (nâ¯=â¯5040 embryos; 1786 treatments) conducted at two Swedish assisted reproductive technology centres between 2016 and 2021. Fresh single embryo transfer was carried out on days 2 or 3 after fertilization. Embryo evaluation using iDAScore v2.0 was compared with manual assessment of numbers of cells and grade of fragmentation, analysed by video sequences. RESULTS: Data from embryos transferred on days 2 and 3 showed that having three or fewer cells compared with four or fewer cells on day 2, and six or fewer cells versus seven to eight cells on day 3, correlated significantly with a difference in iDAScore (medians 2.4 versus 4.0 and 2.6 versus 4.6 respectively; both P < 0.001). The iDAScore for 0-10% fragmentation was significantly higher compared with the groups with higher fragmentation (P < 0.001). When combining cell numbers and fragmentation, iDAScore values decreased as fragmentation increased, regardless of cell number. iDAScore discriminated between embryos that resulted in live birth or no live birth (AUC of 0.627 and 0.607), compared with the morphological model (AUC of 0.618 and 0.585) for day 2 and day 3, respectively. CONCLUSIONS: The iDAScore v2.0 values correlated significantly with cell numbers and fragmentation scored manually for cleavage-stage embryos on days 2 and 3. iDAScore had some predictive value for live birth, conditional that embryo selection was based on morphology.
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Aprendizaje Profundo , Transferencia de Embrión , Humanos , Embarazo , Femenino , Transferencia de Embrión/métodos , Embarazo Múltiple , Embrión de Mamíferos , Nacimiento Vivo , Estudios Retrospectivos , Recuento de Células , Fertilización In Vitro/métodosRESUMEN
STUDY QUESTION: Can use of a commercially available time-lapse algorithm for Day 5 blastocyst selection improve pregnancy rates compared with morphology alone? SUMMARY ANSWER: The use of a time-lapse selection model to choose blastocysts for fresh single embryo transfer on Day 5 did not improve ongoing pregnancy rate compared to morphology alone. WHAT IS KNOWN ALREADY: Evidence from time-lapse monitoring suggests correlations between timing of key developmental events and embryo viability. No good quality evidence exists to support improved pregnancy rates following time-lapse selection. STUDY DESIGN, SIZE, DURATION: A prospective multicenter randomized controlled trial including 776 randomized patients was performed between 2018 and 2021. Patients with at least two good quality blastocysts on Day 5 were allocated by a computer randomization program in a proportion of 1:1 into either the control group, whereby single blastocysts were selected for transfer by morphology alone, or the intervention group whereby final selection was decided by a commercially available time-lapse model. The embryologists at the time of blastocyst morphological scoring were blinded to which study group the patients would be randomized, and the physician and patients were blind to which group they were allocated until after the primary outcome was known. The primary outcome was number of ongoing pregnancies in the two groups. PARTICIPANTS/MATERIALS, SETTING, METHODS: From 10 Nordic IVF clinics, 776 patients with a minimum of two good quality blastocysts on Day 5 (D5) were randomized into one of the two study groups. A commercial time-lapse model decided the final selection of blastocysts for 387 patients in the intervention (time-lapse) group, and blastocysts with the highest morphological score were transferred for 389 patients in the control group. Only single embryo transfers in fresh cycles were performed. MAIN RESULTS AND THE ROLE OF CHANCE: In the full analysis set, the ongoing pregnancy rate for the time-lapse group was 47.4% (175/369) and 48.1% (181/376) in the control group. No statistically significant difference was found between the two groups: mean difference -0.7% (95% CI -8.2, 6.7, P = 0.90). Pregnancy rate (60.2% versus 59.0%, mean difference 1.1%, 95% CI -6.2, 8.4, P = 0.81) and early pregnancy loss (21.2% versus 18.5%, mean difference 2.7%, 95% CI -5.2, 10.6, P = 0.55) were the same for the time-lapse and the control group. Subgroup analyses showed that patient and treatment characteristics did not significantly affect the commercial time-lapse model D5 performance. In the time-lapse group, the choice of best blastocyst changed on 42% of occasions (154/369, 95% CI 36.9, 47.2) after the algorithm was applied, and this rate was similar for most treatment clinics. LIMITATIONS, REASONS FOR CAUTION: During 2020, the patient recruitment rate slowed down at participating clinics owing to coronavirus disease-19 restrictions, so the target sample size was not achieved as planned and it was decided to stop the trial prematurely. The study only investigated embryo selection at the blastocyst stage on D5 in fresh IVF transfer cycles. In addition, only blastocysts of good morphological quality were considered for transfer, limiting the number of embryos for selection in both groups: also, it could be argued that this manual preselection of blastocysts limits the theoretical selection power of time-lapse, as well as restricting the results mainly to a good prognosis patient group. Most patients were aimed for blastocyst stage transfer when a minimum of five zygotes were available for extended culture. Finally, the primary clinical outcome evaluated was pregnancy to only 6-8 weeks. WIDER IMPLICATIONS OF THE FINDINGS: The study suggests that time-lapse selection with a commercially available time-lapse model does not increase chance of ongoing pregnancy after single blastocyst transfer on Day 5 compared to morphology alone. STUDY FUNDING/COMPETING INTEREST(S): The study was financed by a grant from the Swedish state under the ALF-agreement between the Swedish government and the county councils (ALFGBG-723141). Vitrolife supported the study with embryo culture dishes and culture media. During the study period, T.H. changed his employment from Livio AB to Vitrolife AB. All other authors have no conflicts of interests to disclose. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov registration number NCT03445923. TRIAL REGISTRATION DATE: 26 February 2018. DATE OF FIRST PATIENT'S ENROLMENT: 11 June 2018.
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COVID-19 , Algoritmos , Blastocisto , Femenino , Humanos , Embarazo , Índice de Embarazo , Estudios Prospectivos , Imagen de Lapso de TiempoRESUMEN
PURPOSE: miRNAs have been suggested as biomarkers of embryo viability; however, findings from preliminary studies are divergent. Furthermore, the presence of other types of small RNA molecules remains to be investigated. The purpose of this study was to perform a comprehensive analysis of small non-coding RNA levels in spent and unconditioned embryo culture media, along with miRNA levels in blastocoelic fluid samples from human embryos. METHODS: miRNAs in unconditioned culture medium from 3 different manufacturers, along with miRNA from day 5 conditioned culture medium, control medium, and corresponding blastocoel fluid from 10 human blastocysts were analyzed with array-based q-PCR analysis. Subsequently, deep sequencing of total and small RNA in day 5 spent culture medium from 5 human blastocysts and corresponding controls was performed. RESULTS: In spite of using state-of-the-art sensitive detection methods, no miRNAs were found to be reliably present in the spent culture medium or the blastocoel fluid. Ct values were above the recommended limit for detection in the array-based analysis, a finding that was confirmed by deep sequencing. The majority of miRNAs identified by deep sequencing were expressed in all samples including control media and seem to originate from sources other than conditioned IVF media. CONCLUSIONS: Our findings question the use of miRNAs as a reliable biomarker and highlight the need for a critical methodological approach in miRNA studies. Interestingly, tiRNA fragments appear to be overexpressed in conditioned IVF media samples and could potentially be a novel biomarker worthy of investigation.
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Blastocisto/metabolismo , Medios de Cultivo Condicionados/metabolismo , MicroARNs/aislamiento & purificación , ARN Pequeño no Traducido/aislamiento & purificación , Biomarcadores/metabolismo , Técnicas de Cultivo de Embriones , Implantación del Embrión/genética , Transferencia de Embrión/métodos , Femenino , Fertilización In Vitro/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , MicroARNs/genética , ARN Pequeño no Traducido/genéticaRESUMEN
Globally, IVF patients are routinely offered and charged for a selection of adjunct treatments and tests or 'add-ons' that they are told may improve their chance of a live birth, despite there being no clinical evidence supporting the efficacy of the add-on. Any new IVF technology claiming to improve live birth rates (LBR) should, in most cases, first be tested in an appropriate animal model, then in clinical trials, to ensure safety, and finally in a randomized controlled trial (RCT) to provide high-quality evidence that the procedure is safe and effective. Only then should the technique be considered as 'routine' and only when applied to the similar patient population as those studied in the RCT. Even then, further pediatric and long-term follow-up studies will need to be undertaken to examine the long-term safety of the procedure. Alarmingly, there are currently numerous examples where adjunct treatments are used in the absence of evidence-based medicine and often at an additional fee. In some cases, when RCTs have shown the technique to be ineffective, it is eventually withdrawn from the clinic. In this paper, we discuss some of the adjunct treatments currently being offered globally in IVF laboratories, including embryo glue and adherence compounds, sperm DNA fragmentation, time-lapse imaging, preimplantation genetic screening, mitochondria DNA load measurement and assisted hatching. We examine the evidence for their safety and efficacy in increasing LBRs. We conclude that robust studies are needed to confirm the safety and efficacy of any adjunct treatment or test before they are offered routinely to IVF patients.
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Medicina Basada en la Evidencia , Fertilización In Vitro/normas , Técnicas Reproductivas Asistidas/tendencias , Fragmentación del ADN , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/tendencias , Humanos , Nacimiento Vivo , Masculino , Embarazo , Índice de Embarazo , Técnicas Reproductivas Asistidas/normas , Espermatozoides , Imagen de Lapso de TiempoRESUMEN
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 .
RESUMEN
Near-infrared (NIR) spectroscopic metabolomic profiling of spent embryo-culture media has been used to calculate a viability score for individual embryos. These scores have been found to correlate to the reproductive potential of cleavage-stage embryos. In this study, 137 spent blastocyst media samples were collected after single-embryo transfer and analysed by NIR spectroscopy to generate an algorithm and calculate viability scores. To blindly validate the algorithm development process, another algorithm was trained on 47 preselected samples from clinic 1 and then used to predict the outcome of 42 samples from clinic 2. The overall pregnancy rate from the two clinical sites was 50.4%. A positive correlation (R(2)=0.82, P=0.03) was observed with the increasing viability score quintiles and their associated implantation rates. Cross-validation of an algorithm generated from NIR analysis of media samples at one clinical setting blindly was shown to predict implantation potential of blastocysts cultured at another clinic in a different culture media and culture volume. This study demonstrates that metabolomic profiling by NIR spectroscopic analysis of day-5 spent embryo-culture media can predict the implantation potential of blastocysts. Furthermore, this method may not be restricted to a specific set of culturing conditions. The successes of IVF treatment cycles are in part limited by the ability to select the best single embryo from a cohort of patient embryos for transfer back to the woman. Routine procedures of embryo selection are based on morphology, including cell number and size, and the timing of cell division. These methods are favoured because they are quick and easy to assess. Human embryos are grown in culture solutions, which are specific for their stage of development. Recent studies analysing the culture solution in which the embryo are grown, by near infrared (NIR) spectroscopic analysis, have been able to predict if an embryo will implant or not. As culture conditions often vary between IVF laboratories the questions remained if the NIR technique could be used to independently predict the implantation potential of an embryo cultured at one laboratory using an algorithm trained on embryos at a second clinic, a so-called cross-validation. The results of this study show that NIR spectroscopy can predict the ability of embryos to implant even when grown in different IVF laboratories and in two different culture solutions. This information supports the idea that NIR spectroscopy can be used globally not relying on specific culture conditions or media.
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Transferencia de Embrión/métodos , Espectroscopía Infrarroja Corta/métodos , Adulto , Algoritmos , Técnicas de Cultivo de Embriones , Femenino , Humanos , Metabolómica , Valor Predictivo de las Pruebas , Embarazo , Índice de EmbarazoRESUMEN
PURPOSE: Assessment of embryo viability is a key component of in vitro fertilization (IVF) and currently relies largely on embryo morphology and cleavage rate. In this study, we used receiver operating characteristic (ROC) analysis to compare the Viability Score (generated by metabolomic profiling of spent embryo culture media using near infrared (NIR) spectroscopy) to morphologic grading for predicting pregnancy in women undergoing single embryo transfer (SET) on day 5. METHODS: A total of 198 spent embryo culture media samples were collected in four IVF centers located in the USA, Europe and Australia. First, 137 samples (training set) were analyzed by NIR to develop an algorithm that generates a Viability Score predictive of pregnancy for each sample. Next, 61 samples (validation set) were analyzed by observers blinded to embryo morphology and IVF outcome, using the Day 5 algorithm generated with the training set. Pregnancy was defined as fetal cardiac activity (FCA) at 12 weeks of gestation. RESULTS: The Area Under the Curve (AUC) was greater for the metabolomic Viability Score compared to Morphology [Training set: 0.75 versus 0.55, p = 0.0011; Validation set: 0.68 versus 0.50, P = 0.021], and for a Composite score (obtained using a model combining Viability Score with morphologic grading), compared to morphology alone [0.74 versus 0.50, p = 0.004]. CONCLUSIONS: Our findings suggest that Viability Score alone or in combination with morphologic grading has the potential to be a better classifier for pregnancy outcome than morphology alone in women undergoing SET on day 5.
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Implantación del Embrión/fisiología , Embrión de Mamíferos/anatomía & histología , Fertilización In Vitro , Metabolómica/estadística & datos numéricos , Curva ROC , Desarrollo Embrionario , Femenino , Humanos , Embarazo , Pronóstico , Transferencia de un Solo EmbriónRESUMEN
AIM: To investigate whether human embryonic stem cells (hESCs) could be made to attach, grow and differentiate on a human Descemet's membrane (DM). METHODS: Spontaneously differentiated hESCs were transferred onto a human corneal button with the endothelial layer removed using ocular sticks. The cells were cultured on a DM for up to 15 d. The genetically engineered hESC line expressed green fluorescent protein, which facilitated identification during the culture experiments, tissue preparation, and analysis. To detect any differentiation into human corneal endothelial-like cells, we analysed the transplanted cells by immunohistochemistry using specific antibodies. RESULTS: We found transplanted cells form a single layer of cells with a hexagonal shape in the periphery of the DM. The majority of the cells were negative for octamer-binding transcription factor 4 but positive for paired box 6 protein, sodium potassium adenosine triphosphatase (NaKATPase), and Zona Occludens protein 1. In four of the 18 trials, the transplanted cells were found to express CK3, which indicates that the stem cells differentiated into corneal epithelial cells in these cases. CONCLUSION: It is possible to get cells originating from hESCs to become established on a human DM, where they grow and differentiate into corneal endothelial-like cells in vitro.
RESUMEN
Human embryonic stem cells (hESCs) can be coaxed to differentiate into specific cell types, including cardiomyocyte-like cells. These cells express cardiac-specific markers and display functional similarities to their adult counterparts. Based on these properties, hESC-derived cardiomyocytes have the potential to be extremely useful in various in vitro applications and to provide the opportunity for cardiac cell replacement therapies. However, before this can become a reality, the molecular and functional characteristics of these cells need to be investigated in more detail. In the present study we differentiate hESCs into cardiomyocyte-like cells via embryoid bodies (EBs). The fraction of spontaneously beating clusters obtained from the EBs averaged approximately 30% of the total number of EBs used. These cell clusters were isolated, dissociated into single-cell suspensions, and frozen for long-term storage. The cryopreserved cells could be successfully thawed and subcultured. Using electron microscopy, we observed Z discs and tight junctions in the hESC-derived cardiomyocytes, and by immunohistochemical analysis we detected expression of cardiac-specific markers (cTnI and cMHC). Notably, using BrdU labeling we also could demonstrate that some of the hESC-derived cardiomyocytes retain a proliferative capacity. Furthermore, pharmacological stimulation of the cells resulted in responses indicative of functional adrenergic and muscarinic receptor coupling systems. Taken together, these results lend support to the notion that hESCs can be used as a source for the procurement of cardiomyocytes for in vitro and in vivo applications.
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Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Criopreservación , Medios de Cultivo , Células Madre Embrionarias/ultraestructura , Epinefrina/farmacología , Humanos , Inmunohistoquímica , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Norepinefrina/farmacología , Fenilefrina/farmacologíaRESUMEN
Within the past few years the morphological evaluation of in vitro fertilized embryos has been extended to include continuous surveillance, enabled by the introduction of time-lapse incubators developed specifically for IVF treatment. As a result time-lapse monitoring has been implemented in many clinics worldwide. The proposed benefits compared with culture in a standard incubator and fixed time-point evaluation are uninterrupted culture, a flexible workflow in the laboratory, and improved embryo selection. The latter is based on the reasonable assumption that more frequent observations will provide substantially more information on the relationship between development, timing, and embryo viability. Several retrospective studies have confirmed a relationship between time-lapse parameters and embryo viability evaluated by developmental competence, aneuploidy, and clinical pregnancy. Furthermore a much anticipated randomized study has shown improved pregnancy rates (PRs) after culture in a time-lapse incubator combined with selection using a hierarchical time-lapse selection model. At present this is the only randomized study on possible benefits of time lapse in human embryology. Strict evidence may still seem too weak to introduce time lapse in routine clinical setting. This aim of this review is therefore to perform a balanced discussion of the evidence for time-lapse monitoring.
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Transferencia de Embrión/métodos , Imagen de Lapso de Tiempo/métodos , Animales , Blastocisto/fisiología , Técnicas de Cultivo de Embriones/métodos , Técnicas de Cultivo de Embriones/normas , Implantación del Embrión/fisiología , Transferencia de Embrión/normas , Femenino , Humanos , Embarazo , Imagen de Lapso de Tiempo/normasRESUMEN
OBJECTIVE: To study whether a culture medium that allows undisturbed culture supports human embryo development to the blastocyst stage equivalently to a well-established sequential media. DESIGN: Randomized, double-blinded sibling trial. SETTING: Independent in vitro fertilization (IVF) clinics. PATIENT(S): One hundred twenty-eight patients, with 1,356 zygotes randomized into two study arms. INTERVENTION(S): Embryos randomly allocated into two study arms to compare embryo development on a time-lapse system using a single-step medium or sequential media. MAIN OUTCOME MEASURE(S): Percentage of good-quality blastocysts on day 5. RESULT(S): Percentage of day 5 good-quality blastocysts was 21.1% (standard deviation [SD] ± 21.6%) and 22.2% (SD ± 22.1%) in the single-step time-lapse medium (G-TL) and the sequential media (G-1/G-2) groups, respectively. The mean difference (-1.2; 95% CI, -6.0; 3.6) between the two media systems for the primary end point was less than the noninferiority margin of -8%. There was a statistically significantly lower number of good-quality embryos on day 3 in the G-TL group [50.7% (SD ± 30.6%) vs. 60.8% (SD ± 30.7%)]. Four out of the 11 measured morphokinetic parameters were statistically significantly different for the two media used. The mean levels of ammonium concentration in the media at the end of the culture period was statistically significantly lower in the G-TL group as compared with the G-2 group. CONCLUSION(S): We have shown that a single-step culture medium supports blastocyst development equivalently to established sequential media. The ammonium concentrations were lower in the single-step media, and the measured morphokinetic parameters were modified somewhat. CLINICAL TRIAL REGISTRATION NUMBER: NCT01939626.
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Blastocisto/fisiología , Medios de Cultivo/química , Técnicas de Cultivo de Embriones , Fertilización In Vitro , Infertilidad/terapia , Imagen de Lapso de Tiempo , Compuestos de Amonio/metabolismo , Blastocisto/metabolismo , Medios de Cultivo/metabolismo , Método Doble Ciego , Implantación del Embrión , Transferencia de Embrión , Desarrollo Embrionario , Femenino , Fertilidad , Humanos , Infertilidad/diagnóstico , Infertilidad/fisiopatología , Nacimiento Vivo , Masculino , Morfogénesis , Embarazo , Índice de Embarazo , Estudios Prospectivos , Suecia , Factores de Tiempo , Estados UnidosRESUMEN
PURPOSE: The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial-like cells. METHODS: Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman's membrane for up to 9 days, and the culture dynamics documented in a time-lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial-like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. RESULTS: The transplanted cells established and expanded on Bowman's membrane, forming a 1-4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3. CONCLUSION: This shows that it is possible to transplant cells originating from hESCs onto Bowman's membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial-like cells in vitro.
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Lesiones de la Cornea , Células Madre Embrionarias/trasplante , Lesiones Oculares/terapia , Trasplante de Células Madre , Heridas no Penetrantes/terapia , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Madre Embrionarias/citología , Epitelio Corneal/citología , Epitelio Corneal/metabolismo , Lesiones Oculares/metabolismo , Proteínas del Ojo/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Queratina-15/metabolismo , Queratina-3/metabolismo , Limbo de la Córnea/citología , Limbo de la Córnea/metabolismo , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Adhesión del Tejido , Fijación del Tejido , Heridas no Penetrantes/metabolismoRESUMEN
With the introduction of numerous new technologies, human reproduction has undergone considerable development during the last 25 years. The possibilities for treating both female and male infertility are today less of a medico-technical and more of a socio-economic problem. The high incidence of multiple pregnancy is the problem that causes most concern, with impact upon the infertile couple, offspring and society. Attempts to solve this problem have been made but more work is needed.
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Transferencia de Embrión , Infertilidad/terapia , Complicaciones del Embarazo , Embarazo Múltiple , Servicios de Salud Reproductiva/normas , Adulto , Envejecimiento , Transferencia de Embrión/efectos adversos , Transferencia de Embrión/normas , Femenino , Pruebas Genéticas , Humanos , Masculino , Síndrome de Hiperestimulación Ovárica/inducido químicamente , Embarazo , Complicaciones del Embarazo/etiología , Complicaciones del Embarazo/prevención & control , Reducción de Embarazo Multifetal , Servicios de Salud Reproductiva/economía , Servicios de Salud Reproductiva/tendenciasRESUMEN
This paper reports a case where a normal-sized human oocyte has been documented having two polar bodies and two metaphase spindles. This finding suggests that the chromosomal content in oocytes of normal size may occasionally be duplicated and offers an additional explanation for the origin of triploid zygotes in humans. We speculate that this duplication may be caused by oocyte fusion at an early stage.
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Oocitos/citología , Oocitos/ultraestructura , Huso Acromático/ultraestructura , Adulto , Tamaño de la Célula , Femenino , Humanos , Metafase , Microscopía Fluorescente , Valores de ReferenciaRESUMEN
PURPOSE: To document the DNA content of blastomeres/fragments from early human preembryos and to determine if there is a "cutoff" diameter at which a cell should be considered an anucleate fragment rather than a blastomere. METHODS: Surplus embryos from in vitro fertilization were used. Individual cells were measured, fixated, and stained for DNA. RESULTS: In day 2 preembryos, only 2% of cells with a diameter <45 microm contained DNA, compared with 67% of those > or =45 microm. In day 3 preembryos, 3% of cells <40 microm contained DNA, compared with 66% of those > or =40 microm. CONCLUSIONS: It is suggested that cells <45 microm in day 2 preembryos, and <40 microm in day 3 preembryos should be classified as fragments, and cells larger than this, as blastomeres. This may influence the embryo scoring system for in vitro fertilization. We therefore recommend that cells within this critical range should be measured when scoring preembryos for embryo transfer.
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Blastocisto/citología , Blastómeros/citología , ADN/metabolismo , Blastocisto/metabolismo , Blastómeros/metabolismo , Núcleo Celular/metabolismo , Femenino , HumanosRESUMEN
PURPOSE: The aim of this study was to design an experimental set-up for the study of human corneal epithelial wound healing in a controlled in vitro situation. METHODS: A time-lapse set-up was used. This allowed for pictures to be captured with a magnification ranging from x 80 to x 1800. Pictures were captured at 1-min intervals during the observation period, which lasted up to 4 days. Human corneal tissue was obtained from the Eye Bank or from surgery. A small, rounded lesion was produced in the corneal epithelium with a miniature drill. The specimens were placed in a mini-incubator; the camera focused on the epithelial lesion and continuously observed using the time-lapse set-up. RESULTS: The healing process of human corneal epithelium could be followed for several days. The initial healing response could be divided into a slow, a rapid and a consolidating phase. The first two phases lasted about 12 hours, and by then, epithelial cells covered the lesion. Depending on the origin of the tissue and the placement of the lesion, variations in the healing response could be seen. CONCLUSION: The time-lapse technique makes it possible to study epithelial wound healing over time at the cellular level. Data collected in this way can fill the gap between in vivo studies, where, by nature, human wound healing studies are restricted, and cell culture techniques, where cellular responses in many cases differ from the in vivo situation.
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Epitelio Corneal/citología , Cicatrización de Heridas/fisiología , Humanos , Técnicas de Cultivo de Órganos , Fotograbar/métodos , Factores de TiempoRESUMEN
BACKGROUND: IVF laboratories performing embryo transfer at day 2 or 3 after fertilization are currently discarding pre-embryos considered suboptimal using morphological criteria. The objective of this study was to investigate whether blastocysts, cultured from such pre-embryos (surplus), were chromosomally and morphologically normal. As a control group we used morphologically good quality embryos (GQE), cultured to the blastocyst stage. METHODS: Human pre-embryos considered suboptimal were cultured to the blastocyst stage. As a control group, frozen-thawed pre-embryos of good quality were cultured under identical conditions. The chromosomal status of the blastocysts obtained was studied by multi-colour fluorescence in-situ hybridization for chromosomes 13, 16, 18, 21, 22, X and Y. RESULTS: There is, on average, a significantly higher degree of chromosomal aberrations in blastocysts derived from surplus pre-embryos compared to blastocysts derived from GQE, and the chromosomal aberrations are generally found in a higher number of blastomeres per blastocyst. In addition, blastocysts from surplus pre-embryos had significantly poorer morphology compared to GQE. Improvement in morphology and/or developmental rate in surplus pre-embryos between day 2 and day 3 did not predict a morphologically/chromosomally normal blastocyst. However, this study shows that close to half of the surplus pre-embryos that reach the blastocyst stage can be considered chromosomally normal when assessed for these seven chromosomes. Furthermore, we found that chromosomal aberrations were more concentrated in a particular cell population within blastocysts derived from GQE, compared with surplus blastocysts. CONCLUSIONS: The study suggests that even if the IVF laboratory is on average making the correct decision about the potential of a pre-embryo, surplus pre-embryos that might become chromosomally normal blastocysts are still being discarded.