RESUMO
Vitrification of immature germinal vesicle-stage oocytes is a promising method in assisted reproduction but is associated with reduced developmental potential and low birth rates. Cumulus-oocyte complexes (COCs) express several connexins that form hexameric hemichannels, which interact head to head to create a gap junction or exist as unopposed free hemichannels. The latter are normally closed but open under stress conditions and may exert detrimental effects. We determined whether minimizing hemichannel opening and cell death during vitrification could improve COC quality. Bovine immature COCs underwent vitrification, storage and warming, followed by dye uptake to assess hemichannel opening and TUNEL staining to detect cell death. Based on these scores, we optimized the procedure by tuning the equilibration time, temperature, cryoprotectant concentration and extracellular Ca2+ concentration and assessed its impact on maturation, cleavage and blastocyst formation after parthenogenetic activation. We found that the major stressor resides in the cooling/warming phase of the vitrification procedure and observed that hemichannel opening and cell death in cumulus cells measure different aspects of cell stress. Optimization of the hemichannel and cell death readouts demonstrated that combined minimal hemichannel opening/cell death gave the highest cleavage rates but had no effect on maturation and blastocyst formation. Neither hemichannel nor cell death optimization performed better than the non-optimized protocol, leading to the conclusion that cell stress factors other than those detected by hemichannel dye uptake or TUNEL positivity are involved.
Assuntos
Morte Celular/fisiologia , Conexinas/metabolismo , Células do Cúmulo , Oócitos , Vitrificação , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Bovinos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Conexinas/análise , Criopreservação/métodos , Criopreservação/veterinária , Crioprotetores/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/fisiologia , Feminino , Preservação da Fertilidade/efeitos adversos , Preservação da Fertilidade/métodos , Preservação da Fertilidade/veterinária , Junções Comunicantes/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
Patients presenting with abnormally high numbers of immature oocytes at retrieval are more likely to exhibit maturation resistant oocytes. However, the clinical relevance of such events remains unknown. We investigated nuclear maturation competence of immature oocytes from patients showing >40% of collected immature oocytes (Study group) and Controls, in which a normal number of mature oocytes (≥60%) was retrieved. Following in-vitro culture, oocytes were classified as maturation resistant or in-vitro matured (IVM). Treatment outcomes were evaluated in Study and Control groups based on presence of maturation resistant oocytes. Overall, similarly high spindle and chromosome abnormality rates were observed in maturation resistant oocytes from both Study and Control groups. IVM oocytes from the Study group revealed significantly higher percentages of misaligned chromosomes compared with Controls (P < 0.05). Remarkably, Study group patients with at least one maturation resistant oocyte showed significantly reduced cumulative pregnancy and live birth rates compared with Control group maturation resistant patients (P < 0.05). When further investigating the aetiology, a maturation resistant mouse model revealed defective Ca2+ signalling of maturation resistant oocytes at germinal vesicular breakdown and parthenogenetic activation. In conclusion, appropriate treatment strategies, including clinical utilization of IVM oocytes from Study group patients, warrant further investigation.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Meiose/fisiologia , Oócitos/citologia , Indução da Ovulação , Adulto , Animais , Cálcio/metabolismo , Feminino , Humanos , Camundongos , Recuperação de Oócitos , Oócitos/metabolismo , Gravidez , Resultado da Gravidez , Falha de TratamentoRESUMO
Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the presence of Gap26 that targets GJA1 (Cx43) and GJA4 (Cx37). Further work showed that HCs from blastocysts produced after oocyte maturation in the presence of serum were open shortly after vitrification/warming, and this was prevented by Gap26. Gap26, applied for the exposure times used, inhibited Cx43 and Cx37 HCs while it did not have an effect on GJs. Interestingly, Gap26 had no effect on blastocyst degeneration or cell death. We conclude that blocking HCs protects embryos during vitrification and warming by a functional effect not linked to cell death.
Assuntos
Blastocisto/fisiologia , Bovinos/embriologia , Conexinas/antagonistas & inibidores , Técnicas de Cultura Embrionária/veterinária , Vitrificação , Animais , Bovinos/fisiologia , Criopreservação , Desenvolvimento Embrionário , Células HeLa , HumanosRESUMO
STUDY QUESTION: Is it important that end-users know the composition of human embryo culture media? SUMMARY ANSWER: We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. WHAT IS KNOWN ALREADY: Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. STUDY DESIGN, SIZE, DURATION: A review of the literature was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. MAIN RESULTS AND THE ROLE OF CHANCE: The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. LIMITATIONS, REASONS FOR CAUTION: There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. WIDER IMPLICATIONS OF THE FINDINGS: Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the composition of embryo culture media. STUDY FUNDING/COMPETING INTERESTS: This work was funded by The European Society for Human Reproduction and Embryology. No competing interests to declare.
Assuntos
Meios de Cultura , Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/fisiologia , Técnicas de Reprodução Assistida , Fertilização in vitro/métodos , HumanosRESUMO
STUDY QUESTION: Does prospective embryo selection using the results from the Eava Test (Early Embryo Viability Assessment) in combination with standard morphology increase the pregnancy rate of IVF and ICSI patients compared to embryo selection based on morphology only? SUMMARY ANSWER: Embryo selection using the Eeva Test plus standard morphology on Day 3 results in comparable pregnancy rates as conventional morphological embryo selection. WHAT IS KNOWN ALREADY: Time-lapse monitoring of embryo development may represent a superior way to culture and select embryos in vitro. The Eeva Test records the development of each embryo with a cell-tracking system and predicts the likelihood (High, Medium or Low) that an embryo will form a blastocyst based on an automated analysis of early cell division timings. STUDY DESIGN, SIZE, DURATION: This trial was designed as a prospective, observational, two-center pilot study with a propensity matched control group. The analysis involved 280 of 302 enrolled patients who were included in the Eeva Test group in 2013 and 560 control patients who were treated in the years 2011-2013. The majority of transfers (98%) were single embryo transfers. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic hospitals (VUmc Amsterdam and UZ Gent) enrolled patients <41 years old, with <3 previous attempts and ≥5 normally fertilized eggs. Propensity matching was used to identify a propensity matched control group from a cohort of 1777 patients based on age, cycle number, oocyte number and number of fertilized oocytes. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference in patient baseline characteristics between the two groups. The ongoing pregnancy rate (OPR) of patients enrolled in the Eeva Test group (34.3%; 96/280) did not differ significantly from the OPR in the propensity matched control group (34.6%, 194/560; P = 0.92). However, significantly less top quality embryos (eight-cell embryos with ≤25% fragmentation) were transferred in the Eeva Test group compared to the propensity matched control group (70.4% vs. 82.3%; P < 0.001). The transfer of Eeva High and Medium embryos resulted in a significantly higher OPR of 36.8% (89/242) compared to 18.4% (7/38) for Eeva Low embryos (P = 0.02). LIMITATION, REASONS FOR CAUTION: This pilot study is limited by its nonrandomized design with a concurrent and historical control. WIDER IMPLICATIONS OF THE FINDINGS: Our pilot data did not reveal significant differences between time-lapse based and conventional embryo selection. Interestingly, the pregnancy rates were comparable in both groups even though the morphological quality of the transferred embryos was significantly lower in the Eeva Test group compared to the propensity matched control group. A sufficiently powered three-armed randomized controlled trial (RCT) with a solid design should be performed to generate decisive evidence in the future. STUDY FINDING/COMPETING INTERESTS: Progyny Inc., formerly Auxogyn provided the Eeva scopes, software and technical support for this study. The funding sources did neither influence data collection, management, analysis and interpretation of the data, nor the preparation of the manuscript. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov: NCT01671644.
Assuntos
Técnicas de Cultura Embrionária/métodos , Desenvolvimento Embrionário/fisiologia , Fertilização in vitro/métodos , Adulto , Transferência Embrionária/métodos , Feminino , Humanos , Projetos Piloto , Gravidez , Estudos Prospectivos , Imagem com Lapso de TempoRESUMO
Recently, new culture devices such as Corral and Primo Vision dishes have been designed for the culture of human embryos to allow the combination of group culture plus follow-up of individual embryos. Bovine inseminated oocytes were allocated to Primo Vision dishes, Corral dishes, individual culture or classical group culture. Blastocyst development in Primo Vision dishes was similar to classical group culture (34.3 and 39.0% respectively), and better than Corral dishes or individual culture (28.9 and 28.5% respectively). In Primo Vision dishes, a higher number of 'slow' embryos developed to the blastocyst stage compared with their individually cultured counterparts, while no differences were observed for 'fast' embryos. 'Slow' embryos in a 'standard drop' had a higher chance of becoming a blastocyst compared with individual culture (OR: 2.3), whereas blastulation of 'fast' embryos was less efficient in a 'delayed drop' than in individual culture (OR: 0.3). The number of non-cleaved embryos in Primo Vision dishes did not negatively influence blastocyst development. Likewise, removing non-cleaved embryos (NC removed) and regrouping the cleaved embryos afterwards (ReGR) did not affect blastocyst development and quality compared with group culture in Primo Vision dishes (CTRL, 31.6%, NC removed, 29.3% and ReGR, 29.6%). The experiments revealed that group culture of bovine embryos in Primo Vision dishes is superior to individual culture, primarily because of the higher blastocyst rate achieved by slow embryos. Non-cleaved or arrested embryos do not hamper the ability of co-cultured bovine embryos to reach the blastocyst stage in group culture.
Assuntos
Blastocisto/fisiologia , Técnicas de Cocultura/veterinária , Técnicas de Cultura Embrionária/veterinária , Animais , Bovinos , Técnicas de Cocultura/instrumentação , Técnicas de Cultura Embrionária/instrumentação , Desenvolvimento Embrionário , Desenho de Equipamento , Feminino , Fertilização in vitro/veterinária , Masculino , Razão de Chances , Fatores de TempoRESUMO
The aim of this study was to assess the ability of three individual blastocyst morphology parameters - expansion and hatching (EH) stage, inner cell mass (ICM) grade and trophectoderm grade - to predict outcome of a cycle with single-blastocyst transfer. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 intracytoplasmic sperm injection patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist cycle with compulsory single-blastocyst transfer on day 5 were included. In the simple logistic regression analysis, all three blastocyst morphology parameters were statistically significantly (P<0.005 for each) associated with positive human chorionic gonadotrophin, clinical and ongoing pregnancy rates and live birth rates, while only the ICM grade was significantly (P=0.033) associated with early pregnancy loss rate. Blastocyst EH stage was the only significant predictor of live birth (P=0.002) in the multiple logistic regression. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the EH stage. Transfer of a blastocyst with ICM grade A may reduce the risk of early pregnancy loss. Choosing the embryo(s) with the best implantation potential is essential for securing each couple the highest chance of achieving pregnancy after assisted reproduction. The selection of embryo(s) for transfer at the blastocyst stage is based on morphology parameters of expansion and hatching stage, inner cell mass grade and trophectoderm grade. The aim of this study was to assess the relative impact of each parameter in predicting the probability of a successful outcome. The study was a secondary analysis of data prospectively collected in a large multicentre trial. A total of 618 patients who underwent single-blastocyst transfer on day 5 were included. Statistical analysis showed that all three blastocyst morphology parameters were significantly associated with positive human chorionic gonadotrophin (ßHCG), clinical and ongoing pregnancy rates and live birth rates. Only the inner cell mass grade was significantly associated with early pregnancy loss between the positive ßHCG test and confirmation of ongoing pregnancy 10-11weeks after transfer. The expansion and hatching stage was the only significant predictor of live birth in the multiple logistic regression analysis. In conclusion, although all three blastocyst morphology parameters were related to treatment outcome of fresh single-blastocyst cycles, selection of high-quality blastocysts for transfer should consider first the expansion and hatching stage. Transfer of a blastocyst with inner cell mass grade A may reduce the risk of early pregnancy loss.
Assuntos
Blastocisto/citologia , Transferência de Embrião Único , Adulto , Massa Celular Interna do Blastocisto/ultraestrutura , Feminino , Humanos , Modelos Logísticos , Estudos Multicêntricos como Assunto , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Digital microfluidics (DMF) holds great potential for the alleviation of laboratory procedures in assisted reproductive technologies (ARTs). The electrowetting on dielectric (EWOD) technology provides dynamic culture conditions in vitro that may better mimic the natural embryo microenvironment. Thus far, EWOD microdevices have been proposed for in vitro gamete and embryo handling in mice and for analyzing the human embryo secretome. This article presents the development of the first microfluidic chip utilizing EWOD technology designed for the manipulation of bovine embryos in vitro. The prototype sustains the cell cycles of embryos manipulated individually on the chips during in vitro culture (IVC). Challenges related to the chip fabrication as well as to its application during bovine embryo IVC in accordance with the adapted on-chip protocol are thoroughly discussed, and future directions for DMF in ARTs are indicated.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Animais , Bovinos , Humanos , Camundongos , Microfluídica/métodos , Eletroumectação/métodos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Controversy exists about the risk of microbiological contamination from direct contact with unsterile liquid nitrogen during oocyte vitrification. The aim of this observational study was to evaluate the effectiveness of oocyte vitrification using a high-security closed vitrification system in a donation programme. Oocyte vitrification was performed using CBS High Security closed straws (Cryo Bio System) with DMSO/ethylene glycol/sucrose as the cryoprotectant (Irvine Scientific freeze kit). A total of 123 vitrified metaphase-II oocytes were warmed in 20 recipient cycles (6.2 warmed oocytes per recipient); of these, 111 oocytes (90.2%) survived vitrification and warming. All surviving oocytes were microinjected and 86 (77.5%) were normally fertilized, of which 53 (61.6%) developed up to good-quality day 3. Ten embryo transfers resulted in a clinical pregnancy (50.0%) and an ongoing clinical pregnancy rate of 45%. Five revitrified embryos were warmed in three warming cycles (survival rate 100%). These transfers resulted in an additional ongoing twin pregnancy, leading to a cumulative ongoing pregnancy rate per patient of 50% (10/20). The ongoing implantation rate per warmed oocyte and per injected oocyte was 10.6% (13/123) and 11.7% (13/111). The present data demonstrate that oocyte vitrification using a closed vitrification device yields excellent oocyte survival, fertilization and embryo development.
Assuntos
Doação de Oócitos/métodos , Oócitos , Vitrificação , Criopreservação/métodos , Transferência Embrionária , Feminino , Fertilização in vitro/métodos , Humanos , Gravidez , Taxa de GravidezRESUMO
BACKGROUND: Single blastocyst transfer has the advantage of maximizing the fresh single pregnancy rate. However, in patients with a low number of good quality embryos on day 3, it remains unclear whether immediate embryo transfer or further embryo culture with blastocyst transfer is the most preferable option. METHODS: A retrospective cohort study was carried out in which the outcome of 590 fresh in vitro fertilization (IVF) cycles over a 15 months period and their cryo cycles were analyzed. A total of 341 patients cycles had an elective day 5 strategy independent of intermediate embryo evaluation while another 249 patients underwent a day 5 embryo transfer only if at least four embryos were available on day 3. Blastocyst vitrification was performed using a closed high security system. RESULTS: Demographics, stimulation parameters and embryological data were comparable in the two groups. Patients in the elective day 5 group had a lower fresh transfer rate (90.62% vs. 95.18%, p < 0.05) as compared to patients with a day 3 or day 5 embryo transfer policy. No difference was observed in the fresh live birth rate and multiple pregnancy rate per initiated cycle (32.84% vs. 28.92%; 1.17% vs 0%) The projected cumulative ongoing pregnancy rate compensating for double counting in case subjects have more than one pregnancy is not different (42.58% vs. 39.84%). CONCLUSIONS: Despite lower fresh transfer rates, elective single blastocyst transfer yields a similar projected cumulative ongoing pregnancy rate as in a policy with cleavage stage or blastocyst transfer depending on a good quality embryo count on day 3.
Assuntos
Tomada de Decisões/fisiologia , Transferência Embrionária/métodos , Embrião de Mamíferos/citologia , Fertilização in vitro/legislação & jurisprudência , Adulto , Fase de Clivagem do Zigoto/citologia , Estudos de Coortes , Feminino , Humanos , Masculino , Políticas , Gravidez , Taxa de Gravidez , Controle de Qualidade , Estudos Retrospectivos , Fatores de TempoRESUMO
The 'ESHRE Guidelines for Good Practice in IVF Laboratories' were drawn up by the Special Interest Group (SIG) in Embryology and published in the year 2000, and since then they constitute the minimal requirements for any laboratory offering assisted reproduction techniques (ART). In the understanding that the embryologist has a responsibility for the correct and justified application of ART in the laboratory, the implementation of these guidelines requires a quality management programme to be in place that encompasses and integrates the operative units, the processes and procedures that represent the core of ART clinics. In March 2004, the European Parliament issued the Directive 2004/23/EC 'On setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells'. The Directive applies to human tissues and cells, including fresh or frozen reproductive cells for application to the human body, and is mainly concerned with increasing quality and safety through the implementation of a quality management system. Therefore, the European Society of Human Reproduction and Embryology (ESHRE) undertook a series of initiatives aiming to promote assurance of good laboratory practice and to define the concept of qualified embryologists. One ESHRE initiative was to revise the guidelines for good practice in IVF laboratories, not only in response to the need of embryologists for support and guidance in their duties, but also as a complement to the requirements issued by the Tissue and Cell Directive. The SIG in Embryology hopes that this document may assist the laboratory staff to operate according to the requirements of harmonization, implementation, inspection and certification that are now common to all European member states.
Assuntos
Fertilização in vitro/normas , Laboratórios Hospitalares/normas , Guias de Prática Clínica como Assunto , Humanos , Prática Profissional , Controle de QualidadeAssuntos
Anatomia Artística , Atlas como Assunto , Blastocisto/citologia , Oócitos/citologia , HumanosRESUMO
OBJECTIVE: To establish the value of array comparative genomic hybridization (CGH) for preimplantation genetic diagnosis (PGD) in embryos of translocation carriers in combination with vitrification and frozen embryo transfer in nonstimulated cycles. DESIGN: Retrospective data analysis study. SETTING: Academic centers for reproductive medicine and genetics. PATIENT(S): Thirty-four couples undergoing PGD for chromosomal rearrangements from October 2013 to December 2015. INTERVENTION(S): Trophectoderm biopsy at day 5 or day 6 of embryo development and subsequently whole genome amplification and array CGH were performed. MAIN OUTCOME MEASURE(S): This approach revealed a high occurrence of aneuploidies and structural rearrangements unrelated to the parental rearrangement. Nevertheless, we observed a benefit in pregnancy rates of these couples. RESULT(S): We detected chromosomal abnormalities in 133/207 embryos (64.2% of successfully amplified), and 74 showed a normal microarray profile (35.7%). In 48 of the 133 abnormal embryos (36.1%), an unbalanced rearrangement originating from the parental translocation was identified. Interestingly, 34.6% of the abnormal embryos (46/133) harbored chromosome rearrangements that were not directly linked to the parental translocation in question. We also detected a combination of unbalanced parental-derived rearrangements and aneuploidies in 27 of the 133 abnormal embryos (20.3%). CONCLUSION(S): The use of trophectoderm biopsy at the blastocyst stage is less detrimental to the survival of the embryo and leads to a more reliable estimate of the genomic content of the embryo than cleavage-stage biopsy. In this small cohort PGD study, we describe the successful implementation of array CGH analysis of blastocysts in patients with a chromosomal rearrangement to identify euploid embryos for transfer.
Assuntos
Blastocisto/patologia , Aberrações Cromossômicas , Transtornos Cromossômicos/diagnóstico , Cromossomos Humanos , Hibridização Genômica Comparativa , Fertilização in vitro/efeitos adversos , Rearranjo Gênico , Testes Genéticos , Diagnóstico Pré-Implantação/métodos , Biópsia , Transtornos Cromossômicos/etiologia , Transtornos Cromossômicos/genética , Criopreservação , Técnicas de Cultura Embrionária , Transferência Embrionária , Feminino , Predisposição Genética para Doença , Humanos , Nascido Vivo , Fenótipo , Ploidias , Valor Preditivo dos Testes , Gravidez , Taxa de Gravidez , Estudos Retrospectivos , Resultado do Tratamento , VitrificaçãoRESUMO
Current whole genome amplification (WGA) methods lead to amplification bias resulting in over- and under-represented regions in the genome. Nevertheless, certain WGA methods, such as SurePlex and subsequent arrayCGH analysis, make it possible to detect copy number alterations (CNAs) at a 10 Mb resolution. A more uniform WGA combined with massive parallel sequencing (MPS), however, could allow detection at higher resolution and lower cost. Recently, MALBAC, a new WGA method, claims unparalleled performance. Here, we compared the well-established SurePlex and MALBAC WGA for their ability to detect CNAs in MPS generated data and, in addition, compared PCR-free MPS library preparation with the standard enrichment PCR library preparation. Results showed that SurePlex amplification led to more uniformity across the genome, allowing for a better CNA detection with less false positives compared to MALBAC amplified samples. An even more uniform coverage was observed in samples following a PCR-free library preparation. In general, the combination of SurePlex and MPS led to the same chromosomal profile compared to a reference arrayCGH from unamplified genomic DNA, underlining the large potential of MPS techniques in CNA detection from a limited number of DNA material.
Assuntos
Genoma Humano , Linhagem Celular , Cromossomos Humanos/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Feminino , Dosagem de Genes , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência de DNA , Análise de Célula ÚnicaRESUMO
OBJECTIVE: To add evidence that massive parallel sequencing (MPS) is a valuable substitute for array comparative genomic hybridization (arrayCGH) with a resolution that is more appropriate for preimplantation genetic diagnosis (PGD) in translocation carriers. DESIGN: Study of diagnostic accuracy. SETTING: University hospital. PATIENT(S): Fifteen patients with a balanced structural rearrangement were included in the study: eight reciprocal translocations, four Robertsonian translocations, two inversions, and one insertional translocation. INTERVENTION(S): Trophectoderm biopsy was performed on 47 blastocysts. MAIN OUTCOME MEASURE(S): In the current study, shallow whole genome MPS on a NextSeq500 (Illumina) and Ion Proton (Life Technologies) instrument was performed in parallel on 47 whole genome amplified trophectoderm samples. Data analyses were performed using the QDNAseq algorithm implemented in Vivar. RESULT(S): In total, 5 normal and 42 abnormal embryos were analyzed. All aberrations previously detected with arrayCGH could be readily detected in the MPS data using both technologies and were correctly identified. The smallest detected abnormality was a â¼ 4.5 Mb deletion/duplication. CONCLUSION(S): This study demonstrates that shallow whole genome sequencing can be applied efficiently for the detection of numerical and structural chromosomal aberrations in embryos, equaling or even exceeding the resolution of the routinely used microarrays.
Assuntos
Blastocisto/patologia , Transtornos Cromossômicos/genética , Cromossomos Humanos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Diagnóstico Pré-Implantação/métodos , Translocação Genética , Adulto , Biópsia , Transtornos Cromossômicos/patologia , Hibridização Genômica Comparativa , Técnicas de Cultura Embrionária , Feminino , Marcadores Genéticos , Hospitais Universitários , Humanos , Masculino , Valor Preditivo dos Testes , Injeções de Esperma IntracitoplásmicasRESUMO
The duration of administration of GnRH antagonist in cycles stimulated with recombinant FSH for IVF/intracytoplasmic sperm injection does not affect the probability of blastocyst replacement and of achieving an ongoing pregnancy in subsequent frozen-thawed cycles.
Assuntos
Fertilização in vitro/métodos , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Gonadotropina/uso terapêutico , Injeções de Esperma Intracitoplásmicas/métodos , Blastocisto/citologia , Criopreservação , Implantação do Embrião/fisiologia , Transferência Embrionária , Feminino , Humanos , Masculino , Ciclo Menstrual/efeitos dos fármacos , GravidezRESUMO
To investigate the applicability of preimplantation genetic diagnosis (PGD), we used trophectoderm (TE) biopsy to determine the mutation load in a 35-year-old female with mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome (MELAS). Transfer of a mutation-free blastocyst gave birth to a healthy boy with undetectable mutation in any of the analyzed tissues. We found strong correlation among TE cells (r=0.90) within blastocysts and also between cytoplasmic fragments and TE (r=0.95). This is the first case of mutation-free baby born from a MELAS patient after TE biopsy and supports the applicability of blastocyst PGD for patients with mtDNA disorders to establish healthy offspring.
Assuntos
Síndrome MELAS/diagnóstico , Síndrome MELAS/prevenção & controle , Complicações na Gravidez , Diagnóstico Pré-Implantação , Adulto , Biópsia , Transferência Embrionária , Feminino , Humanos , Recém-Nascido , Masculino , GravidezRESUMO
OBJECTIVE: To assess the Ca2+-releasing ability of sperm involved in partial hydatidiform moles. DESIGN: Analysis of the activating and Ca2+-releasing ability of human sperm. SETTING: University hospital research laboratory. PATIENT(S): Patients undergoing intracytoplasmic sperm injection (ICSI) treatment. INTERVENTION(S): Microinjection of mouse and human oocytes with sperm. MAIN OUTCOME MEASURE(S): Measurement of the fertilizing and Ca2+-releasing ability of human sperm. RESULT(S): The mouse oocyte Ca2+ analysis showed that only 19.0% (4/21) of the mouse oocytes injected with sperm involved in molar pregnancies exhibited a normal pattern of Ca2+ oscillations versus 63.2% (36/57) of those injected with control sperm. Further, 83.3% (15/18) of donated in vitro-matured human oocytes injected with deficient sperm did not exhibit any Ca2+ release, while 76.9% (10/13) failed to show normal pronuclear development. Yet the sperm oocyte activation factor phospholipase C zeta (PLCζ) was present in the majority (96.6%, n=113) of the analyzed sperm at a normal expression level. Eventually, fertilization failure was overcome with assisted oocyte activation in subsequent therapeutic ICSI cycles, which led to normal deliveries. CONCLUSION(S): Sperm that previously provoked recurrent partial hydatidiform mole pregnancies due to dispermic fertilization is not able to activate human oocytes or trigger the normal pattern of Ca2+ oscillations in mouse and human oocytes in vitro.