RESUMO
Vitrification, is an ultra-rapid, manual cooling process that produces glass-like (ice crystal-free) solidification. Water is prevented from forming intercellular and intracellular ice crystals during cooling as a result of oocyte dehydration and the use of highly concentrated cryoprotectant. Though oocytes can be cryopreserved without ice crystal formation through vitrification, it is still not clear whether the process of vitrification causes any negative impact (temperature change/chilling effect, osmotic stress, cryoprotectant toxicity, and/or phase transitions) on oocyte quality, which translates to diminished embryo developmental potential or subsequent clinical outcomes. In this review, we attempt to assess the technique's potential effects and the consequence of these effects on outcomes.
Assuntos
Oócitos , Vitrificação , Temperatura Baixa , Criopreservação , Crioprotetores/farmacologiaRESUMO
PURPOSE: Oocyte donor in vitro fertilization (IVF) represents an ideal model to study the effects of embryo stage on reproductive success, as embryos come from young women with high-quality oocytes. Our study aimed to determine if embryo transfer stage affected outcomes in oocyte donor IVF, including the common scenario where only a limited number of quality embryos are available after culture. METHODS: This retrospective cohort analyzed anonymous vitrified donor oocyte cycles at a single clinic between 2008 and 2015. Overall, 983 recipients underwent 1178 warming cycles resulting in fresh transfer of one-to-two embryos. Our primary outcome was live birth; secondary outcomes included multiple birth, birthweight, and gestational age. Log binomial regression with cluster-weighted generalized estimating equations were used to calculate adjusted risk ratios (aRR) accounting for recipient age, race, and transfer year. RESULTS: Among 132 cleavage and 1046 blastocyst transfer cycles, cleavage transfers were associated with lower probability of live birth (aRR 0.72, 95% CI 0.59-0.88). Subgroup analysis focused on cycles with a limited number of quality embryos 3 days post-fertilization (≤2), as clinically these women were most likely to be considered for cleavage transfers. Among these cycles (120 cleavage, 371 blastocyst), cleavage transfers were still associated with lower live birth rates compared to blastocyst (aRR 0.66, 95% CI 0.51-0.87) CONCLUSIONS: Even in a donor oocyte model with high-quality oocytes, there was a benefit to extended culture and blastocyst transfer, including when only one-to-two quality embryos were available after early culture. This is possibly owed to improved uterine synchronicity or decreased contractility.
Assuntos
Blastocisto/citologia , Transferência Embrionária/métodos , Doadores de Tecidos , Adulto , Peso ao Nascer , Criopreservação , Feminino , Fertilização in vitro , Humanos , Recém-Nascido , Gravidez , Taxa de Gravidez , Gravidez Múltipla , Estudos Retrospectivos , VitrificaçãoRESUMO
BACKGROUND: This prospective, Phase IV, multicenter, observational registry of assisted reproductive technology clinics in the USA studied outcomes of first cycles using thawed/warmed cryopreserved (by slow-freezing/vitrification) oocytes (autologous or donor). METHODS: Patients were followed up through implantation, clinical pregnancy, and birth outcomes. The main outcome measure was live birth rate (LBR), defined as the ratio of live births to oocytes thawed/warmed minus the number of embryos cryopreserved for each cycle, averaged over all thawing cycles. Clinical pregnancy rate (CPR) was also evaluated, and was defined as the presence of a fetal sac with heart activity, as detected by ultrasound scan performed on Day 35-42 after embryo transfer. RESULTS: A total of 16 centers enrolled 204 patients; data from 193 patients were available for analyses. For donor oocytes, in the slow-freezing (n = 40) versus vitrification (n = 94) groups, respectively, CPR and LBR were significantly different: 32.4% versus 62.6%, and 25.0% versus 52.1%; outcomes from Day 3 transfers did not differ significantly. For vitrified oocytes, in the autologous (n = 46) versus donor (n = 94) group, respectively, CPR and LBR were significantly different: 30.0% versus 62.6% and 17.4% versus 52.1%. This was largely due to a significant difference in CPR with Day 5/6 transfers. CONCLUSIONS: In two subgroup data analyses, in women who received cryopreserved oocytes from donors, CPR and LBR were significantly higher in cycles using oocytes cryopreserved via vitrification versus slow-freezing, reflecting differences in methodologies and more Day 5/6 transfers; in women who received vitrified oocytes, CPR and LBR were significantly higher in cycles using donor versus autologous oocytes with Day 5/6 transfers. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00699400 . Registered June 13, 2008.
Assuntos
Criopreservação/métodos , Oócitos/citologia , Sistema de Registros/estatística & dados numéricos , Técnicas de Reprodução Assistida , Adulto , Transferência Embrionária , Feminino , Humanos , Nascido Vivo , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde/métodos , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Gravidez , Taxa de Gravidez , Estudos Prospectivos , Vitrificação , Adulto JovemRESUMO
STUDY QUESTION: Does the type of luteal support affect pregnancy outcomes in recipients of vitrified blastocysts? SUMMARY ANSWER: Luteal support with vaginal progesterone gel or i.m. progesterone (IMP) results in comparable implantation and pregnancy rates in IVF patients receiving vitrified blastocysts. WHAT IS KNOWN ALREADY: In fresh IVF cycles, both IMP and vaginal progesterone have become the standard of care for luteal phase support. Due to conflicting data in replacement cycles, IMP is often considered to be the standard of care. STUDY DESIGN, SIZE, DURATION: Retrospective analysis of 920 frozen embryo transfer (FET) cycles between 1 January 2010 and 1 September 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients from a large, private practice undergoing autologous and donor FET using IMP or vaginal progesterone gel for luteal support were included in the analysis. IMP was used for luteal support in 682 FET cycles and vaginal progesterone gel was used in 238 FET cycles. Standard clinical outcomes of positive serum hCG levels, implantation, clinical pregnancy, spontaneous abortion and live birth were reported. MAIN RESULTS AND THE ROLE OF CHANCE: The IMP and vaginal progesterone gel groups had similar patient demographics for all characteristics assessed. Implantation rates (46.4 versus 45.6%, P = 0.81), clinical pregnancy rates (61.7 versus 60.5%, P = 0.80) and live birth rates (49.1 versus 48.9%, P > 0.99) were not significantly different between IMP and vaginal progesterone gel, respectively. LIMITATIONS, REASONS FOR CAUTION: This study is limited by its retrospective design and by its lack of randomization to the type of luteal support. In addition, because no a priori expected rates of success could be provided for this retrospective investigation, it was not possible to estimate statistical power associated with the various outcomes presented. WIDER IMPLICATIONS OF THE FINDINGS: With the recent trends toward single embryo transfer (SET) and use of vitrified blastocysts in FET cycles, our data with â¼40% of cycles being SET and use of exclusively vitrified blastocysts are more relevant to current practices than previous studies. STUDY FUNDING/COMPETING INTERESTS: Support for data collection and analysis was provided by Actavis, Inc. D.S. has received honoraria for lectures and participation in Scientific Advisory Boards for Actavis, Inc. J.P. is an employee of Actavis, Inc. N.E. has received payment from Actavis, Inc., for her time for data collection. H.H. has received payment from Actavis, Inc., for statistical analyses. Z.P.N. has nothing to disclose.
Assuntos
Fertilização in vitro , Progesterona/administração & dosagem , Feminino , Humanos , Injeções Intramusculares , Gravidez , Resultado da Gravidez , Progesterona/uso terapêutico , Estudos Retrospectivos , Cremes, Espumas e Géis Vaginais/uso terapêuticoRESUMO
STUDY QUESTION: Does conventional blastocyst morphological evaluation correlate with euploidy (as assessed by comprehensive chromosome screening (CCS) of trophectoderm (TE) biopsies) and implantation potential? SUMMARY ANSWER: A moderate relation between blastocyst morphology and CCS data was observed but the ability to implant seems to be mainly determined by the chromosomal complement of preimplantation embryos rather than developmental and morphological parameters conventionally used for blastocyst evaluation. WHAT IS KNOWN ALREADY: Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select euploid embryos for transfer. Understanding the role of morphology in blastocyst stage preimplantation genetic screening (PGS) cycles may help in further optimizing the cycle management and clinical outcomes. STUDY DESIGN, SIZE, DURATION: This is a multicenter retrospective observational study performed between January 2009 and August 2013. The study includes the data analysis of 956 blastocysts with conclusive CCS results obtained from 213 patients following 223 PGS cycles. Single frozen embryo transfer (FET) cycles of 215 euploid blastocysts were performed where it was possible to track the implantation outcome of each embryo transferred. PARTICIPANTS/MATERIALS, SETTING, METHODS: PGS was offered to infertile patients of advanced maternal age (>35 years) and/or with a history of unsuccessful IVF treatments (more than two failed IVF cycles) and/or previous spontaneous abortion (more than two spontaneous miscarriages). Prior to TE biopsy for CCS, blastocyst morphology was assessed and categorized in four groups (excellent, good, average and poor quality). The developmental rate of each embryo reaching the expanded blastocyst stage was defined according to the day of biopsy post-fertilization. Day 5 and Day 6 biopsied blastocysts were defined as faster and slower growing embryos, respectively. A novel blastocyst biopsy method, not requiring the opening of the zona pellucida at the cleavage stage of embryo development, was used. Linear regression models were used to test the relationship between blastocyst morphology and developmental rate CCS data and FET cycle outcomes of euploid blastocysts. MAIN RESULTS AND THE ROLE OF CHANCE: Among the embryological variables assessed (morphology and developmental rate), only blastocyst morphology was predictive of the CCS data. The euploidy rate was 56.4, 39.1, 42.8 and 25.5% in the excellent, good, average and poor blastocyst morphology groups, respectively. A diagnosis of complex aneuploidy was also associated with blastocyst morphology (P < 0.01) with 6.8, 15.2, 17.4 and 27.5% of excellent, good, average and poor quality embryos, respectively, showing multiple chromosome errors. Faster and slower growing embryos showed a similar aneuploidy rate. Regression logistic analysis showed that none of the parameters used for conventional blastocyst evaluation (morphology and developmental rate) was predictive of the implantation potential of euploid embryos. The implantation potential of euploid embryos was the same, despite different morphologies and developmental rates. LIMITATIONS, REASONS FOR CAUTION: The study is limited by its retrospective nature. A higher sample size or a prospective randomized design could be used in future studies to corroborate the current findings. WIDER IMPLICATIONS OF THE FINDINGS: This study provides knowledge for a better laboratory and clinical management of blastocyst stage PGS cycles suggesting that the commonly used parameters of blastocyst evaluation are not good enough indicators to improve the selection among euploid embryos. Accordingly, all poor morphology and slower growing expanded blastocysts should be biopsied and similarly considered for FET cycles. This knowledge will be of critical importance to achieve similar cumulative live birth rates in PGS programs compared with conventional IVF, avoiding the potential for exclusion of low quality but viable embryos from the biopsy and transfer procedures. Future research to identify non-invasive biomarkers of reproductive potential may further enhance selection among euploid blastocysts. STUDY FUNDING/COMPETING INTEREST(S): No funding was obtained for the study. All authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: None.
Assuntos
Aneuploidia , Blastocisto , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/fisiologia , Adulto , Transferência Embrionária/métodos , Feminino , Humanos , Infertilidade/terapia , Gravidez , Estudos RetrospectivosRESUMO
STUDY QUESTION: Does comprehensive chromosome screening (CCS) of cells sampled from the blastocyst trophectoderm (TE) accurately predict the chromosome complement of the inner cell mass (ICM)? SUMMARY ANSWER: Comprehensive chromosome screening of a TE sample is unlikely to be confounded by mosaicism and has the potential for high diagnostic accuracy. WHAT IS KNOWN ALREADY: The effectiveness of chromosome aneuploidy screening is limited by the technologies available and chromosome mosaicism in the embryo. Combined with improving methods for cryopreservation and blastocyst culture, TE biopsy and CCS is considered to be a promising approach to select diploid embryos for transfer. STUDY DESIGN, SIZE, DURATION: The study was performed between January 2011 and August 2011. In the first part, a new ICM isolation method was developed and tested on 20 good morphology blastocysts. In the main phase of the study, fluorescence in situ hybridization (FISH) was used to reanalyse the ICMs and TEs separated from 70 embryos obtained from 26 patients undergoing blastocyst stage array comparative genome hybridization (aCGH) PGS cycles. MATERIALS, SETTING, METHODS: The isolated ICM and TE fractions were characterized by immunostaining for KRT18. Then, non-transferrable cryopreserved embryos were selected for the FISH reanalysis based on previous genetic diagnosis obtained by TE aCGH analysis. Blastocysts either diploid for chromosome copy number (20) or diagnosed as single- (40) or double aneuploid (10) were included after preparing the embryo into one ICM and three equal-sized TE sections. Accuracy of the aCGH was measured based on FISH reanalysis. Chromosomal segregations resulting in diploid/aneuploid mosaicism were classified as 'low-', 'medium-' and 'high-' grade and categorized with respect to their distribution (1TE, 2TE, 3TE, ICM or ALL embryo). Linear regression model was used to test the relationship between the distributions and the proportion of aneuploid cells across the four embryo sections. Fisher's exact test was used to test for random allocation of aneuploid cells between TE and ICM. MAIN RESULTS AND THE ROLE OF CHANCE: All ICM biopsy procedures displayed ICM cells in the recovered fraction with a mean number of ICM cells of 26.2 and a mean TE cell contamination rate of 2%. By FISH reanalysis of previously aCGH-screened blastocysts, a total of 66 aneuploidies were scored, 52 (78.8%) observed in all cells and 14 (21.2%) mosaic. Overall, mosaic chromosomal errors were observed only in 11 out of 70 blastocysts (15.7%) but only 2 cases were classified as mosaic diploid/aneuploid (2.9%). Sensitivity and specificity of aCGH on TE clinical biopsies were 98.0 and 100% per embryo and 95.2 and 99.8% per chromosome, respectively. Linear regression analysis performed on the 11 mosaic diploid/aneuploid chromosomal segregations showed a significant positive correlation between the distribution and the proportion of aneuploid cells across the four-blastocyst sections (P < 0.01). In addition, regression analysis revealed that both the grade and the distribution of mosaic abnormal cells were significantly correlated with the likelihood of being diagnosed by aCGH performed on clinical TE biopsies (P = 0.019 and P < 0.01, respectively). Fisher's exact test for the 66 aneuploidies recorded showed no preferential allocation of abnormal cells between ICM and TE (P = 0.33). LIMITATIONS, REASONS FOR CAUTION: The study is limited to non-transferable embryos, reanalyzed for only nine chromosomes and excludes segmental imbalance and uniparental disomy. The prevalence of aneuploidy in the study group is likely to be higher than in the general population of clinical PGD embryos. WIDER IMPLICATIONS OF THE FINDINGS: This study showed high accuracy of diagnosis achievable during blastocyst stage PGS cycles coupled with 24-chromosomes molecular karyotyping analysis. The new ICM isolation strategy developed may open new possibilities for basic research in embryology and for clinical grade derivation of human embryonic stem cells. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was sought or obtained for this study.
Assuntos
Diagnóstico Pré-Implantação/métodos , Massa Celular Interna do Blastocisto , Transtornos Cromossômicos/diagnóstico , Hibridização Genômica Comparativa/métodos , Humanos , Hibridização in Situ Fluorescente , Mosaicismo , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages. In the present manuscript, the current experience with oocyte donation using cryopreservation technology is reviewed. The outcomes of two recently established donor egg cryobanks at Instituto Valenciano de Infertilidad in Spain and Reproductive Biology Associates in the USA (involving a large number of cases) demonstrate that egg cryo-survival is high and that fertilization, embryo development, implantation and pregnancy rates are similar to those reported after fresh egg donation. It also provides additional advantages of being more efficient, more economical, easier for both donors and recipients and potentially also safer, because eggs can now be quarantined for 6 months (or longer) to retest for infectious diseases in the donors. It is the opinion of the authors, based on several advantages associated with the use of donor egg cryobanking, that in the future there will be fewer traditional egg donations and increasingly more cryo-egg donations.
Assuntos
Criopreservação/métodos , Oócitos , Obtenção de Tecidos e Órgãos , Feminino , Fertilização in vitro , Humanos , Bancos de TecidosRESUMO
Until recently, success in oocyte cryopreservation has been very limited mainly due to poor understanding of the complex physiological processes that lead to cell damage during cryopreservation. In the past three decades, however, a wealth of information has been collected using various different animal models, which has led to development of new technologies and optimization of existing ones. The use of these models has provided the opportunity for research that may not have been possible with human material. Today, results of these studies still continue to form the basis of oocyte cryobiology. This review discusses these studies, especially the physiological impacts of cryopreservation on oocyte biology. It will also focus on the role that animal models have played in improvement strategies, validation before translating new techniques into the human model and the advances made in the human in IVF because of these animal models. Finally, existing investigations and their potential impact in other areas of research will be discussed. Until recently, success in oocyte cryopreservation has been very limited mainly due to poor understanding of the complex physiological processes that lead to cell damage during cryopreservation. In the past three decades, however, a wealth of information has been collected using various different animal models, which has led to development of new technologies and optimization of existing ones. The use of these models provided the opportunity for research that may not have been possible with human material. Today, animal models still continuously provide imperative data that facilitate further advancements in oocyte cryobiology. This review will focus on the physiological impacts, current improvement strategies and future applications of oocyte cryopreservation using animal models as they benefit not only human oocyte cryopreservation procedures, but also the human species through their usefulness in agriculture, medicine and conservation.
Assuntos
Criopreservação , Modelos Animais , Oócitos/fisiologia , Animais , Sinalização do Cálcio , Crioprotetores/farmacologia , Citoesqueleto/ultraestrutura , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/ultraestruturaRESUMO
During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.
Assuntos
Criopreservação , Oócitos , Transição de Fase/efeitos dos fármacos , Fuso Acromático/ultraestrutura , Animais , Cromossomos/efeitos dos fármacos , Crioprotetores/farmacologia , Feminino , Camundongos , Fuso Acromático/efeitos dos fármacosRESUMO
Despite an ongoing debate over its efficacy, preimplantation genetic screening (PGS) is increasingly being used to detect numerical chromosomal abnormalities in embryos to improve implantation rates after IVF. The main indications for the use of PGS in IVF treatments include advanced maternal age, repeated implantation failure, and recurrent pregnancy loss. The success of PGS is highly dependent on technical competence, embryo culture quality, and the presence of mosaicism in preimplantation embryos. Today, cleavage stage biopsy is the most commonly used method for screening preimplantation embryos for aneuploidy. However, blastocyst biopsy is rapidly becoming the more preferred method due to a decreased likelihood of mosaicism and an increase in the amount of DNA available for testing. Instead of using 9 to 12 chromosome FISH, a 24 chromosome detection by aCGH or SNP microarray will be used. Thus, it is advised that before attempting to perform PGS and expecting any benefit, extended embryo culture towards day 5/6 should be established and proven and the clinical staff should demonstrate competence with routine competency assessments. A properly designed randomized control trial is needed to test the potential benefits of these new developments.
Assuntos
Blastocisto/citologia , Diagnóstico Pré-Implantação/métodos , Aneuploidia , Técnicas de Cultura Embrionária , Fertilização in vitro , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
As more reproductive-age women survive cancer at the expense of gonadotoxic therapy, the need for viable fertility preservation options has become paramount. Embryo cryopreservation, often using donor sperm, has been the standard offered these women over the past 20 years. Preservation of unfertilized oocytes now represents an acceptable and often equally viable alternative, particularly for single women, due to technologic advances made in the past decade. Given such, oocyte cryopreservation's experimental designation and need for IRB approval should thus be revisited.
Assuntos
Criopreservação , Oócitos , Antineoplásicos/efeitos adversos , Criopreservação/ética , Criopreservação/métodos , Destinação do Embrião , Transferência Embrionária , Feminino , Fertilização in vitro , Humanos , Recuperação de Oócitos/legislação & jurisprudência , Gravidez , Resultado da Gravidez , Insuficiência Ovariana Primária/induzido quimicamente , Técnicas de Reprodução Assistida/ética , Técnicas de Reprodução Assistida/legislação & jurisprudência , Injeções de Esperma Intracitoplásmicas , Sobreviventes , Terapias em EstudoRESUMO
Cryopreservation has become a central pillar in assisted reproduction, reflected in the exponential increase of "freeze all" cycles in the past few years. Vitrification makes it possible to cool and warm human eggs and embryos with far less cryo-damage than 'slow-freeze' and allows nearly intact survival of embryos with very high survival rates for eggs as well. This has resulted in a complete transformation how we manage treatment for in vitro fertilization patients. Fresh transfers can be avoided without compromising outcomes, and in fact, cumulative pregnancy/delivery rates may be improved by performing sequential elective "frozen" single embryo transfers. Some recent evidence suggests that previously vitrified embryos give better perinatal outcomes than fresh embryo transfers. Frozen embryo transfer, especially when coupled with preimplantation genetic testing allows for highly efficient single embryo transfers that translate to more singleton and therefore safer pregnancies, as well as healthier babies. Additionally, vitrification has also opened new options for patients, most notably fertility preservation (through oocyte cryopreservation), and donor egg banking.
Assuntos
Fertilização in vitro/métodos , Vitrificação , Biópsia , Criopreservação , Transferência Embrionária , Feminino , Fertilização in vitro/efeitos adversos , Humanos , Nascido Vivo , Gravidez , Taxa de GravidezRESUMO
For a long time, the cryopreservation of gametes and embryos remained a major hurdle for the clinicians and scientists in terms of success. However, recent technical advancement in the field of cryobiology has opened up various options for freezing gametes and embryos at different developmental stages. The tendency of the IVF world to switch over to natural-cycle IVF and to elective single-embryo transfer has put cryotechnology in the forefront of research. Still, the intricacies of the cold-induced changes in human gametes and embryos that could affect the intracellular and developmental processes need to be known. The transcriptomics, proteomics and metabolomic platforms hold promise for elucidating these complex processes during cryopreservation processes.
Assuntos
Criopreservação , Embrião de Mamíferos , Oócitos , Proteínas Anticongelantes/metabolismo , Antioxidantes/metabolismo , Metabolismo dos Carboidratos , Sobrevivência Celular , Feminino , Fertilização in vitro , Humanos , Gravidez , Resultado da GravidezRESUMO
The aim of the current study was to evaluate whether near-infrared (NIR) spectroscopy-generated metabolomic data obtained from oocyte culture samples would correlate with nuclear maturity status and derived embryo development. A total of 412 oocyte culture samples were collected from 43 patient cycles. Metabolomic profiles of metaphase I and II oocytes were obtained by NIR spectroscopy and were significantly different from each other and from profiles of prophase I (germinal vesicle) oocytes (P +/- 0.001 at the 95% confidence interval). Additionally, NIR spectroscopic analysis of culture medium of oocytes that developed to grade A embryos on day 3 demonstrated significantly higher viability indices (0.62 +/- 0.23) than those that developed to grades C/D (0.42 +/- 0.26; P < 0.006); and on day 5 grade A (0.37 +/- 0.20) was also higher than grades C/D (0.14 +/- 0.21; P < 0.02). Metabolomic profiles of oocytes that resulted in pregnancy had higher viability indices (0.87 +/- 0.27) than those that did not (0.44 +/- 0.17; P < 0.0001). The results of the current study demonstrate that metabolomic profiling from spent culture medium of the oocyte is related to nuclear maturity, is able to predict embryo development at day 3 and day 5 stages, and relates to embryo viability.
Assuntos
Metabolômica/métodos , Oócitos/metabolismo , Oócitos/fisiologia , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultura/química , Transferência Embrionária , Feminino , Humanos , Masculino , Oócitos/citologia , Gravidez , Taxa de Gravidez , Controle de Qualidade , Projetos de Pesquisa , Sensibilidade e Especificidade , Espectroscopia de Luz Próxima ao Infravermelho , Injeções de Esperma Intracitoplásmicas/métodosRESUMO
Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine.
Assuntos
Criopreservação/métodos , Técnicas de Transferência Nuclear , Oócitos/citologia , Medicina Regenerativa/métodos , Animais , Humanos , Medicina Regenerativa/tendênciasRESUMO
Increasing the efficiency of the IVF procedure by improving pregnancy/implantation rates and at the same time lowering (or avoiding) the risks of multiple gestations are the primary goals of the current assisted reproductive technology. These aims require a much improved gamete/embryo testing and selection procedure, which, using the current approach of microscopy-based morphology evaluation is unlikely to be achieved. Therefore, alternative or additional, non-invasive techniques have been proposed which may be able to detect alterations of the culture environment surrounding gametes/embryos reflective of the (patho-)physiological processes. One of the most recently applied approaches is to measure metabolomic changes in the culture medium of embryos and oocytes ('exometabolomics'). Initial studies have demonstrated that different types of spectrophotometric tests, including Raman and near-infrared (NIR) techniques, are similarly well capable of detecting specific changes of the 'secretome' (exometabolome). These studies have also demonstrated that metabolomic measurements correlate well with embryo development and morphology assessment. Furthermore, viability index on oocytes/embryos established by metabolomic tests may be a stronger predictor for implantation potential than traditional morphological assessment. Although the results of these initial investigations are promising, further prospective studies are required to define clearly the potential benefits and most relevant applications of this novel non-invasive technology in the field of assisted reproduction.
Assuntos
Blastocisto/fisiologia , Meios de Cultura/metabolismo , Viabilidade Fetal , Metabolômica/métodos , Diagnóstico Pré-Implantação/métodos , Blastocisto/metabolismo , Meios de Cultura/química , Técnicas de Cultura Embrionária , Transferência Embrionária , Feminino , Viabilidade Fetal/fisiologia , Humanos , Modelos Biológicos , Gravidez , Resultado da Gravidez , Técnicas de Reprodução Assistida/tendênciasRESUMO
Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P < 0.05). In a separate study, also in human, a total of 48 patients with a cumulative 214 unsuccessful previous IVF cycles were selected for the trials. In subsequent intracytoplasmic sperm injection cycles, oocytes/embryos were cultured individually in the WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.
Assuntos
Blastocisto/citologia , Técnicas de Cultura Embrionária , Oócitos/metabolismo , Adulto , Animais , Blastocisto/metabolismo , Estudos de Casos e Controles , Feminino , Fertilização in vitro , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Injeções de Esperma Intracitoplásmicas/métodos , SuínosRESUMO
This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.
Assuntos
Criopreservação/métodos , Oócitos/crescimento & desenvolvimento , Adulto , Blastocisto/citologia , Fase de Clivagem do Zigoto/citologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário , Feminino , Fertilização in vitro , Humanos , Recém-Nascido , Oócitos/citologia , Gravidez , Resultado da GravidezRESUMO
The aim of this study was to determine whether the denuding procedure causes the polar body to move within the perivitelline space. Only those patients undergoing IVF who had unused in-vitro matured (IVM) oocytes were included in this study. IVM oocytes were initially viewed under a non-invasive, polarized light microscope. A laser was used to mark the location of the polar body on the zona. Oocytes were subjected to the denuding procedure with a 150 microm, 135 microm and 125 mum diameter pipette. After each pipetting, the oocytes were viewed again to determine whether the polar body had moved. After denuding, the oocyte was left to culture overnight and viewed 24 h later. After denuding with the 150 microm, 135 microm and 125 microm pipettes and after 24 h in culture, the average angle between the spindle and polar body was 15.4 +/- 10.4 degrees , 16.1 +/- 10.1 degrees , 20.9 +/- 11.7 degrees , and 26.7 +/- 18.2 degrees , respectively (P = 0.0021). Slight changes in angles were noted between denuding with the different diameter pipettes. The largest changes in angles were seen after 24 h in culture.
Assuntos
Oócitos/citologia , Oogênese/fisiologia , Fuso Acromático/fisiologia , Zona Pelúcida/fisiologia , Técnicas de Cultura de Células , Fracionamento Celular/métodos , Células Cultivadas , Feminino , Humanos , Movimento/fisiologia , Oócitos/ultraestrutura , Injeções de Esperma IntracitoplásmicasRESUMO
OBJECTIVE: To examine the degree to which paternal variables of age, body mass index (BMI), and sperm parameters affect vitrified donor oocyte IVF outcomes. Previous studies examining the impact of male partner characteristics on in-vitro fertilization (IVF) have found conflicting results. Concerns are rising over the potential effects of paternal factors, such as age and obesity, on pregnancy and child health. Frozen donor oocyte IVF offers an ideal model to study these effects. DESIGN: Retrospective chart review. SETTING: Private fertility clinic. PATIENT(S): Nine hundred forty-nine recipients undergoing transfer of blastocyst embryo(s) from a vitrified oocyte donor bank between 2008-2015. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation rate, clinical pregnancy rate, live birth rate, rate of low birth weight singleton infants (≤2500 g), and preterm deliveries (PTD) of singleton infants (<37 wk). RESULTS: After adjusting for covariates known to affect oocyte donation cycle success, male age, BMI and sperm parameters were not associated with differences in IVF outcomes. There were higher PTD rates for men ≥51 years and BMI ≥35 kg/m2, however, these were not significant after adjustment. There were no differences in rates of low birth weight infants with men >35 years or BMI >25 kg/m2. Lastly, there were no differences in rates of PTD or low birth weight infants with abnormal sperm parameters. CONCLUSIONS: Neither advancing male age, elevated BMI, nor poor sperm quality were associated with outcomes in frozen donor oocyte IVF cycles in this study. Intracytoplamic sperm injection and "oocyte quality" likely mitigate some of the effects of male variables on outcomes following cryopreserved oocyte donation.