Inhibition of Phosphoinositide Metabolism or Chelation of Intracellular Calcium Blocks FSH-Induced but Not Spontaneous Meiotic Resumption in Mouse Oocytes

AbstractMammalian oocytes are arrested at the diplotene phase of the first meiotic division until ovulation. In the mouse, germinal vesicle breakdown (GVBD) and progression to metaphase II is thought to be triggered by a positive signal originating in the follicular cells following stimulation by the luteinizing hormone (LH) surge. Isolated, fully grown oocytes can also undergo spontaneous reinitiation of meiosisin vitroin the absence of gonadotrophin stimulation. To investigate the mechanism of meiotic resumption, inhibitors of phosphoinositide metabolism and an intracellular calcium chelator were used during maturationin vitrounder different conditions. In a series of experiments, isolated cumulus cell–oocyte complexes (COCs) maintained in meiotic arrest by hypoxanthine were induced to resume meiosis by treatment with follicle-stimulating hormone (FSH). Under these conditions, both LiCl and neomycin, which inhibit phosphoinositide hydrolysis, produced a dose-dependent inhibitory effect on meiotic resumption. Similar results were obtained when FSH-induced meiotic resumption was observed in the presence of the acetoxymethyl ester form of 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA/AM), an intracellular calcium chelator. In hypoxanthine-arrested oocytes, GVBD induced by epidermal growth factor (EGF), which mimics FSH action inin vitromaturation, was also repressed by LiCl and neomycin. Conversely, meiotic resumption triggered by a pulse of 8-bromo-cyclic adenosine monophosphate (8-Br cAMP) was not affected by these two inhibitors. In experiments in which oocytes were cultured under conditions which permit spontaneous meiotic maturation, resumption of meiosis was not affected by either inhibition of phosphoinositide hydrolysis or chelation of intracellular calcium. Therefore, it appears that meiotic resumption induced by hormone stimulation requires activation of the phosphoinositide pathway and mobilization of intracellular calcium. In contrast, spontaneous maturation probably occurs through a different mechanism because it is not affected by inhibition of this signaling pathway

The enigmatic morula: mechanisms of development, cell fate determination, self-correction and implications for ART

Background: Assisted reproduction technology offers the opportunity to observe the very early stages of human development. However, due to practical constraints, for decades morphological examination of embryo development has been undertaken at a few isolated time points at the stages of fertilisation (day 1), cleavage (day 2-3) and blastocyst (day 5-6). Rather surprisingly, the morula stage (day 3-4) has been so far neglected, despite its involvement in crucial cellular processes and developmental decisions. Objective and rationale: The objective of this review is to collate novel and unsuspected insights into developmental processes occurring during formation of the morula, highlighting the key importance of this stage for a better understanding of preimplantation development and an improvement of ART. Search method: PubMed was used to search the MEDLINE database for peer-reviewed English-language original articles and reviews concerning the morula stage in mammals. Searches were performed by adopting ‘embryo’, ‘morula’, ‘compaction’, ‘cell fate’ and ‘IVF/assisted reproduction’ as main terms, in association with other keywords expressing concepts relevant to the subject (e.g. cell polarity). The most relevant publications, i.e. those concerning major phenomena occurring during formation of the morula in established experimental models and the human species, were assessed and discussed critically.Outcomes: Novel live cell imaging technologies and cell biology studies have extended our understanding of morula formation as a key stage for the development of the blastocyst and determination of the inner cell mass (ICM) and the trophectoderm (TE). Cellular processes, such as dynamic formation of filopodia and cytoskeleton-mediated zippering cell-to-cell interactions, intervene to allow cell compaction (a geometrical requisite essential for development) and formation of the blastocoel, respectively. At the same time, differential orientation of cleavage planes, cell polarity and cortical tensile forces interact and cooperate to position blastomeres either internally or externally, thereby influencing their cellular fate. Recent time lapse microscopy (TLM) observations also suggest that in the human the process of compaction may represent an important checkpoint for embryo viability, through which chromosomally abnormal blastomeres are sensed and eliminated by the embryo.Wider implications: In clinical embryology, the morula stage has been always perceived as a ‘black box’ in the continuum of preimplantation development. This has dictated its virtual exclusion from mainstream ART procedures. Recent findings described in this review indicate that the morula, and the associated process of compaction, as a crucial stage not only for the formation of the blastocyst, but also for the health of the conceptus. This understanding may open new avenues for innovative approaches to embryo manipulation, assessment and treatment

Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis

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Differential regulation of cumulus cell transcription during oocyte maturation in vivo and in vitro

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Good practice recommendations for the use of time-lapse technology†

STUDY QUESTION: What recommendations can be provided on the approach to and use of time-lapse technology (TLT) in an IVF laboratory?SUMMARY ANSWER: The present ESHRE document provides 11 recommendations on how to introduce TLT in the IVF laboratory. WHAT IS KNOWN ALREADY: Studies have been published on the use of TLT in clinical embryology. However, a systematic assessmentof how to approach and introduce this technology is currently missing.STUDY DESIGN, SIZE, DURATION: A working group of members of the Steering Committee of the ESHRE Special Interest Group in Embryology and selected ESHRE members was formed in order to write recommendations on the practical aspects of TLT for the IVF laboratory.PARTICIPANTS/MATERIALS, SETTING, METHODS: The working group included 11 members of different nationalities with internationally recognized experience in clinical embryology and basic science embryology, in addition to TLT. This document is developed according to the manual for development of ESHRE recommendations for good practice. Where possible, the statements are supported by studies retrieved from a PUBMED literature search on ‘time-lapse’ and ART.MAIN RESULTS AND THE ROLE OF CHANCE: A clear clinical benefit of the use of TLT, i.e. an increase in IVF success rates, remains to be proven. Meanwhile, TLT systems are being introduced in IVF laboratories. The working group listed 11 recommendations on what to do before introducing TLT in the lab. These statements include an assessment of the pros and cons of acquiring a TLT system, selection of relevant morphokinetic parameters, selection of an appropriate TLT system with technical and customer support, development of an internal checklist and education of staff. All these aspects are explained further here, based on the current literature and expert opinion.LIMITATIONS, REASONS FOR CAUTION: Owing to the limited evidence available, recommendations are mostly based on clinical and technical expertise. The paper provides technical advice, but leaves any decision on whether or not to use TLT to the individual centres.WIDER IMPLICATIONS OF THE FINDINGS: This document is expected to have a significant impact on future developments of clinical embryology, considering the increasing role and impact of TLT

Influence of thyroid hormone on mouse preantral follicle development in vitro

Objective: To evaluate the role of 3,3',5-triiodothyronine (T-3) on ovarian follicle development in vitro. Design: Experimental study. Setting: University reproductive biology unit. Animal(s): CD1 mice (female). Intervention(s): In vitro culture. Main Outcome Measure(s): Assessment of follicle and oocyte development following a 6-day culture with FSH (100 mU/mL), (Bu)(2)cAMP (0.5 mM), and T-3 (1-100 nM), alone or in combination. Result(s): A high percentage of in vitro grown (IVG) FSH-stimulated follicles formed an antral cavity (AC) while the addition of T-3 (100 nM) significantly reduced this response in a statistically significant manner. A statistically significant recovery of AC formation was obtained by limiting exposure to 4 days of culture. Formation of AC was induced by (Bu)(2)cAMP, which prevented T-3-mediated suppression of AC formation. Under different conditions, high proportions of germinal vesicle-arrested IVG oocytes displayed condensed chromatin configuration. The capacity to undergo germinal vesicle breakdown (GVBD) was similar in all groups, and a statistically significant reduction in the percentage of oocytes with PB1 was recorded when T-3 was added to FSH or (Bu)(2)cAMP. This ability was partially recovered by removing T-3 on day 4 of culture. Conclusion(s): Exposure to high T-3 concentrations can impair preantral follicle development, but this effect can be partly reverted by restoring a physiological hormonal milieu before follicle commitment to antral development. ((C) 2004 by American Society for Reproductive Medicine)

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols

To ascertain possible cell damage from cryopreservation, the ultrastructure of human oocytes cryopreserved by slow cooling was assessed. Cryopreservation was performed through two protocols with one-step or two-step propanediol. Fresh control oocytes were examined for comparison. Samples were processed for transmission electron microscopy analysis. By light microscopy, both fresh and frozen-thawed oocytes appeared regularly rounded, with intact zona pellucida, and homogeneous cytoplasm. By electron microscopy observation, organelles were abundant and uniformly dispersed. Mitochondria-smooth endoplasmic reticulum associations appeared regular. However, both the amount and density of cortical granules appeared abnormally reduced in frozen-thawed samples. Slight to moderate vacuolization was also found in the ooplasm of oocytes of both frozen groups. Slow cooling ensures a good overall preservation of human oocytes. However, cytoplasmic vacuolization and cortical granule loss appears associated with cryopreservation, irrespective of the protocol used

Cytoplasmic movements of the early human embryo: imaging and artificial intelligence to predict blastocyst development

Research question: Can artificial intelligence and advanced image analysis extract and harness novel information derived from cytoplasmic movements of the early human embryo to predict development to blastocyst? Design: In a proof-of-principle study, 230 human preimplantation embryos were retrospectively assessed using an artificial neural network. After intracytoplasmic sperm injection, embryos underwent time-lapse monitoring for 44 h. For comparison, standard embryo assessment of each embryo by a single embryologist was carried out to predict development to blastocyst stage based on a single picture frame taken at 42 h of development. In the experimental approach, in embryos that developed to blastocyst or destined to arrest, cytoplasm movement velocity was recorded by time-lapse monitoring during the first 44 h of culture and analysed with a Particle Image Velocimetry algorithm to extract quantitative information. Three main artificial intelligence approaches, the k-Nearest Neighbour, the Long-Short Term Memory Neural Network and the hybrid ensemble classifier were used to classify the embryos. Results: Blind operator assessment classified each embryo in terms of ability to develop to blastocyst, with 75.4% accuracy, 76.5% sensitivity, 74.3% specificity, 74.3% precision and 75.4% F1 score. Integration of results from artificial intelligence models with the blind operator classification, resulted in 82.6% accuracy, 79.4% sensitivity, 85.7% specificity, 84.4% precision and 81.8% F1 score. Conclusions: The present study suggests the possibility of predicting human blastocyst development at early cleavage stages by detection of cytoplasm movement velocity and artificial intelligence analysis. This indicates the importance of the dynamics of the cytoplasm as a novel and valuable source of data to assess embryo viability