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Purpose: To study the effectiveness of whole-scenario embryo identification using a self-supervised learning encoder (WISE) in in vitro fertilization (IVF) on time-lapse, cross-device, and cryo-thawed scenarios., Methods: WISE was based on the vision transformer (ViT) architecture and masked autoencoders (MAE), a self-supervised learning (SSL) method. To train WISE, we prepared three datasets including the SSL pre-training dataset, the time-lapse identification dataset, and the cross-device identification dataset. To identify whether pairs of images were from the same embryos in different scenarios in the downstream identification tasks, embryo images including time-lapse and microscope images were first pre-processed through object detection, cropping, padding, and resizing, and then fed into WISE to get predictions., Results: WISE could accurately identify embryos in the three scenarios. The accuracy was 99.89% on the time-lapse identification dataset, and 83.55% on the cross-device identification dataset. Besides, we subdivided a cryo-thawed evaluation set from the cross-device test set to have a better estimation of how WISE performs in the real-world, and it reached an accuracy of 82.22%. There were approximately 10% improvements in cross-device and cryo-thawed identification tasks after the SSL method was applied. Besides, WISE demonstrated improvements in the accuracy of 9.5%, 12%, and 18% over embryologists in the three scenarios., Conclusion: SSL methods can improve embryo identification accuracy even when dealing with cross-device and cryo-thawed paired images. The study is the first to apply SSL in embryo identification, and the results show the promise of WISE for future application in embryo witnessing., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

With the rising demand for in vitro fertilization (IVF) cycles, there is a growing need for innovative techniques to optimize procedure outcomes. One such technique is time-lapse system (TLS) for embryo incubation, which minimizes environmental changes in the embryo culture process. TLS also significantly advances predicting embryo quality, a crucial determinant of IVF cycle success. However, the current subjective nature of embryo assessments is due to inter- and intra-observer subjectivity, resulting in highly variable results. To address this challenge, reproductive medicine has gradually turned to artificial intelligence (AI) to establish a standardized and objective approach, aiming to achieve higher success rates. Extensive research is underway investigating the utilization of AI in TLS to predict multiple outcomes. These studies explore the application of popular AI algorithms, their specific implementations, and the achieved advancements in TLS. This review aims to provide an overview of the advances in AI algorithms and their particular applications within the context of TLS and the potential challenges and opportunities for further advancements in reproductive medicine., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Maintaining a stable pH at optimal level in human embryo culture media is crucial for embryo development but poses a challenge for all IVF laboratories. We validate analytically reliable conditions for pH measurement that are as close as possible to the embryo microenvironment during IVF., Methods: This was a multicentric study. A Siemens EPOC portable blood gas analyzer was used. The analytical validation was carried out under the culture medium (Global Total HSA®) conditions of use (microdroplets, under oil overlay, in a IVF incubator with (EmbryoScope®) or without a time lapse system (K system G210+®) and using IVF dishes. The validation included repeatability ("within-run" precision), total precision (between-day precision), trueness based on inter-laboratory comparison, inaccuracy based on external quality assessment and comparison to the reference technique. We also assessed the pre-analytical medium incubation time required to obtain a target value., Results: A measurement after an incubation period of 24 to 48 h is more representative of the pH to which the embryo will be exposed throughout the culture. The "within-run" and "between-day" precision show very low coefficients of variation (CV%): 0.17 to 0.22% and 0.13 to 0.34%, respectively, with IVF culture media. Trueness (% bias) range from - 0.07 to - 0.03%. We demonstrate good correlation between EPOC and reference pH electrode with an overestimation of 0.03 pH units of EPOC., Conclusion: Our method demonstrates good analytical performance for IVF laboratories wishing to implement a robust quality assurance system to monitor pH in embryo culture media. Compliance with stringent pre-analytical and analytical conditions is essential., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The aim of this study was to investigate the use of time-lapse morphokinetic parameters and cumulus cells transcriptomic profile to achieve a more accurate and non-invasive method in embryo evaluation. Two hundred embryos from 20 couples were evaluated based on morphokinetic characteristics using time-lapse. Embryos were divided into the high-quality, moderate-quality, and bad-quality groups. Non-fertilized oocytes were considered as the fourth group. T5 (time to five cells), S2 (time from three to four cells), and CC2 (time from two to three cells) were recorded. Also, the cumulus cells of the respective oocytes were divided into high-quality, moderate-quality, bad-quality, and non-fertilized groups based on the grading of the embryos. Then their transcriptomic profiles were analyzed by RNA-sequencing. Finally, the correlation between differentially expressed genes and embryo time-lapse parameters was investigated. T5 was the only timing that showed a statistically significant difference between high-quality group and other groups. RNA-sequencing results showed that 37 genes were downregulated and 106 genes were upregulated in moderate, bad-quality, and non-fertilized groups compared to high-quality group (q value < 0.05). These genes were involved in the main biological processes such as cell cycle, DNA repair, cell signaling and communication, transcription, and cell metabolism. Embryos graded in different groups showed different transcriptomic profiles in the related cumulus cells. Therefore, it seems that embryo selection using the combination of cytokinetics and cumulus cells gene expression can improve the accuracy of the embryo selection and pregnancy rate in ART clinics., (© 2021. Society for Reproductive Investigation.)

Purpose: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes., Method(s): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um 2 ), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement., Results: Male zygotes had longer time intervals of tPB2&#95;tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um 2 , p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um 2 , p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023)., Conclusion(s): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: The few studies that examined the effect of male and/or female features on early embryo development, notably using the time-lapse system (TL), reported conflicting results. This can be explained by the small number of studies using an adapted model., Methods: We used two original designs to study the female and male effects on embryo development: (1) based on embryos from donor oocytes (TL-DO), and (2) from donor sperm (TL-DS). Firstly, we analyzed the female and male similarities using an ad hoc intraclass correlation coefficient (ICC), then we completed the analysis with a multivariable model to assess the association between both male and female factors, and early embryo kinetics. A total of 572 mature oocytes (TL-DO: 293; TL-DS: 279), fertilized by intracytoplasmic sperm injection (ICSI) and incubated in a TL (Embryoscope®) were included from March 2013 to April 2019; 429 fertilized oocytes (TL-DO: 212; TL-DS: 217) were assessed. The timings of the first 48 h have been analyzed., Results: The similarities in the timings thought to be related to the female component were significant: (ICC in both DO-DS designs respectively: tPB2: 9-18%; tPNa: 16-21%; tPNf: 40-26%; t2: 38-24%; t3: 15-20%; t4: 21-32%). Comparatively, those related to male were lower. Surprisingly after multivariable analyses, no intrinsic female factors were clearly identified. However, in TL-DO design, oligozoospermia was associated with a tendency to longer timings, notably for tPB2 (p = 0.026)., Conclusion: This study quantifies the role of the oocyte in the first embryo cleavages, but without identified specific female factors. However, it also highlights that sperm may have an early embryonic effect., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The purpose of this study was to determine if morphometric parameters that can be measured quantitatively using a time-lapse embryo incubator are associated with aneuploidy. Embryos cultured in a time-lapse incubator and assessed with preimplantation genetic testing for aneuploidy (PGT-A) were analyzed retrospectively. Morphokinetic analysis included timing of cell divisions. Quantitative morphometric measurements included the distance between the second and first polar body, zona pellucida thickness at the pronuclear stage and at the 2-cell stage, and blastomere area at the 2- and 4-cell stages. Symmetry at the 2-cell stage was determined by percent difference between blastomeres; symmetry at the 4-cell stage was the percent difference between the smallest and largest blastomeres. Maternal age, blastocyst grade and day of biopsy were recorded. Euploid embryo characteristics were compared to aneuploid embryos. A receiver operating characteristic (ROC) curve was used to evaluate cell symmetry as a predictor of aneuploidy. Embryos (n = 182) from 21 patients (age 22-43; median = 34) were analyzed. Of the 182 embryos, 45% were euploid. Euploid and aneuploid embryos had similar morphokinetics and morphometry across many measures. As expected, age and blastocyst grade were associated with embryo ploidy. It was notable that, additionally, symmetry at the 4-cell stage (27% vs 31%, p = 0.01) was also associated with embryo ploidy. The optimized cutoff from the ROC curve to predict aneuploidy was determined to be 21%. Embryos with > 21% asymmetry at the 4-cell stage had high rates of aneuploidy while morphokinetic parameters were similar. In conclusion, this suggests that embryo selection models using time-lapse parameters would improve if they incorporate cleavage-stage morphometrics., (© 2021. Society for Reproductive Investigation.)

Objective: To assess the relationship between maternal body mass index (BMI) and embryo morphokinetics on time-lapse microscopy (TLM)., Design: Retrospective cohort study., Methods: All IVF cycles between June 2015 and April 2017 were reviewed. Female BMI prior to egg retrieval was collected through chart review. BMI (kg/m 2 ) classification included underweight (< 18.5), normal weight (18.5-25), overweight (25-30), and obese (≥ 30). Embryos' morphokinetic parameters were assessed with TLM and included time to syngamy, 2-cell, 3-cell, 4-cell, and 8-cell. A generalized linear mixed model was used to control for potential confounders and multiple embryos resulting from a single IVF cycle., Results: A total of 2150 embryos from 589 IVF cycles were reviewed and included in the analysis. Classification based on BMI was as follows: underweight (N = 56), normal weight (N = 1252), overweight (N = 502), and obese (N = 340). After adjusting for race and use of intracytoplasmic sperm injection, the mean time to the 8-cell stage in the underweight group was 4.3 (95% CI: - 8.31, - 0.21) h less than in the normal weight group (P = 0.025) and 4.6 (95% CI: - 8.8, - 0.21) h less than in the obese group (p = 0.022). No significant difference was noted between race and TLM after controlling for possible confounders., Conclusions: Embryos from underweight women were demonstrated to have a faster time to the 8-cell stage than normal weight or obese women. No significant difference was noted for race. This study demonstrates that weight can be a factor contributing to embryo development as observed with TLM., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Our retrospective study is to investigate an end-to-end deep learning model in identifying ploidy status through raw time-lapse video., Methods: By randomly dividing the dataset of time-lapse videos with known outcome of preimplantation genetic testing for aneuploidy (PGT-A), a deep learning model on raw videos was trained by the 80% dataset, and used to test the remaining 20%, by feeding time-lapse videos as input and the PGT-A prediction as output. The performance was measured by an average area under the curve (AUC) of the receiver operating characteristic curve., Result(s): With 690 sets of time-lapse video image, combined with PGT-A results, our deep learning model has achieved an AUC of 0.74 from the test dataset (138 videos), in discriminating between aneuploid embryos (group 1) and others (group 2, including euploid and mosaic embryos)., Conclusion: Our model demonstrated a proof of concept and potential in recognizing the ploidy status of tested embryos. A larger scale and further optimization on the exclusion criteria would be included in our future investigation, as well as prospective approach., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In vitro fertilization has been regarded as a forefront solution in treating infertility for over four decades, yet its effectiveness has remained relatively low. This could be attributed to the lack of advancements for the method of observing and selecting the most viable embryos for implantation. The conventional morphological assessment of embryos exhibits inevitable drawbacks which include time- and effort-consuming, and imminent risks of bias associated with subjective assessments performed by individual embryologists. A combination of these disadvantages, undeterred by the introduction of the time-lapse incubator technology, has been considered as a prominent contributor to the less preferable success rate of IVF cycles. Nonetheless, a recent surge of AI-based solutions for tasks automation in IVF has been observed. An AI-powered assistant could improve the efficiency of performing certain tasks in addition to offering accurate algorithms that behave as baselines to minimize the subjectivity of the decision-making process. Through a comprehensive review, we have discovered multiple approaches of implementing deep learning technology, each with varying degrees of success, for constructing the automated systems in IVF which could evaluate and even annotate the developmental stages of an embryo., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To study the relationship between the migration speed of nucleolus precursor bodies (NPBs) in male and female pronuclei (mPN; fPN) and human embryo development during assisted reproduction., Methods: The migration speed of 263 NPBs from 47 zygotes was quantitated, and embryonic development was observed until the blastocyst stage. The central coordinates of mPN, fPN, and NPBs were noted at multiple timepoints. Then, the distance traveled by the NPBs between two sequential images was measured, and migration speed was calculated. Additionally, we investigated the relationship between NPB migration speed and ploidy status (N = 33) or live birth/ongoing pregnancy (LB/OP) (N = 60) after assisted reproduction., Results: The NPB migration speed in both mPN and fPN was significantly faster in the zygotes that developed into blastocysts (N = 25) than that in the zygotes that arrested (N = 22). The timing of blastulation was negatively correlated with NPB migration speed in the mPN. Faster NPB migration was significantly correlated with LB/OP. In multivariate logistic analysis, NPB migration speed in the mPN was the only morphokinetic parameter associated with LB/OP. In a receiver-operating characteristic curve analysis of LB/OP by the NPB migration speed in the mPN, the cut-off value was 4.56 μm/h. When this cut-off value was applied to blastocysts with preimplantation genetic testing for aneuploidy, 100% of the blastocysts faster than or equal to the cut-off value were euploid., Conclusion: The NPBs migrated faster in zygotes having the potential to develop into a blastocyst, and eventually into a baby. This predictor could be an attractive marker for non-invasive embryo selection., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: While several studies reported the association between morphokinetic parameters and implantation, few predictive models were developed to predict implantation after day 5 embryo transfer, generally without external validation. The objective of this study was to evaluate the respective performance of 2 commercially available morphokinetic-based models (KIDScore™ Day 5 versions 1 and 2) for the prediction of implantation and live birth after day 5 single blastocyst transfer., Methods: This monocentric retrospective study was conducted on 210 ICSI cycles with single day 5 embryo transfer performed with a time-lapse imaging (TLI) system between 2013 and 2016. The association between both KIDScore™ and the observed implantation and live birth rates was calculated, as well as the agreement between embryologist's choice for transfer and embryo ranking by the models., Results: Implantation and live birth rate were both 35.7%. A significant positive correlation was found between both models and implantation rate (r = 0.96 and r = 0.90, p = 0.01) respectively. Both models had statistically significant but limited predictive power for implantation (AUC 0.60). There was a fair agreement between the embryologists' choice and both models (78% and 61% respectively), with minor differences in case of discrepancies., Conclusions: KIDScore™ Day 5 predictive models are significantly associated with implantation rates after day 5 single blastocyst transfer. However, their predictive performance remains perfectible. The use of these predictive models holds promises as decision-making tools to help the embryologist select the best embryo, ultimately facilitating the implementation of SET policy. However, embryologists' expertise remains absolutely necessary to make the final decision.

Purpose: There is clinical evidence that early cleavage timing parameters predictive of blastocyst development also correlate with embryo implantation potential. The aim of this study is to determine the developmental competency of embryos with delayed blastulation., Methods: Retrospective study performed from 2015 to 2016 at the Division of Reproductive Endocrinology and Infertility at Northwestern University., Results: A total of 2,292 embryos from 524 patients were included. Day 6 blastocysts had statistically significant longer times for every time point analyzed than day 5 blastocysts (p < 0.001). We found no statistically significant difference in euploidy rates between day 5 (44%) and day 6 (41%) embryos (p = 0.573). t7 and t8 time points were independent predictors of euploidy after controlling for day of biopsy (p < 0.015 and p < 0.014, respectively). Intrauterine pregnancy (IUP) and live birth (LB) were less likely to occur after transferring day 6 embryos (p = 0.0033 and p = 0.0359) without previous genetic testing. However, in embryos that undergo preimplantation genetic testing for aneuploidy (PGT-A), there were no significant differences in IUP or LB rates., Conclusion: Early time-lapse points can be used to predict embryo development. Day of blastulation may be an independent predictor IUP, with day 6 blastocysts having lower pregnancy and live birth rates. Our data suggests that day 5 and day 6 PGT-A tested embryos show similar rates of euploidy, suggesting that differences in PR seen in the non-PGT-A tested group may be caused by factors other than aneuploidy. Genetic testing technologies in combination with time-lapse microscopy may provide further information to improve IVF outcomes.

Purpose: Although the clinical value of time-lapse imaging (TLI) systems in in vitro fertilization (IVF) cycles is still debated, its prevalence worldwide seems to be expanding. The situation of TLI in the USA has been recently surveyed, but these results might not be transposable to other countries with different IVF regulation and funding such as France. This study evaluated the TLI situation in French IVF laboratories., Methods: An anonymous online cross-sectional survey was sent by email to 210 embryologists in September and October 2017. Laboratories, demographics, TLI clinical use, purchasing plan, and embryologists' opinions were analyzed using logistic regression to calculate odds ratio., Results: Of the 210 lab directors surveyed, 78 responded (37.1%), 43 (55%) working in private IVF laboratories and 35 (45%) in public hospitals. Thirty (38.5%) were TLI users. The odds of TLI possession were not statistically different according to laboratory sector or size. Most embryologists (n = 21, 70%) used TLI for unselected patients. Cost was the main reason given by non-users for not implementing TLI (n = 24, 50%). Most respondents were convinced that TLI is superior to standard morphology (n = 52, 73.2%) and that TLI improves culture conditions (n = 62, 84.9%). However, half (n = 39, 54.9%) indicated that evidence was still lacking to assert TLI clinical usefulness., Conclusion: The prevalence of TLI systems and embryologists' opinion in France was slightly different from the American situation. The different regulation and funding policy might account for some differences in terms of TLI use and perception.

Introduction: Time-lapse technology allows almost continuous noninvasive assessment of embryonic development. It was shown previously that relative kinetics defining cleavage synchronicity are better predictors of blastocyst quality than absolute time points. This study aims to compare relative kinetics in embryos from patients with and without endometriosis., Methods: Time-lapse data were collected retrospectively from 596 patients undergoing infertility treatment for in vitro fertilization from January 2011 to July 2016. Four hundred twenty-eight patients with confounding comorbidities (ie, polycystic ovary syndrome, pathological spermiogram in the included cycle, numerical/structural genetic abnormalities, preimplantation genetic screening performed) or incomplete data sets were excluded. Of the 168 included patients, 72 (42.9%) had endometriosis. Indications for in vitro fertilization of controls were tubal factor, unexplained infertility, or prolonged infertility. Relative kinetics were calculated as defined previously: cleavage synchronicity (CS)2-8=((t3-t2) + (t5-t4))/(t8-t2), CS4-8=(t8-t5)/(t8-t4), CS2-4=(t4-t3)/(t4-t2), DNA replication time ratio (DR)=(t3-t2)/(t5-t3). In women with more than one embryo, the median time was analyzed., Results: Median age, body mass index, smoking status, and AMH levels were similar in both groups. Embryos from patients with endometriosis showed poorer relative kinetics. The relative time CS2-8 was decreased in embryos from patients with endometriosis (0.7 [0.0-0.93] vs 0.8 [0.0-0.94], P < .05) and CS4-8 was increased (0.4 [0.0-1.0] vs 0.3 [0.0-1.0], P < .05). The less powerful diagnostic relative kinetic parameters (CS2-4 and DR) were not significantly different., Conclusions: Embryos from patients with endometriosis presented with altered relative kinetics suggesting poorer embryo quality. These findings support recently published data demonstrating reduced oocyte quality in patients with endometriosis which is one possible explanation for their poor response to fertility treatment.

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF's blood vessel forming potential prior to clinical implantation.

Purpose: Several time-lapse imaging (TLI) systems for non-invasive continuous monitoring of developing embryos are currently available. The present study explored the prevalence, means of acquisition, and clinical application of TLI systems in USA in vitro fertilization (IVF) laboratories., Methods: An online cross-sectional survey of 294 USA IVF laboratory directors was conducted in February and March 2016. Those directing more than one laboratory were asked to complete the survey for their home program and for their smallest laboratory by number of IVF/intracytoplasmic sperm injection (ICSI) cycle starts. Use of TLI was analyzed using logistic regression to calculate odds ratios (OR)., Results: Of 294 directors surveyed, 162 (55%) reported data on 204 laboratories. Thirty-five laboratories (17%) possessed at least one TLI system (median 2, interquartile range 1-4, total range 1-11). The more oocyte retrievals a laboratory performed annually, the more likely the laboratory was to possess a TLI system. Fifteen laboratories (43%) purchased their own systems, while others leased, loaned, or received donated systems. Twenty-five laboratories (71%) reported using TLI for embryo selection; all used TLI always, or usually, in combination with standard morphology evaluation. Twenty laboratories (80%) offered TLI to all patients. Some laboratories charged patients for TLI. Directors with TLI systems were more inclined to believe that TLI has value for embryo selection in clinical IVF., Conclusions: TLI system possession in USA IVF laboratories is low, although positively associated with the number of retrievals performed and with directors' opinions on the technology's utility. Over 70% of laboratories with TLI systems use them clinically, and less than half purchased their systems.

Purpose: This study aimed to test whether there is an association between embryo morphokinetic parameters and ploidy status., Methods: Patients with high risk of aneuploidy were analyzed by time-lapse microscopy combined with preimplantation genetic screening (PGS). Accordingly, 256 blastocysts from 75 patients were subjected to trophectoderm biopsy and microarray comparative genomic hybridization (array-CGH). Blastocyst development process was analyzed using time-lapse images., Results: Morphokinetic parameters: tPNf, t2, t3, t4, t5, t8, t9, tcom, tM, tSB, tB, tEB, CC1, CC2, CC3, S2, S3, t5-t2, and tB-tSB showed no significant difference in euploid embryos compared to aneuploid counterparts. In addition, two risk models based on previously published morphokinetic parameters failed to segregate euploid from aneuploid embryos., Conclusions: Morphokinetic parameters subjected to investigation in the present study failed to improve the chance of selecting euploid embryos.

Purpose: Recently, we established a noninvasive system for selecting human blastocysts with a high pre-transfer implantation potential based on first and second division patterns. The present study was carried out to improve the selection system., Methods: Embryos that completed first and second divisions within 25.90 and 37.88 h after culture, respectively, were selected using a time-lapse incubator. We examined the effects of compaction and blastocyst formation times on pregnancy rates after transferring these embryos at the blastocyst stage., Results: The completion of compaction and blastocyst formation times (79.93 and 97.47 h after culture, respectively) of embryos resulting in pregnancies after transfer were significantly (P < 0.01) shorter than those (86.46 and 100.34 h after culture, respectively) of embryos that failed to induce pregnancies. Embryo selection based on completion of compaction time improved pregnancy rates (40.9 vs. 74.6%, P < 0.01)., Conclusions: Of the embryos that formed two cells during the first division within 25.90 h after culture and four cells during the second division within 37.88 h after culture, those that completed compaction within 79.93 h after culture before reaching the blastocyst stage had a high implantation potential.

Purpose: The objective of this work was to determine which embryonic morphokinetic parameters up to D3 of in vitro development have predictive value for implantation for the selection of embryos for transfer in clinical practice based upon information generated from embryo transfers with known implantation data (KID)., Methods: A total of 800 KID embryos (100% implantation rate (IR) per transfer and 0% IR per transfer) cultured in an incubator with Time-Lapse system were retrospectively analysed. Of them, 140 embryos implanted, whereas 660 did not., Results: The analysis of morphokinetic parameters, together with the embryo morphology assessment on D3, enabled us to develop a hierarchical model that places the classical morphological score, the t4 and t8 morphokinetic values, as the variables with the best prognosis of implantation., Conclusion: In our decision tree, the classical morphological score is the most predictive parameter. Among embryos with better morphological scores, morphokinetics permits deselection of embryos with the lowest implantation potential.

Background: The ability of tumor cells to invade and metastasize is relevant to the process of cancer progression and, as such, it represents an obstacle to cancer cure. So far, limited information is available on interactions between circulating tumor cells and blood cells. It is well-documented that galectin-4 is upregulated in many types of tumor cells and is involved in metastasis. Here, we address the hypothesis that tumor cells may interact with red blood cells (RBCs) via galectin-4., Methods: High galectin-4 expressing colon, normal pancreatic and pancreatic cancer-derived cell lines (n = 5) were incubated with peripheral blood cells from different donors. Their interactions and associated proteins were examined by immunostaining and live cell imaging., Results: We found that (endogenous or exogenous) galectin-4 expressing tumor cells interact directly with RBCs. We also observed an accumulation of galectin-4 and human blood group antigens at the contact sites between these cells. By comparing the number of RBCs attaching to each tumor cell, we found that cells with high pre-incubation expression levels of galectin-4 attached significantly more RBCs than those with low expression levels (p < 1 × 10 -7 ). Conversely, we found that RBC attachment induces galectin-4 expression in tumor cells., Conclusions: From our data we conclude that tumor cells directly interact with red blood cells via galectin-4.

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.

Purpose: This study investigated the prevalence of abnormally cleaved embryos and determined which types of abnormally cleaved embryos (1-3c, 2-4c, 3-5c, 4-6c), might be suitable for transfer based on live birth data., Methods: One hundred seventy-one women (whose transferred embryos were confirmed to be either fully implanted or fully unimplanted) provided 1256 embryos, which were analyzed., Results: Of these embryos, 320 embryos were transferred, of these transferred embryos, 291 embryos were normal and 29 embryos were abnormal, which five embryos were not analyzed because each one was presented one abnormal cleavage type. These 24 embryos were divided into four groups. Inclusion criteria were as follows: women under 37 years of age undergoing first fresh in vitro fertilization (IVF) treatment with a basal antral follicle count of 5-15, body mass index (BMI) of 18-25 kg/m(2), number of retrieved oocytes between 5 and 20, and tubal factors as the cause of infertility. Time-lapse imaging analysis software was used to compare temporal parameters of normal cleavage and abnormal cleavage groups (there were four abnormal groups, based on the prevalence of abnormal cleavage embryos). Cleavage times were analyzed before the abnormal cleavage occurred, and time intervals were analyzed after the abnormal cleavage based upon the types of abnormal cleavage. In addition, the time intervals of t4-t3 and t8-t5 were also analyzed; corresponding time parameters were measured in the normal group as well. Implantation rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate were also measured in the normally cleaved and abnormally cleaved embryos. The prevalence of abnormal cleavage was 15.92% (200/1256). T8-t5 was the most important parameter in the prediction of potential development (production of a live-born baby) of abnormally cleaving embryos., Conclusions: Abnormally cleaving embryos were able to produced live births with T8-t5 the best parameter to predict the developmental potential of abnormally cleaving embryos.

Purpose: The aim of this study was to create a model to predict the implantation of transferred embryos based on information contained in the morphokinetic parameters of time-lapse monitoring., Methods: An analysis of time-lapse recordings of 410 embryos transferred in 343 cycles of in vitro fertilization (IVF) treatment was performed. The study was conducted between June 2012 and November 2014. For each embryo, the following data were collected: the duration of time from the intracytoplasmic sperm injection (ICSI) procedure to further division for two, three, four, and five blastomeres, time intervals between successive divisions, and the level of fragmentation assessed in successive time-points. Principal component analysis (PCA) and logistic regression were used to create a predictive model., Results: Based on the results of principal component analysis and logistic regression analysis, a predictive equation was constructed. Statistically significant differences (p < 0.001) in the size of the created parameter between the implanted group (the median value: Me = -5.18 and quartiles: Q 1= -5.61; Q 3 = -4.79) and the non-implanted group (Me = -5.69, Q 1 = -6.34; Q 3 = -5.16) were found. A receiver operating characteristic (ROC) curve constructed for the considered model showed the good quality of this predictive equation. The area under the ROC curve was AUC = 0.70 with a 95% confidence interval (0.64, 0.75). The presented model has been validated on an independent data set, illustrating that the model is reliable and repeatable., Conclusions: Morphokinetic parameters contain information useful in the process of creating pregnancy prediction models. However, embryo quality is not the only factor responsible for implantation, and, thus, the power of prediction of the considered model is not as high as in models for blastocyst formation. Nevertheless, as illustrated by the results of this study, the application of advanced data-mining methods in reproductive medicine allows one to create more accurate and useful models.

Purpose: Various time-lapse studies have postulated embryo selection criteria based on early morphokinetic markers. However, late paternal effects are mostly not visible before embryonic genome activation. The primary objective of this retrospective study was to investigate whether those early morphokinetic algorithms investigated by time-lapse imaging are reliable enough to allow for the accurate selection of those embryos that develop into blastocysts, while of course taking into account the correlation with the type of injected spermatozoa., Methods: During a period of 18 months, a total of 461 MII oocytes from 43 couples with severe male factor infertility and previous "external" IVF failures after cleavage-stage embryo transfer (ET) were fertilized by intracytoplasmic morphologically selected sperm injection (IMSI). Thereof, 373 embryos were monitored in a time-lapse incubator until ET on day 5. Blastocyst outcome in combination with three previously postulated MKc (cc2: t3-t2, 5-12 h; t3, 35-40 h; t5, 48-56 h) and the morphology of the selected sperm were analyzed., Results: A significant increase in the rate of blastocysts (54.0 vs. 36.3 %; P < 0.01) and top blastocysts (25.3 vs. 10.8 %; P < 0.001) was observed in the group of those meeting all three morphokinetic criteria (MKc3). However, MKc3 were only met in 23.3 % of all embryos. Moreover, TBR was influenced by the type of injected spermatozoa. In both groups, TBR decreased dramatically (MKc3, 35.0 vs. 17.0 %; MKc < 3, 14.2 vs. 8.4 %) when class II/III sperm instead of class I were injected., Conclusion: Early morphokinetic parameters might give some predictive information but fail to serve as a feasible selective tool for the prediction of blastocyst development given the influence of the type of spermatozoa injected.

Purpose: The purpose of our study was to use time-lapse in order to evaluate the impact of sperm origin (fresh ejaculate or surgically retrieved) on embryo morphokinetic parameters and clinical outcome in intracytoplasmic sperm injection (ICSI) cycles., Methods: This retrospective monocentric study was conducted in 485 unselected couples undergoing 604 ICSI cycles with embryo culture in the Embryoscope®. Among them, 445 couples underwent ICSI cycle with fresh ejaculated sperm and 40 with surgically retrieved sperm (26 with testicular sperm and 14 with epididymal sperm). Embryo morphokinetic parameters and clinical cycle outcome were compared between fresh ejaculated sperm and surgically retrieved sperm. A subgroup analysis was also conducted between testicular and epididymal sperm ICSI cycles., Results: Clinical outcome was comparable between groups according to sperm origin. Although most early morphokinetic parameters were comparable between ejaculated and surgical sperm groups, a few parameters were significantly different between both groups, but with a considerable overlap in their distribution. Late cellular events occurred significantly later in the surgical sperm group than in the ejaculated sperm group., Conclusions: Morphokinetic analysis did not allow us to identify clinically relevant differences between fresh ejaculate and surgically retrieved sperm groups. Further studies are needed, especially concerning the relationship between sperm origin and late morphokinetic parameters, such as blastocyst development.

Purpose: The aims of this prospective study were to evaluate whether time-lapse parameters can aid in the prediction of day 5 embryo quality and also to assess their discriminatory capacity., Methods: In this prospective cohort study, we used time-lapse technology to record specific timings of key events for 380 day 5 blastocysts (originating from 108 patients). Generalized estimating equation regression models were used to evaluate the capacity of these markers to identify a top-quality blastocyst. Multivariable regression models were also constructed, aiming to identify the model with the highest capacity to predict a top-quality blastocyst. The discriminatory capacity of single predictors or composite models was assessed with the use of receiver operating characteristic (ROC) analyses., Results: Eight significant predictive parameters of a top-quality blastocyst were identified: s3, t6, t7, t8, tM, tSB, tB and tEB. A ROC analysis of the identified parameters found s3 (area under the curve--AUC 0.585, 95 % CI 0.534-0.635) to have the best individual discriminatory capacity to predict a top-quality blastocyst prior to embryo compaction. The parameter tEB (AUC 0.727, 95 % CI 0.675-0.775) was the best predictor regardless of embryo stage. A model containing s3, t8 and tEB showed a slightly increased discriminatory capacity for top-quality blastocyst prediction (AUC 0.748, 95 % CI 0.697-0.794)., Conclusions: The identified morphokinetic parameters and their cutoffs, albeit of limited clinical value, add to the increasing knowledge concerning the potential predictive markers of a top-quality blastocyst. Additional evidence is necessary before validated time-lapse parameters can be used for embryo selection in IVF laboratories.

Purpose: Polycystic ovarian syndrome (PCOS) is a common cause of female infertility. Factors other than anovulation, such as low embryo quality have been suggested to contribute to the infertility in these women. This 2-year retrospective study used timelapse technology to investigate the PCOS-influence on timing of development in the pre-implantation embryo (primary endpoint). The secondary outcome measure was live birth rates after elective single-embryo transfer., Methods: In total, 313 embryos from 43 PCOS women, and 1075 embryos from 174 non-PCOS women undergoing assisted reproduction were included. All embryos were monitored until day 6. Differences in embryo kinetics were tested in a covariance regression model to account for potential confounding variables: female age, BMI, fertilization method and male infertility., Results: Time to initiate compaction and reach the morula stage as well as the duration of the 4th cleavage division was significantly shorter in PCOS embryos compared with non-PCOS embryos. No other kinetic differences were found at any time-points annotated. The proportion of multi-nucleated cells at the 2-cell stage was significantly higher in PCOS embryos compared with non-PCOS embryos. The live birth rates were comparable between the two groups., Conclusion: The findings suggest that the causative factor for subfertility in PCOS is not related to timing of development in the pre-implantation embryo.

Purpose: The aim of this study was to undertake a critical appraisal of the available evidence for the use of time-lapse imaging for embryo selection in clinical IVF., Methods: A literature search in PubMed, Scopus, Cochrane Central, ClinicalTrials.gov, Current Controlled Trials, and WHO International Clinical Trials Registry Platform was performed to identify randomized controlled trials that investigated the effect of time-lapse embryo selection and/or the time-lapse incubation system on ongoing pregnancy rate. We then performed a systematic review and assessed the relative risks (RRs) and 95 % confidence intervals (CIs) for ongoing pregnancy rates and the risk of bias of the eligible studies., Results: We identified four eligible randomized studies, three of which investigated the effect of both time-lapse incubation system and selection on ongoing pregnancy rate; the pooled result revealed a benefit of this intervention (relative risk (RR) 1.20; 95 % CI 1.05-1.37). However, the evidence was judged to be of low quality due to study limitations; a beneficial effect was observed in only one study deemed to be at high risk of bias. The single study assessing the effect of only the time-lapse incubation system revealed a non-significant negative effect (RR 0.71; 95 % CI 0.49-1.03)., Conclusions: The findings from this systematic review of the current evidence do not support routine use of time-lapse technology in clinical IVF. We therefore believe that the use of time-lapse imaging for embryo selection should remain experimental and that couples should not be subject to a surcharge for having their embryos cultured in a time-lapse imaging system. Future studies evaluating this technology in well-designed trials should be performed.

Purpose: To compare the outcomes of embryos selected via time lapse monitoring (TLM) versus those selected with conventional methods of selection in subfertile women undergoing ICSI., Methods: The study population (239 women) was classified into two groups, based on the monitoring method used: Group 1 (TLM) and Group 2 (conventional monitoring). Groups were compared according to the clinical and ICSI cycle characteristics and reproductive outcomes, while transfers were performed at day 2 or 3. Subgroup analyses were performed, in women of both groups according to age and clinical parameters, and in embryos of Group 1 based on their cellular events., Results: There was a statistically significant difference between the two study groups with regard to the outcome parameters, favoring Group 1 and especially in women >40 years of age. No differences were found in subgroup analyses in participants of both groups, regarding the stimulation protocol used, number of the oocytes retrieved and type of subfertility, while in Group 1 the percentages of "in range" cellular events were higher in certain divisions in ages 35-40, non-smokers, and the GnRH-agonist group, and in embryos that resulted in pregnancy., Conclusion: Morphokinetic parameters of early embryo development via TLM are related to the characteristics of subfertile patients and associated with ICSI outcomes.

Purpose: The aim of the study was to create a predictive model of blastocyst development based on morphokinetic parameters of time-lapse embryoscope monitoring., Methods: Time-lapse recordings of 432 embryos (obtained from 77 patients), monitored in Embryoscope, were involved in the study. Patients underwent in vitro fertilization according to standard procedure between June 2012 and April 2013. A retrospective analysis of morphokinetic features, focused on duration of time from the Intracytoplasmic Sperm Injection (ICSI) procedure to consecutive embryo division for 2, 3, 4 and 5 blastomeres, as well as time intervals between each division, was conducted. All embryos were observed for 5 days., Results: Based on the distribution of analyzed morphokinetic parameters and number of embryos developed to blastocyst, a range denoting the possibility of an embryo reaching blastocyst stage was determined. According to the obtained results, univariate and multivariate logistic regression analyses were performed. Based on the times of division for two and five blastomeres and intervals between the second and third division, a multivariate predictive model was created. The predictive equation was constructed based on the parameters of logistic regression analysis (odds ratios). Statistically significant differences (p < 0.001) in the size of the prediction parameter between the group of embryos developed to blastocyst (the median value: Me = 9.95, and quartiles: Q1 = 7.59, Q3 = 12.30) and embryos that did not develop to the blastocyst stage (Me = 4.66, Q1 = 2.33, Q3 = 8.19) were found. A Receiver Operating Characteristic (ROC) curve was created for the constructed predictive model. The Area Under the Curve was AUC = 0.806 with a 95 % confidence interval (0.747, 0.864). The predictive model constructed in this study has been validated using an independent data set, which indicates that the model is reliable and repeatable., Conclusions: Time-lapse imaging presents a new diagnostic tool for parametric evaluation of embryo development, from the oocyte stage, through fertilization, up to the blastocyst stage. The assessment of morphokinetic parameters can help us to provide more accurate information about the reproductive potential of embryos. It allows for early selection of embryos with high reproductive potential and shortens embryo incubation.

Telocytes (TC) are cells with telopodes (Tp), very long prolongations (up to 100 μm) with an uneven caliber ( www.telocytes.com ). Factors determining the dynamics of cellular prolongations are still unknown, although previous studies showed telopode motility in TC cultures. We comparatively investigated, by time-lapse videomicroscopy, the dynamics of Tp of mouse heart TC seeded on collagen, fibronectin, and laminin. Under our experimental conditions, TC and fibroblasts (cell line L929) behaved differently in terms of adherence, spreading, and prolongation extension. Fibroblasts showed lower spreading on the matrix proteins used. The time needed for spreading was 2-4 h for TC, versus 8-10 h for fibroblasts. The values for final cell surface area after spreading were between 200 and 400 μm(2) for fibroblasts and 800-2,000 μm(2) for TC. TC showed a more than three times higher ability to spread on the tested matrix proteins. An extremely low capacity to extend prolongations with lengths shorter than cell bodies was noted for fibroblasts, while TC extended prolongations longer than the cell body length, with a moniliform appearance. The stronger adherence and spreading were noted for TC seeded on fibronectin, while the lowest were on laminin. Collagen determined an intermediate adherence and spreading for TC, but the highest dynamics in Tp extensions. In conclusion, TC behave differently than fibroblasts in terms of adherence, spreading, and cell prolongation extension when seeded on various matrix proteins in cell culture.

Purpose: Since many transferred, good morphology embryos fail to implant, technologies to identify embryos with high developmental potential would be beneficial. The Eeva™ (Early Embryo Viability Assessment) Test, a prognostic test based on automated detection and analysis of time-lapse imaging information, has been shown to benefit embryo selection specificity for a panel of three highly experienced embryologists (Conaghan et al., 2013). Here we examined if adjunctive use of Eeva Test results following morphological assessment would allow embryologists with diverse clinical backgrounds to consistently improve the selection of embryos with high developmental potential., Methods: Prospective, double-blinded multi-center study with 54 patients undergoing blastocyst transfer cycles consented to have embryos imaged using the Eeva System, which automatically measures key cell division timings and categorizes embryos into groups based on developmental potential. Five embryologists of diverse clinical practices, laboratory training, and geographical areas predicted blastocyst formation using day 3 morphology alone and day 3 morphology followed by Eeva Test results. Odds ratio (OR) and diagnostic performance measures were calculated by comparing prediction results to true blastocyst outcomes., Results: When Eeva Test results were used adjunctively to traditional morphology to help predict blastocyst formation among embryos graded good or fair on day 3, the OR was 2.57 (95 % CI=1.88-3.51). The OR using morphology alone was 1.68 (95 % CI=1.29-2.19). Adjunct use of the Eeva Test reduced the variability in prediction performance across all five embryologists: the variability was reduced from a range of 1.06 (OR=1.14 to 2.20) to a range of 0.45 (OR=2.33 to 2.78)., Conclusions: The Eeva Test, an automated, time-lapse enabled prognostic test, used adjunctively with morphology, is informative in helping embryologists with various levels of experience select embryos with high developmental potential.

Purpose: Morphological assessment of human blastocysts has been effective for selecting embryos with high potential. However, they often show repeated shrinkage and expansion toward their hatching. Here we assessed whether capturing morphological changes over time of vitrified-warmed blastocysts could lead to a better selection of viable embryos from shrunken blastocysts., Methods: The implantation rates of vitrified-warmed blastocysts that were shrunken or expanded (developing) at the time of loading for transfer were compared among 2,729 cycles that were subjected to single blastocyst transfer. Vitrified (107) and fresh blastocysts (17) were donated for the experimental study. To assess the relationship between morphology (expanded vs. shrunken) and the mitochondrial respiration of blastocysts, the oxygen consumption rate (OCR) was analyzed for 55 specimens using an uncoupler of oxidative phosphorylation. The remaining 69 blastocysts were used for recording morphological changes every 15 min for 48 h after warming., Results: Because there were no surplus embryos, 7 % of the vitrified-warmed blastocysts were shrunken and transferred. The shrunken embryos had sufficient implantation ability (40 %). The OCR of the shrunken embryos was significantly lower than that of their expanded counterparts. Upon exposure to the uncoupler, the OCR of some shrunken embryos increased to levels similar to the expanded specimens. Time-lapse images revealed some shrunken embryos which formed blastocoel by 5 h following warming exhibited developmental competence to the hatched stage., Conclusions: Data of the present study suggest a group of shrunken blastocysts contains many viable and clinically available embryos and time-lapse observation of vitrified-warmed blastocysts is a potential method to distinguish viable embryos from shrunken blastocysts.

Purpose: Embryo kinetics analysis is an emerging tool for selecting embryo(s) for transfer. The aim of the present study was to determine morphokinetic parameters easily usable in the laboratory and predictive of embryo development and, most importantly, of embryo competence in producing a clinical pregnancy after day 5 transfer., Methods: A retrospective time-lapse monitoring analysis of morphokinetic parameters for 72 fully implanted embryos (group A) were compared to 106 non-implanted embryos (group B), and to 66 embryos with arrested development from the same pool of group A. All the embryos were from 78 patients undergoing ICSI treatment and day 5 embryo transfers., Results: A day 3 embryo will develop into a viable blastocyst if the following ranges of morphokinetic parameters are met: t1 (between 18.4 h and 30.9 h post-ICSI), t2 (21.4-34.8 h), t4 (33.1-57.2 h), t7 (46.1-82.5 h), t8 (46.4-97.8 h), tC-tF (7.7-22.9 h) and s3 (0.7-30.8 h). On day 5 embryos with the highest probability to implant are those with a cc3 between 9.7 h and 21 h., Conclusions: Morphokinetic parameters are helpful to make appropriate decisions for the disposition of each embryo. It is recommended that each laboratory should determine its own ranges of in vitro development (IVD-MKP) and implantation-associated (IMP-MKP) morphokinetic parameters.

The accurate and automated measuring of durations of certain human embryo stages is important to assess embryo viability and predict its clinical outcomes in in vitro fertilization (IVF). In this work, we present a multi-level embryo stage classification method to identify the number of cells at every time point of a time-lapse microscopy video of early human embryo development. The proposed method employs a rich set of hand-crafted and automatically learned embryo features for classification and avoids explicit segmentation or tracking of individual embryo cells. It was quantitatively evaluated using a total of 389 human embryo videos, resulting in a 87.92% overall embryo stage classification accuracy.

This paper proposes a vision-based method for detecting apoptosis (programmed cell death), which is essential for non-perturbative monitoring of cell expansion. Our method targets non-adherent cells, which float or are suspended freely in the culture medium-in contrast to adherent cells, which are attached to a petri dish. The method first detects cell regions and tracks them over time, resulting in the construction of cell tracklets. For each of the tracklets, visual properties of the cell are then examined to know whether and when the tracklet shows a transition from a live cell to a dead cell, in order to determine the occurrence and timing of a cell death event. For the validation, a transductive learning framework is adopted to utilize unlabeled data in addition to labeled data. Our method achieved promising performance in the experiments with hematopoietic stem cell (HSC) populations, which are currently in clinical use for rescuing hematopoietic function during bone marrow transplants.

Objective: To compare the dynamics of early development between embryos cultured in single and sequential media., Design: Randomized, comparative study., Setting: Private IVF centre., Patients: A total of 446 metaphase II oocytes from 51 couples who underwent oocyte retrieval procedure for intracytoplasmic sperm injection. Forty-nine resulted in embryo transfer., Intervention: Oocytes were split between single and sequential media produced by the same manufacturer and cultured in a time-lapse incubator., Main Outcome Measures: Morphokinetic parameters until the embryos reached the 5-cell stage (t5), utilization, clinical pregnancy and implantation rates., Results: Embryos cultured in single media were advanced from the first mitosis cycle and reached 2- to 5-cell stages earlier. There was not any difference between the durations for cell cycle two (cc2 = t3-t2) and s2 (t4-t3). The utilization, clinical pregnancy and implantation rates did not differ between groups. The proportion of cryopreserved day 6 embryos to two pronuclei oocytes was significantly higher in sequential than in single media., Conclusions: Morphokinetics of embryo development vary between single and sequential culture media at least until the 5-cell stage. The overall clinical and embryological parameters remain similar regardless of the culture system.

Purpose: To analyze the fertilization process related to polyspermy block in human oocytes using an in vitro culturing system for time-lapse cinematography., Methods: We had 122 oocytes donated for this study from couples that provided informed consent. We recorded human oocytes at 2,000 to 2,800 frames every 10 s during the fertilization process and thereafter every 2 min using a new in vitro culture system originally developed by the authors for time-lapse cinematography. We displayed 30 frames per second for analysis of the polyspermy block during fertilization., Results: Three oocytes showed the leading and following sperm within the zona pellucida in the same microscopic field. The dynamic images obtained during the fertilization process using this new system revealed that once a leading sperm penetrated the zona pellucida and attached to the oocyte membrane, a following sperm was arrested from further penetration into the zona pellucida within 10 s., Conclusions: The present results strongly suggest the existence of a novel mechanism of polyspermy block that takes place at the zona pellucida immediately after fertilization. These findings are clearly different from previous mechanisms describing polyspermy block as the oocyte membrane block to sperm penetration and the zona reaction. The finding presented herein thus represents a novel discovery about the highly complicated polyspermy block mechanism occurring in human oocytes.

Ribosomal proteins and ribosomal associated proteins are complicated subjects to target and study because of their high conservation through evolution which led to highly structured and regulated proteins. Tagging of ribosomal proteins may allow following of protein synthesis in vivo and isolating translated mRNAs. HaloTag® is a new technology which allows detection in living cells, biochemical purification, and localization studies. In the present work, we tested HaloTag®-based ribosomal tagging. We focused on eIF6 (eukaryotic Initiation Factor 6 free 60S ribosomal marker), RACK1 (Receptor for Activated C Kinase 1; 40S and polysomes, not nuclear), and rpS9 (40S ribosomes, both in the nucleus and in the cytoplasm). Experiments performed on HEK293 cells included ribosomal profiles and Western blot on the fractions, purification of HaloTag® proteins, and fluorescence with time-lapse microscopy. We show that tagged proteins can be incorporated on ribosomes and followed by time-lapse microscopy. eIF6 properly accumulates in the nucleolus, and it is redistributed upon actinomycin D treatment. RACK1 shows a specific cytoplasmic localization, whereas rpS9 is both nucleolar and cytoplasmic. However, efficiency of purification varies due to steric hindrances. In addition, the level of overexpression and degradation may vary upon different constructs. In summary, HaloTag® technology is highly suitable to ribosome tagging, but requires prior characterization for each construct.

Tumor growth, invasiveness, and metastasis are dynamic processes involving cancer interactions with the extracellular matrix, the vasculature, and various types of non-cancerous host cells that form the tumor stroma. An often-present stromal component is the immune cells, such as tumor-associated myeloid and lymphocytic infiltrates, yet endogenous anti-tumor immune responses are typically ineffective in tumor rejection and may even contribute to the progression of some cancers. How exactly cancer cells interact with the stroma and invade healthy tissues while avoiding anti-tumor immune responses, and which interactions should be targeted for anti-tumor therapy, can now be studied by minimally invasive observation using multiphoton and other low impact confocal microscopy techniques and fluorescent animal tumor models. Intravital video microscopy has already been instrumental in defining the roles and modes of cellular motility in the angiogenic process and during tissue invasion at the tumor margin. In the hands of cancer immunologists, intravital video microscopy is beginning to unravel the complexity of effector and suppressory lymphocytic interactions in tumors and in the draining lymphoid organs. As the intravital microscopy approach is beginning to move beyond fundamental description and into analyzing the molecular underpinnings of cell's dynamics, future technical advances will undoubtedly provide yet deeper insight while stitching together a systems dynamics view of cancer-host interactions that will keep on inspiring cancer researchers and therapists.

During the last several years, live tissue imaging, in particular using two-photon laser microscopy, has advanced our understanding of leukocyte trafficking mechanisms. Studies using this technique are revealing distinct molecular requirements for leukocyte migration in different tissue environments. Also emerging from the studies are the ingenious infrastructures for leukocyte trafficking, which are produced by stromal cells. This review summarizes the recent imaging studies that provided novel mechanistic insights into in vivo leukocyte migration essential for immunosurveillance.