Spindle Imaging Research Articles

Ex ovo omnia-why don't we know more about egg quality via imaging?

Determining egg quality is the foremost challenge in assisted reproductive technology (ART). Although extensive advances have been made in multiple areas of ART over the last 40 years, oocyte quality assessment tools have not much evolved beyond standard morphological observation. The oocyte not only delivers half of the nuclear genetic material and all of the mitochondrial DNA to an embryo but also provides complete developmental support during embryonic growth. Oocyte mitochondrial numbers far exceed those of any somatic cell, yet little work has been done to evaluate the mitochondrial bioenergetics of an oocyte. Current standard oocyte assessment in in vitro fertilization (IVF) centers include the observation of oocytes and their surrounding cell complex (cumulus cells) via stereomicroscope or inverted microscope, which is largely primitive. Additional oocyte assessments include polar body grading and polarized light meiotic spindle imaging. However, the evidence regarding the aforementioned methods of oocyte quality assessment and IVF outcomes is contradictory and non-reproducible. High-resolution microscopy techniques have also been implemented in animal and human models with promising outcomes. The current era of oocyte imaging continues to evolve with discoveries in artificial intelligence models of oocyte morphology selection albeit at a slow rate. In this review, the past, current, and future oocyte imaging techniques will be examined with the goal of drawing attention to the gap which limits our ability to assess oocytes in real time. The implications of improved oocyte imaging techniques on patients undergoing IVF will be discussed as well as the need to develop point of care oocyte assessment testing in IVF labs.

CTCF is essential for proper mitotic spindle structure and anaphase segregation.

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E.

P-248 The meiotic spindle is preserved during freezing but post-thawing rehydration causes its destabilization in human eggs

Abstract Study question Is the human egg spindle integrity impaired by vitrification and thawing procedures? Summary answer Cryoprotective-caused dehydration stabilizes the MII spindle, but the re-entry of water during thawing induces depolymerization of microtubules, thus promoting instability of the division apparatus. What is known already Freezing sperm and preimplantation embryos have become routine IVF procedures with excellent clinical results, whereas cryopreservation of oocytes remains problematic. The major factor underlying the human oocytés notorious propensity to cryoinjury is the temperature sensitivity of the meiotic spindle, the fidelity of which is critical for faithful post-fertilization development. Most studies focus on evaluating vitrified-thawed oocytes' post-fertilization outcome, but behavior of the meiotic spindle during the freezing process has received only a little attention. Study design, size, duration The experimental study involved a total of 165 human oocytes donated for research. The presence and morphology of the meiotic spindle were examined by polarized light microscopy (PLM) and fluorescent confocal imaging to map out the MII spindle dynamics during the vitrification-thawing procedure. Participants/materials, setting, methods A total of 114 women gave informed consent for their surplus immature oocytes to be used in this project. Oocytes that completed maturation in vitro were subjected to spindle imaging (Oosight) and vitrified using a Kitazato/Cryotop open system. The PLM-positive/negative oocytes were fixed at 6 steps of the vitrification-thawing protocol and 2 hours after thawing (15 oocytes in each subgroup). Microtubules and DNA were fluorescently labeled to inspect chromosome-spindle configuration. Main results and the role of chance Time-course experiments showed that PLM-detectable bipolar spindle remained intact in all but one oocyte (44/45) fixed during pre-vitrification equilibration. Notably, the PLM signal became more prominent after adding a cryoprotectant that displaced intracellular water. In contrast, the MII spindle signal progressively disappeared during thawing. Specific tubulin labeling revealed that the microtubule mass was still present in most (41/45) oocytes from the vitrified-thawed sample group. However, the division apparatus tended to lose its bipolarity and disintegrate (8/45) during the washing steps. This trend was accented in the group of oocytes that lacked PLM-detectable spindles before freezing. Here, only a minority of cells (11/45) undergoing rehydration displayed characteristic chromosome-spindle configuration. The difference in eggś capability to ensure spindle bipolarity was apparent even 2 hours after thawing when 13/15 of initially PLM-positive but only 7/15 PLM-negative oocytes exhibited normal-shaped spindles. Based on these data, we hypothesize that the transient disappearance of the PLM spindle signal during warming may be attributed to the restoration of microtubule dynamics and spindle destabilization. The speed and efficiency of spindle reconstruction seem to depend on the overall egǵs fitness. Limitations, reasons for caution This research study involved a limited number of hormonally-primed oocytes that extruded a polar body in vitro shortly after retrieval. The obtained imaging data represent snapshots of the process. A technically challenging live imaging study is needed to complement the description of the oocyte spindle dynamics during post-vitrification recovery. Wider implications of the findings Our results demonstrate that meiotic spindles in frozen-thawed eggs are not products of de novo spindle assembly but are built on preexisting microtubule mass. Suboptimal handling and reducing the interval between thawing and ICSI may enhance unavoidable spindle instability and thus compromise the developmental potential of vitrified eggs. Trial registration number not-applicable

Value of Meiotic Spindle Imaging on Fertilization and Embryo Development Following Human Oocyte Piezo-ICSI

Background: The objective of this study was to investigate accurately the effect of the meiotic spindle imaging of human oocytes on their fertilization and embryo development by using the Piezo-ICSI.Methods: We retrospectively assessed 529 oocytes with the first polar body retrieved from 124 infertile couples (147 cycles) who attended our clinic between May 2016 and December 2018. Of these, 489 oocytes (92.4%) with visible meiotic spindle comprised the Spindle (+) group and 40 oocytes (7.6%) not observed meiotic spindle comprised the Spindle (−) group. The meiotic spindle was imaged using polarized light microscopy. The rates of fertilization, high-quality day-3 embryo, day-5 blastocyst, and high-quality day-5 blastocyst were evaluated in both groups.Results: The fertilization rates of Spindle (+) and Spindle (−) were 92.0% (450/489) and 70.0% (28/40), respectively; high-quality day-3 embryo rates were 62.9% (283/450) and 35.7% (10/28); day-5 blastocyst rates were 53.7% (205/382) and 32.1% (9/28); high-quality day-5 blastocyst rates were 29.8% (114/382) and 3.6% (1/28). Fertilization, high-quality day-3 embryo, day-5 blastocyst, and high-quality day-5 blastocyst rates were significantly higher in the Spindle (+) group than in the Spindle (−) group.Conclusion: Spindle imaging (i.e., oocytes with a visible meiotic spindle or with not observed meiotic spindle) influences the outcome of Piezo-ICSI in human oocytes, including fertilization and embryo development. To our knowledge, this study is the first to evaluate the effect of meiotic spindle imaging on fertilization or embryo development among oocytes after Piezo-ICSI. The meiotic spindle imaging could be the indicator for the quality management of medical doctors and embryologists.

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This, in turn, implies that rotational forces, i.e., torques, exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this work, we describe a simple method for measuring and evaluating the shapes of microtubule bundles by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allows us to trace the entire microtubule bundle. For each traced bundle, we first fit a plane and then fit a circle lying in that plane. With this robust method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.

The Xenopus spindle is as dense as the surrounding cytoplasm.

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function.

Live birth achieved despite the absence of ejaculated spermatozoa and mature oocytes retrieved: a case report

The most common reason for in vitro fertilization (IVF) cycle cancelation is a lack of quality gametes available for intracytoplasmic sperm injection (ICSI). Here we present the successful fertility treatment of the couple affected by obstructive azoospermia combined with suboptimal response to controlled ovarian stimulation. Since the conventional approach appeared ineffective to overcome both partnersˈ specific problems, the targeted interventions, namely, (1) pharmacological enhancement of sperm motility and (2) polarized light microscopy (PLM)-guided optimization of ICSI time, were applied to rescue the cycle with only immature oocytes and immotile testicular sperm retrieved. The treatment with theophylline aided the selection of viable spermatozoa derived from cryopreserved testicular tissue. When the traditional stimulation protocol failed to produce mature eggs, non-invasive spindle imaging was employed to adjust the sperm injection time to the maturational stage of oocytes extruding a polar body in vitro. The fertilization of 12 late-maturing oocytes yielded 5 zygotes, which all developed into blastocysts. One embryo was transferred into the uterus on day 5 post-fertilization, and another 3 good quality blastocysts were vitrified for later use. The pregnancy resulted in a full-term delivery of a healthy child. This case demonstrates that the individualization beyond the standard IVF protocols should be considered to maximize the chance of poor-prognosis patients to achieve pregnancy with their own gametes.

Oocyte Selection for In Vitro Embryo Production in Bovine Species: Noninvasive Approaches for New Challenges of Oocyte Competence.

Simple SummaryThe efficiency of producing embryos using in vitro technologies in cattle species remains lower when compared to mice, indicating that the proportion of female gametes that fail to develop after in vitro manipulation is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting embryo production, the precise identification of noninvasive markers that predict oocyte competence is of major interest. The aim of this review was to explore the current literature on different noninvasive markers associated with oocyte quality in the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, a large slightly expanded investment without dark areas, large oocyte diameter (>120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated with better embryonic development. In addition, the combination of oocyte and zygote selection, spindle imaging, and the anti-Stokes Raman scattering microscopy together with studies decoding molecular cues in oocyte maturation have the potential to further optimize the identification of oocytes with better developmental competence for in vitro technologies in livestock species.The efficiency of producing embryos using in vitro technologies in livestock species rarely exceeds the 30–40% threshold, indicating that the proportion of oocytes that fail to develop after in vitro fertilization and culture is considerably large. Considering that the intrinsic quality of the oocyte is one of the main factors affecting blastocyst yield, the precise identification of noninvasive cellular or molecular markers that predict oocyte competence is of major interest to research and practical applications. The aim of this review was to explore the current literature on different noninvasive markers associated with oocyte quality in the bovine model. Apart from some controversial findings, the presence of cycle-related structures in ovaries, a follicle size between 6 and 10 mm, large number of surrounding cumulus cells, slightly expanded investment without dark areas, large oocyte diameter (>120 microns), dark cytoplasm, and the presence of a round and smooth first polar body have been associated with better competence. In addition, the combination of oocyte and zygote selection via brilliant cresyl blue (BCB) test, spindle imaging, and the anti-Stokes Raman scattering microscopy together with studies decoding molecular cues in oocyte maturation have the potential to further optimize the identification of oocytes with better developmental competence for in-vitro-derived technologies in livestock species.

Fertilization and Cleavage Axes Differ In Primates Conceived By Conventional (IVF) Versus Intracytoplasmic Sperm Injection (ICSI)

With nearly ten million babies conceived globally, using assisted reproductive technologies, fundamental questions remain; e.g., How do the sperm and egg DNA unite? Does ICSI have consequences that IVF does not? Here, pronuclear and mitotic events in nonhuman primate zygotes leading to the establishment of polarity are investigated by multidimensional time-lapse video microscopy and immunocytochemistry. Multiplane videos after ICSI show atypical sperm head displacement beneath the oocyte cortex and eccentric para-tangential pronuclear alignment compared to IVF zygotes. Neither fertilization procedure generates incorporation cones. At first interphase, apposed pronuclei align obliquely to the animal-vegetal axis after ICSI, with asymmetric furrows assembling from the male pronucleus. Furrows form within 30° of the animal pole, but typically, not through the ICSI injection site. Membrane flow drives polar bodies and the ICSI site into the furrow. Mitotic spindle imaging suggests para-tangential pronuclear orientation, which initiates random spindle axes and minimal spindle:cortex interactions. Parthenogenetic pronuclei drift centripetally and assemble astral spindles lacking cortical interactions, leading to random furrows through the animal pole. Conversely, androgenotes display cortex-only pronuclear interactions mimicking ICSI. First cleavage axis determination in primates involves dynamic cortex-microtubule interactions among male pronuclei, centrosomal microtubules, and the animal pole, but not the ICSI site.

Effects of meiotic spindle imaging in human oocytes following PIEZO-ICSI on oocyte fertilization and embryo development

Effects of meiotic spindle imaging in human oocytes following PIEZO-ICSI on oocyte fertilization and embryo development

Impact of meiotic spindle imaging on fertilization, embryo development, clinical outcome and morphokinetic parameters: an analysis of 415 in-vivo matured and 317 in-vitro matured human oocyte siblings

To evaluate the relationship between meiotic spindle imaging of in-vivo and in-vitro matured human oocytes and intracytoplasmic sperm injection (ICSI) outcomes. This study was a retrospective observational study conducted at Kyono ART Clinic in Japan from September 2012 to January 2019. This study included a total of 259 ICSI cycles in which were retrieved six or fewer mature oocytes and at least one immature oocyte. ICSI was performed on matured oocytes immediately after denudation. After denudation, MⅠ oocytes were cultured for 4 hours to allow oocyte maturation. We categorized each sibling MII oocyte into an in-vivo matured oocyte group (n=415 oocytes) and an in-vitro matured oocyte group (n=317 oocytes). Both groups, the oocytes’ meiotic spindles were visualized with a Polscope before ICSI. We compared fertilization rate, embryo development and clinical pregnancy rate between the two groups with or without a spindle. Furthermore, 196 embryos (96 in-vivo matured oocytes with spindle, and 7 without spindle; 39 in-vitro matured oocytes withspindle, and 44 without spindle) were analyzed for morphokinetic parameters and incidence of direct unequal cleavage (DUC) by time-lapse imaging (TLS, EmbryoScope+TM). Statistical comparisons between the experimental groups were performed through Fisher’s exact test. Statistical difference was considered to be significant at P<0.05. The mean patient age was 39.0±4.1years (range: 25-45years). The spindle was detected in 85.3% (325/381) and 22.1% (70/317) of the in-vivo and in-vitro matured oocytes, respectively. In both groups, fertilization, blastocyst formation, and good-quality blastocyst rates were significantly higher when spindles were detected (Table). When the spindle was detected, there were no significant differences in fertilization rate or embryo development competence between in-vivo matured and in-vitro matured oocytes. Also, there was no significant difference in clinical pregnancy rate in each group (Table). In the morphokinetic parameters analysis, there were no significant differences in time points of cell division (tPNf to t8), interval of cell cleavage (CC2 and S2), or incidence of DUC. Meiotic spindle imaging may be useful for prediction in both in vitro and in vitro-matured oocyte development. When meiotic spindle is detected in matured oocytes, developmental competence may not be influenced by whether maturation occurs in vivo or in vitro.TableFertilization rate, embryo development and clinical outcomesMeiotic spindle in in-vivo matured oocytesMeiotic spindle in in-vitro matured oocytesDetectedNot detectedDetectedNot detectedNo. of oocytes3255670247Fertilization rate73.2%* (273/373)45.2% (19/42)69.4% * (50/72)49.4% (121/245)Blastocyst rate45.6% * (93/204)28.6% (4/14)34.3% * (12/35)17.7% (17/96)Good quality blastocyst20.1% * (41/204)0.0% (0/14)8.6% * (3/35)0.0% (0/96)No. of vitrified oocytes8331214No. of transferred oocytes6371021Clinical pregnancy rate (fresh and vitrified)28.2% (29/103)25.0% (2/8)16.7% (3/18)8.0% (2/25)*P<0.05 Open table in a new tab

Human Egg Maturity Assessment and Its Clinical Application.

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg's developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg's readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte's developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

Egg maturity assessment prior to ICSI prevents premature fertilization of late-maturing oocytes

ProposeThe presence of metaphase II (MII) spindle together with the polar body (PB) indicates completion of oocyte maturation. This study was designed to explore if spindle imaging can be used to optimize timing of intracytoplasmic sperm injection (ICSI).MethodsThe study involved 916 oocytes from 234 conventionally stimulated ICSI cycles with an unexpectedly poor ovarian response. All PB-displaying oocytes were subjected to polarized light microscopy (PLM) prior to ICSI. When MII spindle was absent in the majority of oocytes, ICSI was postponed and performed after additional spindle imaging. Fertilization, embryo development, and clinical outcome were evaluated with respect to the observed spindle pattern.ResultsThe visible spindle was absent in 32.64% of PB-displaying oocytes. The late-maturing oocytes extruding PB in vitro were less likely to exhibit a spindle signal than in vivo matured MII oocytes (38.86% vs. 89.84%). When fertilization was postponed, 59.39% of initially spindle-negative oocytes developed detectable MII spindle. Spindled eggs had significantly higher developmental potential, and the presence of the spindle has been identified as an independent measure for predicting the formation of the blastocyst. Embryos derived from spindle-positive oocytes also showed a higher chance to implant and develop to term. Notably, 11 children were conceived by finely timed fertilization of late-maturing oocytes which are normally discarded.ConclusionsThe study confirms the prognostic value of spindle imaging and demonstrates that immature oocytes can be clinically utilized and give rise to live births when the timing of ICSI is adjusted to their developmental stage.

Automated classification and characterization of the mitotic spindle following knockdown of a mitosis-related protein

BackgroundCell division (mitosis) results in the equal segregation of chromosomes between two daughter cells. The mitotic spindle plays a pivotal role in chromosome alignment and segregation during metaphase and anaphase. Structural or functional errors of this spindle can cause aneuploidy, a hallmark of many cancers. To investigate if a given protein associates with the mitotic spindle and regulates its assembly, stability, or function, fluorescence microscopy can be performed to determine if disruption of that protein induces phenotypes indicative of spindle dysfunction. Importantly, functional disruption of proteins with specific roles during mitosis can lead to cancer cell death by inducing mitotic insult. However, there is a lack of automated computational tools to detect and quantify the effects of such disruption on spindle integrity.ResultsWe developed the image analysis software tool MatQuantify, which detects both large-scale and subtle structural changes in the spindle or DNA and can be used to statistically compare the effects of different treatments. MatQuantify can quantify various physical properties extracted from fluorescence microscopy images, such as area, lengths of various components, perimeter, eccentricity, fractal dimension, satellite objects and orientation. It can also measure textual properties including entropy, intensities and the standard deviation of intensities. Using MatQuantify, we studied the effect of knocking down the protein clathrin heavy chain (CHC) on the mitotic spindle. We analysed 217 microscopy images of untreated metaphase cells, 172 images of metaphase cells transfected with small interfering RNAs targeting the luciferase gene (as a negative control), and 230 images of metaphase cells depleted of CHC. Using the quantified data, we trained 23 supervised machine learning classification algorithms. The Support Vector Machine learning algorithm was the most accurate method (accuracy: 85.1%; area under the curve: 0.92) for classifying a spindle image. The Kruskal-Wallis and Tukey-Kramer tests demonstrated that solidity, compactness, eccentricity, extent, mean intensity and number of satellite objects (multipolar spindles) significantly differed between CHC-depleted cells and untreated/luciferase-knockdown cells.ConclusionMatQuantify enables automated quantitative analysis of images of mitotic spindles. Using this tool, researchers can unambiguously test if disruption of a protein-of-interest changes metaphase spindle maintenance and thereby affects mitosis.

Combined classical spindle cell/pleomorphic lipoma spectrum imaging and clinical data.

Compile the largest study to date on the imaging and clinical features of the classic spindle cell/pleomorphic lipoma spectrum and suggest this diagnosis be included in the differential for benign and malignant macroscopic fat-containing soft tissue masses regardless of the mass location or patient demographics. An institutional search was performed to identify all available classic-type spindle cell/pleomorphic lipomas with available demographic and imaging data. Images and reports were analyzed by one MSK-trained radiologist and radiographic, anatomic and clinical data were recorded. Additionally, a literature search was performed to identify studies describing the spindle cell lipoma spectrum imaging features and were combined with institutional data. Forty-two institutional cases were identified, 37 of which had MRIs performed among which 21 had images available (T1- and T2-weighted pulse sequences) for review while the remainder had outside reports detailing the mass imaging features. There was a mean age of 57 with 79% of cases occurring in males. Contrary to prior reports, 57% of masses were subcutaneous, and the neck and back region accounted for 26% of cases. When the institutional cases were combined with available data in the literature, there was a new sample size of 91 masses, 74 of which had MRI and/or CT data. Eighty-seven percent of masses were heterogeneous, 51% were composed of less than 75% fat, 65% were in the back, neck or shoulder region, 27% of masses were deep and 91% demonstrated enhancement. Eighty-two percent of patients were males with a mean age of 58 at excision. Imaging features, patient demographics and tumor location alone are not enough to differentiate tumors of the spindle cell lipoma spectrum from other macroscopic fat-containing benign and malignant tumors, and these entities should be included in the same imaging differential diagnosis.

Polarization microscopy imaging for the identification of unfertilized oocytes after short-term insemination

To develop a unique approach using polarization microscopy (PM) to determine whether the presence of a spindle can be used as an indicator associated with fertilization failure 5hours after short-term insemination. Observational study. Assisted reproduction center. Eighty-five patients undergoing short-term insemination. Oocytes imaged via PM at 4, 5, and 6hours after standard insemination. Spindle visualization and fertilization rate, with rescue intracytoplasmic sperm injection (ICSI) results determined by rates of normal fertilization, abnormal fertilization, and good-quality embryo formation. After standard insemination, comparisons of spindle visualization at three time points indicated that the predictive accuracy rates were 84.30% at 5hours, 86.80% at 6hours, and 62.20% at 4hours, with the rates at 5 and 6hours statistically significantly higher than at 4hours. A spindle was present in 242 of the 788 metaphase-II oocytes 5hours after insemination, and there were 204 failed fertilizations on day 1. The positive predictive value was 0.84. After rescue ICSI, the abnormal fertilization rate of the polar body group (assessed using the polar body visualization method) was statistically significantly higher than that of the PM group (assessed using the spindle visualization method) and the regular ICSI group (9.37%, 5.88%, and 4.87%, respectively). The presence of a spindle 5hours after insemination in invitro fertilization is an accurate indicator of unfertilized oocytes. Spindle imaging combined with rescue measures effectively prevents fertilization failure and decreases the polyspermy rate.

Predictive value of spindle retardance in embryo implantation rate.

We evaluated the relationship between meiotic spindle characteristics and in vitro fertilization cycle outcome. Five hundred sixty-nine oocytes from 86 in vitro fertilization cycles were analyzed for fertilization and subsequent implantation rates. Oocytes were assessed for maturation status. The oocytes and embryos were cultured in sequential and nonsequential media (G Series, Vitrolife, Sweden) and incubated in 6% CO2, 5% O2 at 37°C. Two hours following oocyte decumulation (38-39h post-hCG/GnRH administration) and prior to microinjection, the structure of the meiotic spindle was assessed using the Oosight Imaging System (CRI, UK). Four hundred fifty-six oocytes (80.5%) had a visible meiotic spindle, 82 (14.7%) had no meiotic spindle, and 31 (5.5%) were in telophase I. Oocytes exhibiting a meiotic spindle had a significantly higher fertilization rate and a lower rate of abnormal fertilization. Implantation data were obtained for 195 of the embryos transferred. The implantation rate for embryos derived from oocytes with a meiotic spindle was 32.9%, while in embryos originating from oocytes without a meiotic spindle and oocytes in telophase, this value dropped significantly (8.8 and 0%, respectively). To determine the correlation between retardance values and implantation rate for each oocyte, we established four groups, finding a range of retardance values with significantly higher implantation rates (27.5, 21, 29.3, and 53.8%, respectively). Meiotic spindle imaging may be a valuable tool for prediction of oocyte quality, and retardance values of meiotic spindles, together with classical morphological classification, can be useful to select embryos with a higher implantation potential.

Relationship between meiotic spindles visualization and intracytoplasmic sperm injection outcomes in human oocytes.

In assisted reproductive techniques, the operator attempts to select morphologically best embryos to predict embryo viability. Development of polarized light microscope, which evaluates the oocytes' spindles according to birefringence of living cells, had been helpful in oocyte selection. The aim of this study is evaluating the relationship between meiotic spindles visualization and intracytoplasmic sperm injection (ICSI) outcomes in human oocytes. In this study, 264 oocytes from 24 patients with an average age of 30.5±7.5 years with infertility duration of 1 to 10 years were collected. The oocytes were randomly allocated to the control injection group (n=126) and the oocyte imaging group (spindle-aligned group) (n=138). In the spindle-aligned group, the meiotic spindle was identified by means of polarized light microscope to align the spindle at 6 or 12 o'clock. Then the spindle-aligned group was divided into three sub-groups based on spindle morphology: fine, average, and (poor). After ICSI, embryos were checked every 24 hours and scored; 72 hours later, high-grade embryos were transferred intravaginally to uterus. This study showed that the fertilization rate in the spindle-aligned group was higher than the control group (P<0.05). After cleavage, a positive correlation was observed between spindle morphology and embryo morphology. Among the sub-groups of spindle-aligned group, the embryos' morphology of the fine group was better than the other subgroups and embryos of the poor group had lower quality and more fragmentation. The results revealed that the selection of oocytes based on meiotic spindle imaging can significantly improve the rate of fertilization and embryo cleavage and certainly increase the rate of implantation.

Combination of spindle and first polar body chromosome images for the enhanced prediction of developmental potency of mouse metaphase II oocytes.

The objective of this study was to classify spindle and first polar body (PB1) chromosome images in ovulated mouse oocytes over time to predict the developmental competence of metaphase II (MII) oocytes. Oocytes were collected at 12, 15, 20, and 25 h after human chorionic gonadotropin (hCG) injection, and stained for spindle tubulin, chromosomes, and PB1 chromosomes. MII spindle morphology was classified as tapered type or barrel type and PB1 chromosomes were categorized as aggregated, separated, dot, or collapsed. To determine whether differences in spindle and PB1 images in MII oocytes are associated with fertilization success, we performed in vitro fertilization (IVF) at various times after hCG injection. Barrel-type spindles and aggregate-type PB1 were dominant at 12 h after hCG injection. Oocyte spindles collected 1 h after injection were tapered, and PB1 chromosomes were separated. At 20 and 25 h after treatment, spindle and PB1 images were classified as collapsed. The rate of development to 2-cell embryos after IVF did not differ between the 12 h and 15 h treatments; however, it was significantly lower for the 25 h treatment than for other treatments. The rates of development to blastocysts at 12, 15, 20, and 25 h after hCG injection were 61, 46, 42, and 9%, respectively. MII oocytes with barrel-type spindles and aggregate-type PB1 had high rates of fertilization and blastocyst development, and spindle and PB1 characteristics were correlated with the outcomes of IVF and embryo culture. These results suggested that images of spindles combined with those of PB1 chromosomes enable the prediction of oocytic and/or embryonic quality.

ICSI with the Assistance of Meiotic Spindle Imaging for the Production of High Quality Embryos

Abstract To avoid multiple pregnancies, it is important to select a single high quality embryo for transfer. Observation of meiotic spindles in oocytes is one way of evaluating embryo developmental competence. The spindle is non-invasively visualized by polarized light microscopy. It is possible to perform ICSI more safely and with optimal timing during meiosis by visualizing the meiotic spindle just before ICSI. In some human studies, it has been reported that oocytes with a spindle show significantly higher fertilization, blastocyst formation and pregnancy rates. Recently, it has been reported that morphological spindle normality is related to the live birth rate. In mice, the areas of the spindles in aged oocytes (23 h after hCG injection) are larger than those in young oocytes (15 h after hCG injection), and the blastocyst formation rate of aged oocytes is lower than that of young oocytes. Addition of MG132, a ubiquitin-mediated proteasome proteolysis inhibitor, to the medium prevents the spindle from...