Limited implantation success of direct-cleaved human zygotes: a time-lapse study

2012 ◽  
Vol 98 (6) ◽  
pp. 1458-1463 ◽  
Author(s):  
Irene Rubio ◽  
Reidun Kuhlmann ◽  
Inge Agerholm ◽  
John Kirk ◽  
Javier Herrero ◽  
...  
Keyword(s):  
Time Lapse ◽  
Human Zygotes ◽  
Implantation Success

P–215 The degree of perivitelline space (PS) at the pronuclear stage affects subsequent embryonic development in human zygotes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
K Yumoto ◽  
T Shimura ◽  
M Sugishima ◽  
M Nakaoka ◽  
Y Mio
Keyword(s):  
Embryonic Development ◽  
Time Lapse ◽  
Perivitelline Space ◽  
Blastocyst Development ◽  
Intact Group ◽  
Registration Number ◽  
Group 3 ◽  
Group 2 ◽  
Human Zygotes ◽  
Group 1

Abstract Study question Was embryonic development affected by the degree of perivitelline space (PS) at the pronuclear stage in human zygotes? Summary answer Zygotes with a fully surrounding PS showed less cytoplasmic fragmentation and a higher blastocyst development rate (BDR) than zygotes with a partially surrounding PS. What is known already We previously used abnormally-fertilized oocytes (zygotes with three pronuclei; 3PN), donated by ART patients in our clinic who gave written consent for the research. The zona pellucida (ZP) was artificially removed from these oocytes at the pronuclear stage, termed ZP-free culture. The resultant ZP-free 3PN embryos showed less cytoplasmic fragmentation and a higher rate of good-quality embryos (GQE) compared with ZP-intact embryos. Furthermore, in our clinical setting, the rate of GQE and BDR of normally-fertilized embryos were clearly improved by ZP-free culture in patients with recurrent failure of ART treatments due to severe cytoplasmic fragmentation at the early cleavage stage. Study design, size, duration This study included 49 patients who gave written informed consent for our study and were treated with ART in our clinic between March and December 2020. Embryonic development was compared between zygotes with a fully surrounding PS [PS(+)] with those with a partially surrounding PS [PS(-)] at the pronuclear stage. Furthermore, the ZP of PS(-) embryos were artificially removed at the pronuclear stage, and the rate of GQE and BDR were compared with ZP-intact embryos. Participants/materials, setting, methods The degree of PS in 128 zygotes was confirmed by hypertonic preparation using 0.125M sucrose-containing HEPES medium. PS(+) and PS(-) embryos were both cultured as ZP-intact, and the rate of GQE was compared. Furthermore, 223 zygotes were divided into three groups: 1) PS(-)/ZP-intact, 2) PS(-)/ZP-free, and 3) PS(+)/ZP-intact, and cultured in an incubator equipped with time-lapse monitoring up to Day 7, and the rate of GQE, BDR and useable embryos were compared between each groups. Main results and the role of chance The degree of PS was confirmed by a hypertonic preparation (shrinkage of the ooplasm) in 128 normally-fertilized zygotes obtained from 44 cases. There were 86 PS(-) (67.2%) and 42 PS(+) (32.8%) zygotes. The mean maternal age was 35.9 in PS(-) and 40.5 in PS(+) (P < 0.01), and the rate of GQE was significantly higher in PS(+) [64.3% (27/42)] than in PS(-)[38.4% (33/86)] (P < 0.01). In addition, of 223 normally-fertilized zygotes obtained from 41 cases, there were 51 PS(-)/ZP-intact (Group 1), 132 PS(-)/ZP-free (Group 2) and 40 PS(+)/ZP-intact (Group 3) zygotes. The rate of GQE was significantly lower in Group 1 [29.4% (15/51)] compared with Group 2 [59.8% (79/132)] and Group 3 [62.5% (25/40)] (P < 0.01). BDR was also significantly lower in Group 1 [51.3% (10/39)] compared with Group 2 [75.0% (99/132)] and Group 3 [65.0% (13/20) (P < 0.01). Limitations, reasons for caution Although the artificial removal of ZP at the pronuclear stage (ZP-free culture) clearly increased the rate of GQE, embryonic development was not improved in all cases. It seems that this procedure is only effective in embryos with a viable ooplasm. Wider implications of the findings: The degree of PS at the pronuclear stage affects subsequent embryonic development in human zygotes. The artificial removal of ZP at the pronuclear stage (ZP-free culture) helps to suppress fragmentation and leads to an increase in GQE and BDR, and eventually, improves pregnancy rate in cases with severe fragmentation. Trial registration number non

O-219 Detailed morpho-kinetic analysis of the first cleavage can help in evaluating the viability of direct-cleaved human zygotes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
T Shimura ◽  
K Yumoto ◽  
M Sugishima ◽  
Y Mio
Keyword(s):  
Live Birth ◽  
Birth Rate ◽  
Live Birth Rate ◽  
Time Lapse ◽  
Cell Group ◽  
Group 3 ◽  
Group 2 ◽  
Time Lapse Imaging ◽  
Human Zygotes ◽  
Group 1

Abstract Study question Why do some direct-cleaved human zygotes still lead to a live birth? Summary answer Direct-cleaved zygotes which have undergone the 2-cell stage can lead to a live birth, while zygotes cleaved from 1-cell to ≥ 3-cell do not. What is known already In recent years, zygotes that develop from 2-cell to 3-cell within 5 hours after the first cleavage have been evaluated as “direct-cleaved” zygotes, because normal cleavage takes approximately 12 hours to complete. It was reported that their implantation rate was significantly lower than zygotes with normal cleavage pattern, and eliminating direct-cleaved zygotes from transfer could improve the implantation rate. However, some direct-cleaved zygotes at the first cleavage could still lead to a live birth. Few reports have examined the difference between a cleavage from 1-cell to ≥ 3-cell and 2-cell to ≥ 3-cell within 5 hours after the first cleavage. Study design, size, duration A retrospective study involving 2,077 cycles of IVF/ICSI between July 2012 and July 2019. A total of 5,991 normally fertilized zygotes (2PN/2PB) were included. Of those, 3,508 were evaluated as usable good/fair quality embryos on Day2/3, and the rest (n = 2,483) were evaluated as poor quality and rejected from transfer or cryopreservation after 7 days of culture. Of 3,508 usable embryos, 884 were selected based on the availability of results of live birth for this study. Participants/materials, setting, methods Time-lapse imaging (5 slices along Z-axis every 10 minutes) was performed in EmbryoScopeTM. Zygotes were morphokinetically analyzed in detail and classified into four groups by their cleavage patterns: Group1 (1-cell→2-cell); Group 2 (1-cell→3-cell); Group 3 (1-cell→2-cell→≥3-cell within 5 hours after the first cleavage); and Group 4 (1-cell→2-cell→≥5-cell). The proportion, mean maternal age and live birth rate of each group were examined. Main results and the role of chance The proportion of Groups 1-4 was 83.6% (n = 739), 3.8% (n = 34), 5.9% (n = 52), and 6.7% (n = 59), respectively. 0f 884 zygotes examined in this study, the mean maternal age was significantly higher in Group 2 and 4 than in Group 1 (P < 0.05; 37.4±4.9 in Group1, 39.1±5.2 in Group 2, 38.6±6.0 in Group 3, and 38.7±5.1 in Group 4). The rate of confirmed gestational sac was significantly lower in Group 2 and 4 than in Group 1 [P < 0.01; 36.3% (n = 268/739), 0% (n = 0/34), 25.0% (n = 13/52), and 18.6% (n = 11/59) in Groups 1-4, respectively]. Furthermore, the live birth rate was significantly higher in Group 1 than in Groups 2, 3 and 4 [P < 0.01; 28.4% (n = 210/739), 0% (n = 0/34), 13.5% (n = 7/52), and 15.3% (n = 9/59) in Groups 1-4, respectively]. Above all, while zygotes in Group 2 showed no pregnancy and live birth at all, zygotes in Group 3 showed a live birth rate of 13.5%. However, they had a significantly higher miscarriage rate (42.9%, n = 6) compared to zygotes in Group 1 (19.5%, n = 55). Limitations, reasons for caution It is very difficult to capture cleavage patterns by routine observations because the timings of developmental events are different between embryos. A time-lapse imaging and culturing system is essential to solve this problem, however, it cannot visualize the distribution of chromosomes, and no chromosomal analysis was conducted in this study. Wider implications of the findings This study revealed that zygotes previously classified as “direct-cleaved” and eliminated from transfer included viable zygotes which could lead to a live birth. Therefore, it is crucial to optimize the use of time-lapse imaging of human zygotes in order to precisely evaluate the first cleavage. Trial registration number not applicable

scholarly journals Collapse of blastocysts is strongly related to lower implantation success: a time-lapse study

2015 ◽  
Vol 30 (11) ◽  
pp. 2501-2508 ◽  
Author(s):  
Julian Marcos ◽  
Sonia Pérez-Albalá ◽  
Amparo Mifsud ◽  
Marta Molla ◽  
Jose Landeras ◽  
...  
Keyword(s):  
Time Lapse ◽  
Implantation Success

scholarly journals Contraction of blastocyst is strongly related with lower implantation success; a time-lapse study

2013 ◽  
Vol 100 (3) ◽  
pp. S501
Author(s):  
J. Marcos Alises ◽  
S. Perez ◽  
D. Gumbao ◽  
M. Mollá Silva ◽  
M. Meseguer ◽  
...  
Keyword(s):  
Time Lapse ◽  
Implantation Success

P–247 Application of deep learning for automated measurement of key morphological features of human zygotes for IVF

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Le ◽  
I Miyatsuka ◽  
J Otsuki ◽  
M Shiotani ◽  
N Enatsu ◽  
...  
Keyword(s):  
Deep Learning ◽  
Confidence Score ◽  
Time Lapse ◽  
Morphological Features ◽  
Training Dataset ◽  
Percentage Error ◽  
Male And Female ◽  
The Mean ◽  
Over Time ◽  
Human Zygotes

Abstract Study question Can deep learning (DL) algorithms trained on time-lapse videos be used to detect and track the size and gender of pronuclei in developing human zygotes? Summary answer Our DL algorithm not only outperforms state-of-the-art models in detecting the pronuclei but can also accurately identify and track its gender and size over time. What is known already Recent researches have explored the use of DL to extract key morphological features of human embryos. Existing studies, however, focus either on blastocysts’ morphological measurements (Au et al. 2020) or on embryos’ general developmental stages classification (Gingold et al. 2018, Liu et al. 2019, Lau et al. 2019). So far, only one paper attempted to evaluate zygotes’ morphological components but stopped short of identifying the existence and location of their pronuclei (Leahy et al. 2020). We address this research gap by training a DL model that can detect, classify the gender, and quantify the size of zygotes’ pronuclei over time. Study design, size, duration A retrospective analysis using 91 fertilized oocytes from infertile patients undergoing IVF or ICSI treatment at Hanabusa Women’s Clinic between January 2011 and August 2019 was conducted. Each embryo was time-lapse monitored using Vitrolife which records an image every 15 minutes at 7 focal planes. For our study, we used videos of the first 1–2 days of the embryo from its 3 central focal planes, corresponding to 70–150 images per focal plane. Participants/materials, setting, methods All 273 timelapse videos were split into 30,387 grayscale still images at a 15-minute interval. Each image was checked and annotated by experienced embryologists where every pixel of the image was classified into 3 categories: male pronuclei, female pronuclei, and others. Images were converted into grayscale, resized into 500x500 pixels, and then fed into a neural network with the Mask R-CNN architecture and a ResNet101 backbone to produce a pronuclei instance segmentation model. Main results and the role of chance The 91 embryos were split into training (∼70% or 63 embryos) and validation (∼30% or 28 embryos). Our pronuclei model takes as input a single image and outputs a bounding box, mask, category, confidence score, and size measured in terms of pixel for each detected candidate. For prediction, we run the model on the 3 middle focal planes and merge candidates by using the one with the highest confidence score. We used the mean-average precision (mAP) score to evaluate our model’s ability to detect pronuclei and used the mean absolute percentage error (MAPE) between the actual size (as annotated by the embryologist) and the predicted one to check the model’s performance in tracking the pronuclei’s size. The mAP for detecting pronuclei, regardless of its gender, achieved by our model was 0.698, higher than the 0.680 value reported in the Leahy et al. paper (2020). Breakdown by gender, our model’s mAP for male and female pronuclei are 0.734 and 0.661 respectively. The overall MAPE for tracking pronuclei’s size is 21.8%. Breakdown by gender, our model’s MAPE for male and female pronuclei are 19.4% and 24.3% respectively. Limitations, reasons for caution Samples were collected from one clinic with videos recorded from one time-lapse system which can limit our results’ reproducibility. The accuracy of our DL model is also limited by the small number of embryos that we used. Wider implications of the findings: Even with a limited training dataset, our results indicate that we can accurately detect and track the gender and the size of zygotes’ pronuclei using time-lapse videos. In future models, we will increase our training dataset as well as include other time-lapse systems to improve our models’ accuracy and reproducibility. Trial registration number Not applicable

P–191 Time-lapse videography reveals morphometric and morphokinetic differences in the pronuclei of male and female human zygotes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
L S Orevich ◽  
K Watson ◽  
K Ong ◽  
I Korman ◽  
R Turner ◽  
...  
Keyword(s):  
Logistic Regression ◽  
Live Birth ◽  
Polar Body ◽  
Relative Size ◽  
Time Lapse ◽  
Area Measurement ◽  
Size Difference ◽  
Male And Female ◽  
Stage 1 ◽  
Human Zygotes

Abstract Study question Do morphometric and morphokinetic profiles of pronuclei (PN) following intracytoplasmic sperm injection (ICSI) vary between male and female human zygotes? Summary answer Male and female zygotes displayed different PN morphometrics and morphokinetics. Additionally, variations were identified between sperm-originated (SPN) and oocyte-originated (OPN) pronuclei. What is known already Previous studies have investigated the use of PN-associated parameters via static observations as indicators of zygote viability, including size equality or juxtaposition. However, recent clinical application of time-lapse videography (TLV) provides a novel opportunity to assess these pronuclear events with greater accuracy and precision of morphometric and morphokinetic measurement. A number of recent TLV studies have also investigated potential live birth prediction by such PN associated measures, however whether or not there are gender associated differences in such measures which could in turn confound live birth prediction is unknown. Study design, size, duration: This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles (either fresh or frozen) performed between January 2019 and March 2020. Only ICSI cycles (maternal age <40 years) leading to a singleton live birth (43 males and 51 females) were included for analysis. All oocytes were placed in the EmbryoScope incubator for culture immediately post sperm injection with all annotation performed retrospectively by one embryologist (L-SO). Participants/materials, setting, methods Timings included 2nd polar body extrusion (tPb2), SPN(tSPNa)/OPN(tOPNa) appearance (differentiated by proximity to Pb2) and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2) and 0 hour before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolus precursor body (NPB) arrangement. Means ± standard deviation were compared using student t test or logistic regression as odds ratio (OR) and 95% confidence interval (CI), and proportional data by chi-squared analysis. Main results and the role of chance Logistic regression indicated that male zygotes had longer time intervals of tPb2_tSPNa than female zygotes (4.8±1.5 vs 4.2±1.0 h, OR = 1.442, 95% CI 1.009–2.061, p = 0.044), but not tPb2_tOPNa (4.7±1.8 vs 4.5±1.3 h, OR = 1.224, 95% CI 0.868–1.728, p = 0.250) and tPb2_tPNF (19.9±2.8 vs 19.1±2.3 h, OR = 1.136, 95% CI 0.957–1.347, p = 0.144). SPN increased in size from stage 1 through 2 to 3 (435.3±70.2, 506.7±77.3, and 556.3±86.4 um2, p = 0.000) and OPN did similarly (399.0±59.4, 464.3±65.2, and 513.8±63.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). However, relative size difference between SPN and OPN was similar between male and female zygotes at 3 stages (33.6±61.7 vs 38.6±50.8 um2, p = 0.664; 38.5±53.1 vs 45.7±71.9 um2, p = 0.585; 38.4±77.4 vs 45.8±63.9 um2, p = 0.615; respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (77% vs 51%, p = 0.010), stage 2 (98% vs 86%, p = 0.048) and stage 3 (98% vs 86%, p = 0.048). Furthermore, more OPN showed aligned NPBs than in SPN at stage 1 (45% vs 29%, p = 0.023), but similar proportions at stage 2 (64% vs 50%, p = 0.056) and stage 3 (76% vs 72%, p = 0.618). There were no gender associated differences detected in NPB alignment in either SPN or OPN (p > 0.05 respectively). Limitations, reasons for caution The retrospective design does not allow for control of unknown confounders. Sample size is considered relatively small. PN area measurement may not truly represent volume as PN may not be perfectly spherical. Findings were based on women <40 years old so may not apply to older population. Wider implications of the findings: These findings augment and extend previous studies investigating PN parameters via static observations. The reported variations between male and female embryos may confound live birth prediction when using pronuclei morphometrics and morphokinetics. Larger scaled studies are warranted to verify these findings. Trial registration number Not applicable

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

High-voltage Electron Microscopy of astroglial cell whole mounts

1986 ◽  
Vol 44 ◽  
pp. 316-317
Author(s):  
J.N. Turner ◽  
W.G. Shain ◽  
V. Madelian ◽  
R.A. Grassucci ◽  
D.L. Forman
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Time Lapse ◽  
Astroglial Cells ◽  
High Voltage Electron Microscopy ◽  
Rat Glioma ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Nervous System Function ◽  
Beta Adrenergic Agonist

Homogeneous cultures of astroglial cells have proved useful for studying biochemical, pharmacological, and toxicological responses of astrocytes to effectors of central nervous system function. LRM 55 astroglial cells, which were derived from a rat glioma and maintained in continuous culture, exhibit a number of astrocyte properties (1-3). Stimulation of LRM 55s and astrocytes in primary cell cultures with the beta-adrenergic agonist isoproterenol results in rapid changes of morphology. Studies with time lapse video light microscopy (VLM) and high-voltage electron microscopy (HVEM) have been correlated to changes in intracellular levels of c-AMP. This report emphasizes the HVEM results.

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