P–804 Morphokinetic development by time-lapse imaging of conceptuses generated from artificial oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Trout ◽  
P Xie ◽  
A Petrini ◽  
Z Rosenwaks ◽  
G Palermo
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Blastocyst Development ◽  
Time Lapse Imaging

Abstract Study question What are the ideal culture conditions to enhance full preimplantation development of embryos generated by FVB somatic cell haploidization (SCH) in the mouse model? Summary answer The presence of a histone deacetylase inhibitor yielded the best morphokinetic development of expanded blastocysts generated by FVB SCH, comparable to control blastocysts. What is known already Various culture conditions and medium supplements have been proposed to promote preimplantation development of embryos generated by SCH, including supplementation with trichostatin A (TSA), fasudil, scriptaid, and RAD–51 stimulatory compound–1 (RS–1). TSA and scriptaid, both histone-deacetylase inhibitors, have been found to improve embryo development following nuclear transfer by enhancing histone acetylation and cellular reprogramming. Additionally, fasudil is a Rho-associated kinase inhibitor that has been shown to reduce apoptosis and promote cell proliferation. Finally, RS–1 stimulates RAD51 activity, which promotes the repair of DNA damage and increases the efficacy of somatic cell reprogramming. Study design, size, duration B6D2F1 mouse metaphase II (MII) oocytes underwent enucleation and nuclear transfer, or were ICSI inseminated serving as controls. Reconstituted oocytes showing development of a meiotic-like spindle demonstrated successful SCH, and were ICSI inseminated. SCH conceptuses were cultured in one of three groups: KSOM, KSOM supplemented with TSA (TSA), or KSOM supplemented with fasudil, scriptaid, and RS–1 (Cocktail). ICSI controls (ICSIC) were cultured in KSOM medium. Fertilization and full preimplantation development were compared among all groups. Participants/materials, setting, methods Ooplasts were generated from MII oocytes by removing spindle complexes under OosightÔ visualization and cytochalasin B exposure. A single FVB mouse cumulus cell was transferred into the perivitelline space and fused with the ooplast, facilitated by Sendai virus. Reconstructed oocytes with novel pseudo-meiotic spindles underwent piezo-ICSI and were cultured in different media conditions in a time-lapse imaging system up to 96h. TSA and Cocktail embryos had media changed to regular KSOM 10 hours after insemination. Main results and the role of chance A total of 274 B6D2F1 MII oocytes were enucleated, resulting in a 95.9% survival rate. All ooplasts survived nuclear transfer and 62.1% successfully haploidized after 2 hours. ICSIC and reconstituted SCH oocytes survived piezo-ICSI at rates of 81.5% and 57.0%, respectively (P < 0.01). SCH embryos were then allocated into KSOM, TSA supplied, and Cocktail media. Fertilization rates for ICSIC, KSOM, and TSA embryos were 92.4%, 90.7%, and 94.4%, respectively, while the rate for embryos cultured in Cocktail was only 71.9% (P < 0.03). While embryos cultured in Cocktail had a comparable 2-cell timing to ICSIC, embryos in TSA reached developmental milestones with a closer timing to the ICSIC, having minor delays at the 3-, 4-, and 6-cell stages (P < 0.05). KSOM- and Cocktail-cultured embryos were delayed at most of the stages (P < 0.01), except for the two-pronuclei appearance. Although the TSA group displayed the best embryo developmental pattern, the final rate of blastocyst development was somewhat homogeneous with rates of 15.4%, 23.5%, and 13.0% for the KSOM, TSA, and Cocktail groups, respectively (P < 0.001), and remarkably lower than the ICSIC (81.6%). Limitations, reasons for caution Although live pups have been obtained using BDF cumulus cells, embryos generated by FVB cumulus cells show a remarkably lower blastocyst development, but maintain morphokinetic characteristics similar to ICSIC in the presence of TSA. Wider implications of the findings: While using different strains to enhance genetic variance, the morphokinetic analysis of preimplantation embryos in ideal culture conditions is paramount to the progress of neogametogenesis. The implementation of this technique may soon help create genotyped oocytes for women with compromised ovarian reserve. Trial registration number N/A

38 EFFECT OF OOCYTE MATURATION DURATION ON BLASTOCYST RATES AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 112 ◽  
Author(s):  
Y. H. Choi ◽  
I. C. Velez ◽  
B. Macías-García ◽  
K. Hinrichs
Keyword(s):  
Somatic Cell ◽  
Oocyte Maturation ◽  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Direct Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Donor Cells

In equine cloning, the scarcity of equine oocytes places emphasis on development of the most efficient nuclear transfer (NT) methods possible. In other species, using oocytes matured for the shortest duration needed to reach metaphase II has increased NT efficiency. In the present study, we examined the effect of duration of oocyte maturation at the time of enucleation on equine cloned blastocyst production. Oocytes were collected from live mares by transvaginal ultrasound-guided aspiration of all visible follicles ≥5 mm in diameter. The oocytes were held overnight (16–22 h) at room temperature, matured in vitro, and reconstructed with donor cells as described in our previous study (Choi et al. 2013 Theriogenology 79, 791–796). In Experiment 1, oocytes were divided into 2 groups and matured for 20 or 24 h. After enucleation, oocytes were reconstructed by direct injection of donor cells. Reconstructed oocytes were held for 5 h and then activated by treatment with 5 μM ionomycin for 4 min, then injection with sperm extract, followed by incubation in 2 mM 6-DMAP for 4 h. The activated reconstructed oocytes were cultured in global human embryo culture medium under 5% CO2, 6% O2, and 89% N2 at 38.2°C for 7 to 11 days (20 mM glucose was added at Day 5) and blastocyst rate was recorded. Because a low maturation rate was found at 20 h in Experiment 1, in Experiment 2 oocytes were denuded at 20 h and those that were mature were enucleated and used for NT; those that had not cast out a polar body at 20 h were cultured for an additional 3 h (20 + 3h) and then evaluated for polar body formation and used for NT, which was conducted as in Experiment 1. Data were analysed by Fisher's exact test. In Experiment 1, 203 oocytes were collected in 46 aspiration sessions. The rate of oocyte maturation to metaphase II was significantly lower for oocytes cultured for 20 h (35/116, 30%), than for those cultured for 24 h (47/80, 59%). However, the rate of blastocyst development was significantly higher for oocytes cultured for 20 h (11/27, 41%) than for 24 h (2/38, 5%). In Experiment 2, 89 oocytes were collected in 18 aspiration sessions. After 20 h of maturation culture, 22 oocytes were mature (25%). After an additional 3 h of culture, 21 additional oocytes had matured. There were no significant differences between the two treatments (20 and 20 + 3h) in reconstruction rates (77%, 17/22, and 90%, 19/21, respectively) or blastocyst rates (24%, 4/17, and 32%, 6/19, respectively). These results indicate that duration of in vitro maturation, or the duration of presence of cumulus cells, influences blastocyst development after somatic cell NT in the horse. This appears to be due to a benefit of using oocytes immediately after they reach metaphase II; if this is ensured as in Experiment 2, the duration of maturation itself had no effect.This work was supported by the American Quarter Horse Foundation, the Link Equine Research Endowment Fund, Texas A&M University, and by Ms. Kit Knotts.

scholarly journals The transcriptomic architecture of mouse Sertoli cell clone embryos reveals temporal–spatial-specific reprogramming

Reproduction ◽  
2013 ◽  
Vol 145 (3) ◽  
pp. 277-288 ◽  
Author(s):  
Feng Cao ◽  
Atsushi Fukuda ◽  
Hiroshi Watanabe ◽  
Tomohiro Kono
Keyword(s):  
Somatic Cell ◽  
Sertoli Cell ◽  
Nuclear Transfer ◽  
Cell Clone ◽  
Chromosome Mapping ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Differentiated Cells ◽  
Spatial Changes

Somatic cell nuclear transfer, a technique used to generate clone embryos by transferring the nucleus of a somatic cell into an enucleated oocyte, is an excellent approach to study the reprogramming of the nuclei of differentiated cells. Here, we conducted a transcriptomic study by performing microarray analysis on single Sertoli cell nuclear transfer (SeCNT) embryos throughout preimplantation development. The extensive data collected from the oocyte to the blastocyst stage helped to identify specific genes that were incorrectly reprogrammed at each stage, thereby providing a novel perspective for understanding reprogramming progression in SeCNT embryos. This attempt provided an opportunity to discuss the possibility that ectopic gene expression could be involved in the developmental failure of SeCNT embryos. Network analysis at each stage suggested that in total, 127 networks were involved in developmental and functional disorders in SeCNT embryos. Furthermore, chromosome mapping using our time-lapse expression data highlighted temporal–spatial changes of the abnormal expression, showing the characteristic distribution of the genes on each chromosome. Thus, the present study revealed that the preimplantation development of SeCNT embryos appears normal; however, the progression of incorrect reprogramming is concealed throughout development.

scholarly journals Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 559-567 ◽  
Author(s):  
Irina Lagutina ◽  
Giovanna Lazzari ◽  
Roberto Duchi ◽  
Silvia Colleoni ◽  
Nunzia Ponderato ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Reconstruction Method ◽  
Blastocyst Development ◽  
Adult Fibroblasts

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

scholarly journals Involvement of Nlrp9a/b/c in mouse preimplantation development

Reproduction ◽  
2020 ◽  
Vol 160 (2) ◽  
pp. 181-191 ◽  
Author(s):  
Satoko Kanzaki ◽  
Shiori Tamura ◽  
Toshiaki Ito ◽  
Mizuki Wakabayashi ◽  
Koji Saito ◽  
...  
Keyword(s):  
Asymmetric Cell Division ◽  
Culture Conditions ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Dependent Manner ◽  
Blastocyst Development ◽  
Triple Mutant ◽  
Cell Stage

Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing proteins (NRLPs) are central components of the inflammasome. Accumulating evidence has shown that a reproductive clade of NRLPs is predominantly expressed in oocyte to cleavage stage embryos and participates in mammalian preimplantation development as a component of a multiprotein complex known as the subcortical maternal complex (SCMC). Nlrp9s belong to the reproductive class of NLRPs; Nlrp9b is unique in acting as an inflammasome against rotavirus in intestines. Here we generated mice carrying mutations in all three members of the Nlrp9a/b/c gene (Nlrp9 triple mutant (TMut) mice). When crossed with WT males, the Nlrp9 TMut females were fertile, but deliveries with fewer pups were increased in the mutants. Consistent with this, blastocyst development was retarded and lethality to the preimplantation embryos increased in the Nlrp9 TMut females in vivo. Under in vitro culture conditions, the fertilized eggs from the Nlrp9 TMut females exhibited developmental arrest at the two-cell stage, accompanied by asymmetric cell division. By contrast, double-mutant (DMut) oocytes (any genetic combination) did not exhibit the two-cell block in vitro, showing the functional redundancy of Nlrp9a/b/c. Finally, Nlrp9 could bind to components of the SCMC. These results show that Nlrp9 functions as an immune or reproductive NLRP in a cell-type-dependent manner.

Advances in equine cloning by somatic cell nuclear transfer (SCNT) technique in horses: a review

2016 ◽  
Vol 5 (12) ◽  
pp. 5124
Author(s):  
Rajesh Wakchaure ◽  
Subha Ganguly*
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Genetic Material ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Oocyte Activation

Cloning a horse means using the genetic material (DNA) from a donor horse to produce a genetically identical foal. This technique involves collecting the DNA from the donor and inserting that DNA into an egg from another mare whose, DNA content has been removed, fusing donor nucleus with enucleated recipient oocytes, which then develops as an embryo, in vitro culture of embryo and lastly transfer cultured embryo into the uterus of a recipient mare. The modification of the in vitro culture conditions which can be suitable for equine oocyte activation, oocyte maturation and embryo development are the fundamental steps for a successful in vitro procedure for somatic cell nuclear transfer (SCNT) in the horse to avoid the embryo losses. In general, few studies are available in the literature on equine in vitro embryo production and it is only recently that reports have been published on completely in vitro production of equine preimplantation embryos by means of in vitro oocyte maturation The present review discusses the latest developments in the field of equine cloning technique with the employment of SCNT. The basic understanding of SCNT for in vitro culture conditions is relevant to the increased efficiency of cloning. The available genotype can be used by SCNT which can enhance the vigour of a particular infertile or low fertile animal to produce normal fertility.

scholarly journals Preimplantation embryo programming: transcription, epigenetics, and culture environment

Reproduction ◽  
2008 ◽  
Vol 135 (2) ◽  
pp. 141-150 ◽  
Author(s):  
Veronique Duranthon ◽  
Andrew J Watson ◽  
Patrick Lonergan
Keyword(s):  
Gene Expression ◽  
Embryo Culture ◽  
Nuclear Transfer ◽  
Preimplantation Embryo ◽  
Later Life ◽  
Short Review ◽  
Basic Program ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Negative Consequences

Preimplantation development directs the formation of an implantation- or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and fetal development can be sustained. The preimplantation embryo exhibits a form of autonomous development fueled by products provided by the oocyte and also from activation of the embryo's genome. Despite this autonomy, the preimplantation embryo is highly influenced by factors in the external environment and in extreme situations, such as those presented by embryo culture or nuclear transfer, the ability of the embryo to adapt to the changing environmental conditions or chromatin to become reprogrammed can exceed its own adaptive capacity, resulting in aberrant embryonic development. Nuclear transfer or embryo culture-induced influences not only affect implantation and establishment of pregnancy but also can extend to fetal and postnatal development and affect susceptibility to disease in later life. It is therefore critical to define the basic program controlling preimplantation development, and also to utilize nuclear transfer and embryo culture models so that we may design healthier environments for preimplantation embryos to thrive in and also minimize the potential for negative consequences during pregnancy and post-gestational life. In addition, it is necessary to couple gene expression analysis with the investigation of gene function so that effects on gene expression can be fully understood. The purpose of this short review is to highlight our knowledge of the mechanisms controlling preimplantation development and report how those mechanisms may be influenced by nuclear transfer and embryo culture.

P–201 The beneficial effects of ZP-free culture on cytoplasmic fragmentation in human embryos. : An innovative trial using 3PN zygotes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
M Sugishima ◽  
K Yumoto ◽  
T Shimura ◽  
Y Mio
Keyword(s):  
Embryo Development ◽  
Imaging System ◽  
Culture Method ◽  
Cumulus Cells ◽  
Time Lapse ◽  
Human Embryos ◽  
Beneficial Effects ◽  
Molecular Biological Analysis ◽  
A Prospective Study ◽  
Time Lapse Imaging

Abstract Study question Is it possible to culture ZP-free embryos to eliminate perivitelline threads, which are known to be involved in generating cytoplasmic fragments at the first cleavage? Summary answer ZP-free culturing, an innovative system that decreases the amount of cytoplasmic fragments without disrupting the blastomeres, using incubators with time-lapse imaging. What is known already A study in 2017 observed perivitelline threads in more than 50% of cleavage-stage human embryos using time-lapse imaging, and the rate of cytoplasmic fragmentation (at the first cleavage) was significantly decreased in embryos without perivitelline threads (P &lt; 0.001). While it has been proposed that perivitelline threads play an important role in crosslinking the cumulus cells and oocyte during maturation, the mechanism underlying such a role remains unclear. It is also unknown whether the threads still function in mature MII oocytes. Study design, size, duration A prospective study was conducted using 2,852 normal (2PN/2PB) embryos from c-IVF/ICSI and 113 abnormal (3PN) embryos obtained from c-IVF between 2017 and 2019. The zona pellucida (ZP) of 71 abnormal embryos was removed at the pronuclear stage (“ZP-free”), and the rest (n = 42) were cultured as “ZP-intact”. Normal and abnormal embryos were cultured for five days in bench-top incubators (MINC, COOK) and an incubator equipped with a time-lapse imaging system. Participants/materials, setting, methods Embryos used in this study were donated by 412 couples who underwent c-IVF cycles in our clinic between 2017 and 2019. For ZP removal, 3PN embryos were placed in 0.125M sucrose-containing HEPES media drops to reduce the ooplasm size. Then, ooplasms were completely separated from ZPs by a laser and pipetting. Embryo development and morphology of the three groups (normal, ZP-intact and ZP-free abnormal) were compared based on the degree of cytoplasmic fragmentation. Main results and the role of chance The first cleavage occurred in 97.8% (n = 2,790/2,852) of 2PN/2PB, 83.3% (n = 35/42) of ZP-intact 3PN and 97.2% (n = 69/71) of ZP-free 3PN. Normal (2PN/2PB), ZP-intact and ZP-free 3PN embryos were classified into three groups based on the modified Veeck’s criteria thus: &lt;20% fragmented compared to the total volume of cytoplasm at the first cleavage (Grade 1 and 2, Good); 20–39% fragmented (Grade 3, Fair) and ≧40% fragmented (Grade 4, Poor). Of 69 cleaved ZP-free 3PN embryos, 68.1% (n = 47) showed less than 20% fragments which was significantly higher than 2PN/2PB (43.7%, n = 1,218/2,790) and ZP-intact 3PN (45.7%, n = 16/35; P &lt; 0.05). Furthermore, 24.6% (n = 17/69) of ZP-free 3PN embryos showed 20–39% fragments which was significantly lower than 2PN/2PB (45.9%, n = 1,281/2,790; P &lt; 0.05). In addition, 50.7% of ZP-free 3PN embryos (n = 36) developed to the morula stage after the third cleavage, and 29.6% (n = 21) formed blastocoel and became blastocysts. Thus, removing the ZP before the first cleavage did not adversely affect embryo development and decreased the cytoplasmic fragmentation. Limitations, reasons for caution Due to ethical and clinical limitations, we only examined abnormally fertilized embryos in this study. Moreover, since the relationship between the perivitelline threads and cytoplasmic fragments is unclear, we plan to conduct molecular biological analysis of the perivitelline threads in further studies. Wider implications of the findings: This study revealed that ZP is not always necessary after the pronuclear stage because ZP-free embryos studied herein developed normally and maintained cell adhesion well. This innovative culture method might provide the breakthrough needed for patients to improve embryo quality who obtain embryos with severe fragmentation caused by perivitelline threads. Trial registration number Not applicable

scholarly journals Can novel early non-invasive biomarkers of embryo quality be identified with time-lapse imaging to predict live birth?

2019 ◽  
Vol 34 (8) ◽  
pp. 1439-1449 ◽  
Author(s):  
J Barberet ◽  
C Bruno ◽  
E Valot ◽  
C Antunes-Nunes ◽  
L Jonval ◽  
...  
Keyword(s):  
Live Birth ◽  
Time Lapse ◽  
Added Value ◽  
High Grade ◽  
Medical Systems ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Time Lapse Imaging

AbstractSTUDY QUESTIONCan time-lapse imaging systems make it possible to identify novel early non-invasive biomarkers to predict live birth?SUMMARY ANSWERFrom mostly high-grade embryos, out of 35 morphometric, morphologic and morphokinetic variables, only pronuclei (PN) position at time of PN juxtaposition and the absence of multinucleated blastomeres at the 2-cell stage (MNB2cell), were potentially associated with live birth.WHAT IS KNOWN ALREADYPrevious studies indicate that some kinetic markers may be predictive of blastocyst development and embryonic implantation. Certain teams have suggested including some of them in decisional algorithms for embryo transfers.STUDY DESIGN, SIZE, DURATIONUsing a time-lapse incubator (EmbryoScope, Unisense FertiliTech), we retrospectively explored the associations between the morphometric, morphologic and morphokinetic parameters of oocytes, zygotes and embryos, and their associations with live birth. This study assessed 232 embryos from single embryo transfers after ICSI cycles performed between January 2014 and December 2017.PARTICIPANTS/MATERIALS, SETTING, METHODSThe morphometric, morphologic and morphokinetic parameters (18, 4 and 13, respectively) of oocytes, zygotes and early embryos were studied retrospectively. The associations between these parameters were examined using a Spearman’s correlation, Mann–Whitney or chi-squared test as appropriate. We examined whether these parameters were associated with outcomes in univariate and multivariate logistic regression analyses.MAIN RESULTS AND THE ROLE OF CHANCECentral PN juxtaposition was associated with a 2-fold increase in the odds of live birth (OR = 2.20; 95% CI, [1.26–3.89]; P = 0.006), while the presence of MNB2cell was associated with half the odds of live birth (OR = 0.51; 95% CI, [0.27–0.95]; P = 0.035). These two parameters were independent of embryo kinetics. The 33 remaining parameters had no significant association with the capacity of transferred embryos to develop to term.LIMITATIONS, REASONS FOR CAUTIONEven though the population size was relatively small, our analyses were based on homogeneous cycles, i.e. young women whose transferred embryos were found to be high-grade according to conventional morphology evaluation. In addition, our conclusions were established from a specific, highly selected population, so other study populations, such as women in an older age bracket, may yield different results. Finally, because we assessed day 2/3 transfers, our findings cannot be generalized to embryos cultured up to the blastocyst stage.WIDER IMPLICATIONS OF THE FINDINGSIt would be interesting to explore, prospectively, whether PN localisation is a relevant measure to predict embryo development when added into further algorithms and whether this parameter could be suitable for use in other IVF clinics. Further studies are needed, notably to explore the added value of timing evaluation in cohorts of embryos with low or intermediate morphology grade, as well as in other maternal populations (i.e. older women).STUDY FUNDING/COMPETING INTEREST(S)No external funding was used for this study. P. Sagot received funding from the following commercial companies: Merck Serono, Finox Biotech, Ferring, MSD France SAS, Teva Sante ́ SAS, Allergan France, Gedeon Richter France, Effik S.A., Karl Storz Endoscopie France, GE Medical Systems SCS, Laboratoires Genevrier, H.A.C. Pharma and Ipsen.All the authors confirm that none of this funding was used to support the research in this study. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the journal policies on sharing data and materials.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

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