Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

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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

Reproduction Fertility and Development ◽

10.1071/rd19223 ◽

2019 ◽

Vol 31 (12) ◽

pp. 1862 ◽

Cited By ~ 3

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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175 BOVINE EMBRYO DEVELOPMENT AND MRNA EXPRESSION IN BLASTOCYSTS CULTURED IN ISOLATED MOUSE OVIDUCTS MAINTAINED IN SOF OR KSOM

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab175 ◽

2006 ◽

Vol 18 (2) ◽

pp. 195

Author(s):

D. Rizos ◽

B. Pintado ◽

J. de la Fuente ◽

P. Lonergan ◽

A. Gutierrez-Adan

Keyword(s):

Embryo Development ◽

Relative Abundance ◽

Culture Medium ◽

Embryo Quality ◽

Blastocyst Stage ◽

Glut 1 ◽

Gene Transcripts ◽

Culture Environment ◽

Chi Square Analysis

It is well known that modification of the post-fertilization culture environment of mammalian pre-attachment embryos can affect blastocyst quality, manifested in terms of morphology, cryotolerance, and relative abundance of certain gene transcripts. Culture of in vitro-produced bovine zygotes in the ewe oviduct leads to the development of blastocysts of a quality similar to those derived totally in vitro (Rizos et al. 2002 Biol. Reprod. 66, 589-595). However, such a system has disadvantages from a practical and animal welfare point of view. The isolated mouse oviduct (IMO) culture system is a potential alternative and has been successfully used in the in vitro culture of mouse, rat, hamster, and pig embryos from the one-cell stage to the morula/blastocyst stage. The aim of this study was to examine (1) the development of bovine zygotes in the IMO maintained in two different media (SOF and KSOM) in organ culture, and (2) the quality of the resultant blastocysts assessed in terms of the relative abundance of transcripts for several genes that have been previously implicated in embryo quality. Mouse oviducts were isolated from adult Swiss females (CD1, Harlan) the day after mating with an intact male. Approximately 10-15 presumptive bovine zygotes, produced by in vitro oocyte maturation and fertilization, were transferred to the ampullae of the isolated oviducts and were cultured in Transwell plates (Costar, Corning, NY, USA) over 1.1 mL of culture medium (SOF, n = 241 or KSOM, n = 320) at 39�C in an atmosphere of 5% CO2 in air at maximum humidity. A control group of embryos was cultured in droplets (25 �L) of the same culture medium and conditions in parallel (SOF, n = 278, KSOM, n = 225). Five replicates (=days of bovine ovary collection) were carried out. Following 6 days of culture, embryos were recovered from the oviducts/culture drops and blastocysts were snap-frozen in liquid nitrogen. Quantification of all gene transcripts was carried out by real time quantitative RT-PCR. Data on embryo development were analyzed by chi-square analysis and differences in transcript abundance by ANOVA. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 21.0 vs. 21.9%; KSOM: 22.0 vs. 22.2%). Culture in the IMO in SOF resulted in an increase (P d 0.05) in the abundance of transcripts for Oct-4 and SOX and reduced abundance of Glut-1, Na/K transporter, Cx43, and survivin, compared to control embryos. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and a reduced expression of Na/K transporter and SOX. Transcripts for G6PDH, IFN, and E-Cad were unaffected by culture environment. In conclusion, culture in the IMO leads to alterations in the relative abundance of transcripts that have been previously associated with embryo quality following culture in the ewe oviduct. However, the effect is dependent on the basal medium used.

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197 DOES DURATION OF BOVINE OOCYTE MATURATION IN VITRO AFFECT THE SPEED OF EMBRYO DEVELOPMENT IN VITRO AND SEX RATIO AT THE TWO-CELL OR BLASTOCYST STAGE?

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab197 ◽

2008 ◽

Vol 20 (1) ◽

pp. 177

Author(s):

P. Bermejo-Álvarez ◽

A. Gutiérrez-Adán ◽

P. Lonergan ◽

D. Rizos

Keyword(s):

Sex Ratio ◽

Embryo Development ◽

Blastocyst Stage ◽

Early Embryo ◽

Blastocyst Development ◽

Maturation In Vitro ◽

Maturational Stage ◽

Development In Vitro ◽

Oviduct Fluid

The faster-developing blastocysts in IVC systems are generally considered more viable and better able to survive following cryopreservation or embryo transfer than those that develop more slowly. However, evidence from several species indicates that embryos that reach the blastocyst stage earliest are more likely to be males than females. The aim of this study was to determine whether the duration of maturation could affect early embryo development and, furthermore, the sex ratio of early- or late-cleaved embryos and blastocysts. Cumulus–oocyte complexes were matured in vitro for 16 h (n = 2198) or 24 h (n = 2204). Following IVF, presumptive zygotes from each group were examined every 4 h between 24 and 48 h postinsemination (hpi) for cleavage, and all embryos were cultured to Day 8 in synthetic oviduct fluid to assess blastocyst development. Two-cell embryos at each time point and blastocysts on Days 6, 7, and 8 from both groups were snap-frozen individually for sexing. Sexing was performed with a single PCR using a specific primer BRY. There was a significantly lower number of cleaved embryos from the 16-h compared with the 24-h maturation group at 28 (10.0 � 1.51 v. 28.8 � 3.57%), 32 (35.3 � 1.48 v. 57.6 � 3.33%), 36 (54.8 � 1.76 v. 67.4 � 2.81%), 40 (63.3 � 1.82 v. 72.0 � 2.54%), and 48 (70.6 � 1.78 v. 77.1 � 2.18%) hpi, respectively (mean � SEM; P d 0.05). However, the blastocyst yields on Day 6 (17.1 � 3.11 v. 16.4 � 2.11%), 7 (30.6 � 4.10 v. 34.6 � 3.51%), or 8 (34.1 � 3.90 v. 39.4 � 4.26%) were similar for both groups (mean � SEM; 16 v. 24 h, respectively). Significantly more 2-cell early cleaved embryos (up to 32 hpi) were male compared with the expected 1:1 ratio from both groups (16 h: 1.24:0.76 v. 24 h: 1.17:0.83, P ≤ 0.05); however, the overall sex ratio among 2-cell embryos was significantly different from the expected 1:1 in favor of males only for the 16-h group (1.18:0.82, P ≤ 0.05). The sex ratio of blastocysts on Day 6, 7, or 8 from both groups was not different from the expected 1:1. However, the total number of male blastocysts obtained after 8 days of culture from the 24-h group was significantly different from the expected 1:1 (1.19:0.81, P ≤ 0.05) and approached significance in the 16-h group. These results show that the maturational stage of the oocyte at the time of fertilization has an effect on the kinetics of early cleavage divisions but not on blastocyst yield. Furthermore, irrespective of the duration of maturation, the sex ratio of early-cleaving 2-cell embryos was weighted in favor of males, and this observation was maintained at the blastocyst stage.

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Using Time Lapse Monitoring for Determination of Morphological Defect Frequency in Feline Embryos after in Vitro Fertilization (IVF)

Animals ◽

10.3390/ani10010003 ◽

2019 ◽

Vol 10 (1) ◽

pp. 3 ◽

Cited By ~ 1

Author(s):

Barbara Kij ◽

Joanna Kochan ◽

Agnieszka Nowak ◽

Wojciech Niżański ◽

Sylwia Prochowska ◽

...

Keyword(s):

Time Lapse ◽

Blastocyst Stage ◽

Hatching Rate ◽

Vitro Fertilization ◽

First Results ◽

Morphological Defects ◽

Defect Frequency ◽

Selection Of

Some human, bovine, and mouse in vitro fertilized (IVF) embryos with morphokinetic abnormalities such as fragmentation, direct cleavage, and cytoplasmic vacuoles have the potential to reach the blastocyst stage, which is related to a high potential for implantation. The latest techniques of embryo development observation to enable the evaluation and selection of embryos are based on time lapse monitoring (TLM). The aim of this study was to determine the frequency of morphological defects in feline embryos, their competence to reach the blastocyst stage, and their ability to hatch. Oocyte-cumulus complexes were isolated after the scarification of ovaries and matured in vitro. Matured oocytes were fertilized in vitro by capacitated spermatozoa. Randomly selected oocytes were observed by TLM for seven-to-eight days. Out of 76 developed embryos, 41 were morphologically normal, of which 15 reached the blastocyst stage. Of 35 abnormally developed embryos, 17 reached the blastocyst stage, of which six had single aberrations and 11 had multiple aberrations. The hatching rate (%) was 15.6% in normally cleaving embryos, 6.25% in embryos with single aberrations, and 3.33% in those with multiple aberrations. The present study reports the first results, found by using TLM, about the frequency of the morphological defects of feline embryos, their competence to reach the blastocyst stage, and their ability to hatch.

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Time-lapse imaging of cleavage divisions in embryo quality assessment

Reproduction ◽

10.1530/rep-17-0004 ◽

2017 ◽

Vol 154 (2) ◽

pp. R37-R53 ◽

Cited By ~ 23

Author(s):

Robert Milewski ◽

Anna Ajduk

Keyword(s):

Embryo Quality ◽

Biological Significance ◽

Time Lapse ◽

Reproductive Outcome ◽

Future Perspectives ◽

Morphokinetic Parameters ◽

Current Review ◽

Predictive Algorithms ◽

Time Lapse Imaging

In vitrofertilization (IVF) is one of the most important procedures for treating infertility. As several embryos are usually produced in a single IVF cycle, it is crucial to select only the most viable ones for transfer to the patient. Morphokinetics, i.e. analysis of the dynamics of cleavage divisions and processes such as compaction and cavitation, has provided both biologists and clinicians with a new set of data regarding embryonic behaviour during preimplantation development and its association with embryo quality. In the current review, we focus on biological significance of morphokinetic parameters and show how they can be used to predict a reproductive outcome. We also explain the statistics behind the predictive algorithms and discuss the future perspectives of morphokinetics.

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P–144 Undisturbed culture in time-lapse systems improves embryo development and quality

Human Reproduction ◽

10.1093/humrep/deab130.143 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

T A Vilori. Samochin ◽

M A Valera ◽

L Bori ◽

F Meseguer ◽

J M D Lo. Santos ◽

...

Keyword(s):

Embryo Development ◽

Embryo Culture ◽

Culture Medium ◽

Embryo Quality ◽

Time Lapse ◽

Blastocyst Stage ◽

Culture Conditions ◽

Single Step ◽

Ovum Donation ◽

Blastocyst Rate

Abstract Study question Does culture in integrated time-lapse systems (TLS) improve embryo development and blastocyst quality compared to conventional benchtop incubators (CI), within the same IVF laboratory? Summary answer Under similar conditions, culture in TLS resulted in a significant increase in blastocyst rate, top quality blastocyst rate and proportion of biopsied embryos per treatment What is known already Integrated TLS have the potential of delivering a stable and undisturbed environment throughout the whole embryo culture, avoiding taking them out for assessment. However, there is still lack of quality evidence of the performance of these incubators compared to CI at supporting embryo culture until blastocyst stage. Studies abording this issue are still scarce, heterogeneous and have a small sample size. Although some authors have reported an improvement in embryo development and quality using TLS, global results are inconsistent. To our knowledge, the present study evaluates the effect of TLS on embryo quality on the largest sample size yet. Study design, size, duration Unicentric retrospective cohort study including 14248 ICSI treatments from 2016 to October 2020, with both autologous and donated oocytes. We compared blastocyst rate (BR) and proportion of top-quality blastocysts (TQB=Morphology ASEBIR score A) per treatment between those using TLS (N = 7500) and CI (N = 6748), and the proportion of embryos biopsied (EB) in cycles with pre-implantation genetic testing (PGT-A; N = 2642). We performed a sub-analysis in treatments using single-step culture medium (N-TLS=4398, N-CI=1140) in both types of incubators. Participants/materials, setting, methods Embryo cohorts were cultured until blastocyst stage in one of 3 TLS: EmbryoScope, EmbryoScope Plus (Vitrolife,) and Geri (Genea Biomedx), or in a CI (ASTEC). Embryo quality was assessed following ASEBIR morphological criteria. Culture protocols and media changed during the included time period. For that reason, we did a sub-study in the treatments performed since the implementation of Gems® (Genea Biomedx) single-step (SS) culture medium in all incubators. Statistical analysis was done using ANOVA tests. Main results and the role of chance Treatments were differently distributed and heterogeneous in terms of number of oocytes obtained per patient, so we stratified the analysis according to ovum origin and compared mean rates per cycle instead of total number of embryos per group. BR was statistically higher (P < 0,001) in the TLS group, in both autologous (62,98±29,37% vs 59,49±31,09% in CI) and oocyte donation treatments (69,25±22,07% vs 66,27±23,28% in CI). Proportion of TQB was also significantly higher in the TLS in both types of cycles (P < 0,001): 3,60±12,29% in TLS vs 2,27±9,71% in CI in autologous cycles, 8,68±15,31% in TLS vs 7,32±14,02% CI in ovum donation cycles. Results were corroborated in the SS media sub-study (P < 0,05): BR was 63,87±29,23% in TLS vs 57,53±30,61% in CI with autologous oocytes, and 70,76±21,63% in TLS vs 67,39±22,68% in CI with donated oocytes; TQB rates were 3,66±12,06% in TLS vs 2,05±9,26% in CI in autologous treatments and 8,81±15,21% in TLS vs 6,84±12,91% in CI in ovum donation treatments. Regarding PGT-A treatments, we found no significant difference in the biopsy rate in the total comparison, although the rate significantly increased in the TLS group since the implementation of single-step medium (52,36±24,69% in TLS vs 48,63±22,56% in CI; P = 0,007) Limitations, reasons for caution Not only culture conditions varied over time, but also the number of TLS in the laboratory, which increased lately. Hence, even though the most recent treatments included in the all-SS sub-study are more homogeneous in terms of culture conditions, they are unbalanced regarding the distribution among incubators. Wider implications of the findings: Our results demonstrate the superiority of TLS coupled with single-step culture media against traditional embryo culture systems at supporting embryo development. The optimal environment provided by TLS enhances embryo development until blastocyst stage as well as their quality, increasing the cumulative chances of getting a life-birth for each patien. Trial registration number Not applicable

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Preliminary morphokinetic annotation data for early equine embryo development in vitro using a time-lapse imaging system

Journal of Equine Veterinary Science ◽

10.1016/j.jevs.2016.04.063 ◽

2016 ◽

Vol 41 ◽

pp. 72

Author(s):

Karen Schnauffer ◽

Niamh Lewis ◽

Stephen Troup ◽

Dai Grove-White ◽

Caroline McG. Argo

Keyword(s):

Embryo Development ◽

Imaging System ◽

Time Lapse ◽

Annotation Data ◽

Development In Vitro ◽

Time Lapse Imaging

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The use of electronic health tools from the very beginning of life: Time-lapse embryo monitoring

Medicinski pregled ◽

10.2298/mpns1912389b ◽

2019 ◽

Vol 72 (11-12) ◽

pp. 389-393

Author(s):

Artur Bjelica ◽

Maja Soc ◽

Marijana Despotovic-Zrakic

Keyword(s):

In Vitro Fertilization ◽

Success Rate ◽

Embryo Development ◽

Embryo Quality ◽

Human Life ◽

Time Lapse ◽

Fertilization Success ◽

Vitro Fertilization ◽

Electronic Health

Introduction. In addition to the already widespread use of electronic health services in monitoring the health status of patients, one should also refer to the possibility of using electronic health tools from the very beginning of life by monitoring growth and development of the embryo. In this way, the concept of electronic health would expand to cover the whole human life, from its very beginning to the end. Monitoring the embryo development. The efforts to improve in vitro fertilization success rate have been accompanied by the introduction of various procedures, including the evaluation of the embryo quality. A detailed monitoring of the kinetics of embryo development is achieved by laboratory techniques that provide continuous insight into the embryo development through applying time-lapse monitoring system. Embryo quality evaluation programs based on time-lapse monitoring. Special bioinformatic programs have been developed for automatic analysis of images obtained by time-lapse monitoring which allow a quantitative evaluation of the key moments in the development of the embryo and its morphology. Implementation and limitations of time-lapse embryo monitoring. The majority of authorities in the field of in vitro fertilization consider the use of time-lapse monitoring as a great advancement in the in vitro fertilization technology, obviously leading to a higher success rate. Conclusion. The systems for time-lapse monitoring of embryos represent powerful tools which help clinicians involved in in vitro fertilization and embryologists to select the best embryos, with the aim of improving the in vitro fertilization success rate. Despite all the advantages, these systems also have some shortcomings and limitations.

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111. THE ROLE OF L-PROLINE IN PREIMPLANTATION MOUSE EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/srb10abs111 ◽

2010 ◽

Vol 22 (9) ◽

pp. 29 ◽

Cited By ~ 1

Author(s):

S. Ozsoy ◽

M. B. Morris ◽

M. L. Day

Keyword(s):

Amino Acids ◽

Embryo Development ◽

Preimplantation Embryo ◽

Blastocyst Stage ◽

Low Density ◽

Mouse Embryo Development ◽

Preimplantation Mouse Embryo ◽

Preimplantation Mouse ◽

Development In Vitro

Amino acids are known to play important roles in preimplantation embryo development, including regulation of cell volume and metabolism. Inclusion of l-glutamine, glycine and betaine in embryo culture medium has been shown to improve development in vitro by acting as organic osmolytes, thereby regulating cell volume. The purpose of the present study was to examine the effect of l-proline on preimplantation mouse embryo development in vitro. One-cell stage embryos were cultured in modified HTF, at low density (1 embryo/100 μL) and high density (1 embryo/μL) in the presence and absence of amino acids. Development of the embryos was scored every 24 h until the blastocyst stage. At low density, l-proline significantly increased the proportion of embryos developing to the blastocyst stage. This effect was abolished by culture at high density, suggesting that l-proline was activating a pathway similar to that involved in autocrine signalling by trophic factors in the preimplantation embryo. The improvement in development observed in the presence of l-proline was not due to its action as an organic osmolyte since the osmolality of the medium was 270 mOsm. Furthermore, glycine and betaine, which are known to act as osmolytes in embryos, had no effect on blastocyst development. In embryonic stem cells L-proline is taken up by an amino acid transporter and is involved in the regulation of growth and differentiation (1). The present data suggest that l-proline may have a similar, important role in preimplantation development. (1) JM Washington, J Rathjen, F Felquer, A Lonic, MD Bettess, N Hamra, L Semendric, BSN Tan, J-A Lake, RA Keough, MB Morris and PD Rathjen (2010) Am J Physiol Cell Physiol 298: C982–C992.

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Embryonic disc formation following post-hatching bovine embryo development in vitro

Reproduction ◽

10.1530/rep-20-0243 ◽

2020 ◽

Vol 160 (4) ◽

pp. 579-589 ◽

Cited By ~ 1

Author(s):

Priscila Ramos-Ibeas ◽

Ismael Lamas-Toranzo ◽

Álvaro Martínez-Moro ◽

Celia de Frutos ◽

Alejandra C Quiroga ◽

...

Keyword(s):

Embryo Development ◽

Blastocyst Stage ◽

Embryo Morphology ◽

Bovine Embryo ◽

Pluripotency Marker ◽

Embryonic Disc ◽

Development In Vitro ◽

Apoptosis And Necrosis ◽

Disc Formation

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

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