Harnessing cytoplasmic particles movement of the human early embryo analysed by advanced imaging and artificial intelligence to predict development to blastocyst stage

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p > 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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P–187 Multinucleation and reverse cleavage, sings of early embryo self-repair mechanisms

Human Reproduction ◽

10.1093/humrep/deab130.186 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

A Munuer. Puigvert ◽

V. Montalv Pallès ◽

J Mass. Hernáez ◽

A García-Faura ◽

B Marquè. López-Teijón ◽

...

Keyword(s):

Embryo Development ◽

Culture Media ◽

Live Birth Rate ◽

Time Lapse ◽

Blastocyst Stage ◽

Early Embryo ◽

Control Group ◽

Complete Fusion ◽

Depth Analysis ◽

Self Repair

Abstract Study question Have multinucleation and reverse cleavage any effect on embryo development and clinical outcomes on IVF treatments? Summary answer Embryos capable of repairing dysmorphisms and developing up to blastocyst stage keep intact their ability to become healthy babies. What is known already Time-lapse systems allow IVF laboratories to perform in-depth analysis of embryo development using the continuous monitoring tool. Some events that are impossible to detect with conventional morphologic evaluation, such as reverse cleavage or multinucleation, can be detected using time-lapse. Even though the low scientific evidence, the presence of these events is considered a negative factor when the embryo quality assessment is performed. However, it has been described the possibility that embryos have self-repair intrinsic methods. Study design, size, duration Retrospective study including data from 3,577 cycles with 21,274 embryos cultured until blastocyst stage using one-step culture media in time-lapse incubators (Embryoscope, Vitrolife) up to day 5/6 between 2014 and 2019. Participants/materials, setting, methods Three embryo groups were considered: Control group, embryos without multinucleation or reverse cleavage (CG; n = 16,897); Multinucleation group, embryos with at least one blastomere multinucleated on D + 2/3 (MNC; n = 3,879) and Reverse Cleavage group, embryos undergoing complete fusion of two blastomeres on D + 2/3 (RC; n = 498). Single embryo transfer was performed on blastocyst stage. Clinical outcome rates were compared between groups and analyzed by Chi-square test. Main results and the role of chance As published by other groups, the 2.3% of our embryos showed at least one reverse cleavage event and we observed multinucleation in the 18.2% of the embryos. Blastocyst rate of dysmorphism groups was significantly lower (p < 0.05) than Control group (MNC=20.0%; RC = 27.7%; CG = 58.0%). Once transferred, MNC and RC evolutive embryos showed significantly lower pregnancy (MNC=47.9%; RC = 46.8%; CG = 60.8%; p < 0.05) and clinical pregnancy rates (MNC=39.4%; RC = 40.4% CG = 50.6%; p < 0.05) than the Control group (p < 0.05). However, during the post-implantational development the negative effect of dysmorphisms disappears, reaching values of live birth rate comparable to the Control group (MNC=28.3%; RC = 31.9% CG = 33.8%; p = 0.17). These results prove the importance of blastocyst culture and the inherent capability of the embryos to overcome some abnormal dynamics as multinucleation and reverse cleavage. Thus, these embryos showing the poor-prognosis events can be considered for transfer or vitrify. Limitations, reasons for caution There is a wide difference on sample size between groups despite the fact that the statistical analysis considers that into account. There are some ongoing pregnancies in all groups. Wider implications of the findings: When analyzing the development of embryos undergoing reverse cleavage and multinucleation, we hypothesize that these embryos could be showing a self-correction mechanism for some type of error detected. Embryos capable of repairing and developing up to blastocyst stage keep intact their ability to become healthy babies. Trial registration number Not applicable

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213 HOXB9 PROTEIN IS PRESENT THROUGHOUT EARLY EMBRYO DEVELOPMENT IN THE BOVINE

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab213 ◽

2013 ◽

Vol 25 (1) ◽

pp. 255

Author(s):

C. Sauvegarde ◽

D. Paul ◽

R. Rezsohazy ◽

I. Donnay

Keyword(s):

Embryo Development ◽

Inner Cell Mass ◽

Cell Mass ◽

Mature Oocyte ◽

Blastocyst Stage ◽

Early Embryo ◽

Immature Oocyte ◽

Cell Stage ◽

Morula Stage ◽

Oocyte Stage

Hox genes encode for homeodomain transcription factors well known to be involved in developmental control after gastrulation. However, the expression of some of these genes has been detected during oocyte maturation and early embryo development. An interesting expression profile has been obtained for HOXB9 in the bovine (Paul et al. 2011 Mol. Reprod. Dev. 78, 436): its relative expression increases between the immature oocyte and the zygote, further increases at the 5- to 8-cell stage to peak at the morula stage before decreasing at the blastocyst stage. The main objective of this work is to establish the HOXB9 protein profile from the immature oocyte to the blastocyst in the bovine. Bovine embryos were produced in vitro from immature oocytes obtained from slaughterhouse ovaries. Embryos were collected at the following stages: immature oocyte, mature oocyte, zygote (18 h post-insemination, hpi), 2-cell (26 hpi), 5 to 8 cell (48 hpi), 9 to 16 cell (96 hpi), morula (120 hpi), and blastocyst (180 hpi). The presence and distribution of HOXB9 proteins were detected by whole-mount immunofluorescence followed by confocal microscopy using an anti-human HOXB9 polyclonal antibody directed against a sequence showing 100% homology with the bovine protein. Its specificity to the bovine protein was controlled by Western blot on total protein extract from the bovine uterus and revealed, among a few bands of weak intensities, 2 bands of high intensity corresponding to the expected size. Oocytes or embryos were fixed and incubated overnight with rabbit anti-HOXB9 (Sigma, St. Louis, MO, USA) and mouse anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ, USA) primary antibodies and then for 1 h with goat anti-rabbit Alexafluor 555 conjugated (Cell Signaling Technology, Beverly, MA, USA) and goat anti-mouse FITC-conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Embryos were then mounted in Vectashield containing DAPI. HOXB9 is detected from the immature oocyte to the blastocyst stage. At the immature oocyte stage, it is mainly localised in the germinal vesicle with a weak signal in the cytoplasm. At the mature oocyte stage, HOXB9 labelling is present in the cytoplasm. At the zygote stage, a stronger immunoreactivity is observed in the pronuclei than in the cytoplasm. From the 2-cell stage to the morula stage, the presence of HOXB9 is also more important in the nuclei than in the cytoplasm. HOXB9 is also observed at the blastocyst stage where it is localised in the nuclei of the trophectoderm cells, whereas an inconstant or weaker labelling is observed in the inner cell mass cells. In conclusion, we have shown for the first time the presence of the HOXB9 protein throughout early bovine embryo development. The results obtained suggest the presence of the maternal HOXB9 protein because it is already detected before the maternal to embryonic transition that occurs during the fourth cell cycle in the bovine. Finally, the pattern obtained at the blastocyst stage suggests a differential role of HOXB9 in the inner cell mass and trophectoderm cells. C. Sauvegarde holds a FRIA PhD grant from the Fonds National de la Recherche Scientifique (Belgium).

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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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65 Improvement of bovine early embryo development in vitro by coculture with endometrial epithelial cells

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab65 ◽

2019 ◽

Vol 31 (1) ◽

pp. 158

Author(s):

M. Sponchiado ◽

W. F. A. Marei ◽

P. E. J. Bols ◽

M. Binelli ◽

J. L. M. R. Leroy

Keyword(s):

Epithelial Cells ◽

Primary Culture ◽

Epithelial Mesenchymal Transition ◽

Blastocyst Stage ◽

Early Embryo ◽

Phenol Red ◽

Functional Characteristics ◽

Indirect Contact ◽

Mesenchymal Transition

We optimized a bovine endometrial epithelial cell (BEEC) line as a valuable research model for the study of very early embryo-maternal interactions in vitro. In this study, we aimed to (1) characterise the BEEC monolayers along the primary culture and first passages with respect to the expression of epithelial and mesenchymal cell markers and abundance of functional key transcripts; (2) to test whether direct or indirect contact with endometrial cells alter the quality of the embryos in vitro; and (3) to test the specificity of the effect. In Exp. 1, after isolation from slaughterhouse uteri at the early luteal phase, BEEC were cultured in DMEM/F12 phenol red-free medium supplemented with 10% fetal bovine serum (FBS) from primary culture until subculture 3. Fixed samples were immunostained for cytokeratin and vimentin. Transcript abundances for cellular lineage markers (KRT18 and VIM), oestrogen receptor (ESR1), interferon α/beta receptor 1 (IFNAR1), and prostaglandin G/H synthase 1 (PTGS1) and 2 (PTGS2) were evaluated by real-time quantitative PCR. Statistical analyses were carried out by ANOVA and Tukey test. Immunofluorescence data revealed that the BEEC line co-expresses cytokeratin together with a mesenchymal marker (Vimentin). This indicates that these epithelial cells underwent an epithelial-mesenchymal transition in vitro. Gene expression data showed a 6-fold increased (P<0.001) abundance of VIM mRNA from the primary culture to the subculture 1, which remained constant until subculture 3; however, KRT18, ESR1, IFNAR1, PTGS1, and PTGS2 were similar between the passages, suggesting that the cells conserved their functional characteristics. In Exp. 2, groups of 15 morulas (Day 5.5) were cultured in SOF medium supplemented with 5% FBS in the absence (control) or in the presence (co-culture) of BEEC at passage 2, for 48h. Embryos were placed on direct or indirect contact with a BEEC monolayer using a 96-well insert containing 8μm pores. Developmental rates were compared by chi-square test and P-values were adjusted by Tukey’s test. The percentage of embryos that had developed from morula into blastocyst stage on Day 7.5 was significantly higher in the direct and indirect contact co-culture (65%; P<0.05) groups compared with the control (53%) group. Moreover, 63% of the blastocysts were expanded, hatching, or hatched in the co-culture groups, whereas a rate of 46% was found in the control counterparts (P<0.05). In Exp. 3, the same experimental conditions from Exp. 2 were used, but groups of 15 Day 5.5 morulas were cultured in control, or conditioned medium from BEEC (CondBEEC) or bovine fibroblasts (CondFib). Blastocyst development rate on Day 7.5 was higher in the CondBEEC group (71%; P<0.001) compared with the control (54%) and CondFib (50%) groups. In conclusion, based on the markers studied, BEEC monolayers undergo epithelial-mesenchymal transition in vitro but preserve functional characteristics after few passages. The co-culture system improves bovine embryonic development from morula into blastocyst stage. This support is BEEC specific and does not rely on a direct cell-to-embryo contact.

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Epigenetic asymmetry in the mammalian zygote and early embryo: relationship to lineage commitment?

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1326 ◽

2003 ◽

Vol 358 (1436) ◽

pp. 1403-1409 ◽

Cited By ~ 74

Author(s):

Wolf Reik ◽

Fatima Santos ◽

Kohzoh Mitsuya ◽

Hugh Morgan ◽

Wendy Dean

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Blastocyst Stage ◽

Early Embryo ◽

Lineage Commitment ◽

Imprinted Genes ◽

Mammalian Development ◽

Paternal Genome ◽

Developmental Defects ◽

Set Up

Epigenetic asymmetry between parental genomes and embryonic lineages exists at the earliest stages of mammalian development. The maternal genome in the zygote is highly methylated in both its DNA and its histones and most imprinted genes have maternal germline methylation imprints. The paternal genome is rapidly remodelled with protamine removal, addition of acetylated histones, and rapid demethylation of DNA before replication. A minority of imprinted genes have paternal germline methylation imprints. Methylation and chromatin reprogramming continues during cleavage divisions, but at the blastocyst stage lineage commitment to inner cell mass (ICM) or trophectoderm (TE) fate is accompanied by a dramatic increase in DNA and histone methylation, predominantly in the ICM. This may set up major epigenetic differences between embryonic and extraembryonic tissues, including in X–chromosome inactivation and perhaps imprinting. Maintaining epigenetic asymmetry appears important for development as asymmetry is lost in cloned embryos, most of which have developmental defects, and in particular an imbalance between extraembryonic and embryonic tissue development.

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Effect of protein supplementation on development to the hatching and hatched blastocyst stages of cat IVF embryos

Reproduction Fertility and Development ◽

10.1071/rd01135 ◽

2002 ◽

Vol 14 (5) ◽

pp. 291 ◽

Cited By ~ 26

Author(s):

N. W. Kurniani Karja ◽

Takeshige Otoi ◽

Masako Murakami ◽

Minori Yuge ◽

Mokhamad Fahrudin ◽

...

Keyword(s):

Bovine Serum ◽

Culture Medium ◽

Cell Number ◽

Blastocyst Stage ◽

Early Embryo ◽

Protein Supplementation ◽

Vitro Fertilization ◽

Fetal Bovine ◽

The Mean

The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earle's balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P<0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula or blastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P<0.05) and hatching blastocyst stages (P<0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P<0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.

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Harnessing cytoplasmic particles movement of the human early embryo analysed by advanced imaging and artificial intelligence to predict development to blastocyst stage

10.1101/2020.11.26.399832 ◽

2020 ◽

Author(s):

Giovanni Coticchio ◽

Giulia Fiorentino ◽

Giovanna Nicora ◽

Raffaella Sciajno ◽

Federica Cavalera ◽

...

Keyword(s):

Neural Network ◽

Artificial Intelligence ◽

Performance Metrics ◽

Short Term Memory ◽

Research Question ◽

Time Lapse ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Embryo Viability ◽

Movement Velocity

AbstractResearch QuestionProgress in artificial intelligence (AI) and advanced image analysis offers unique opportunities to develop novel embryo assessment approaches. In this study, we tested the hypothesis that such technologies can extract and harness novel information derived from cytoplasmic movements of the early human embryo to predict development to blastocyst.DesignIn a proof-of principle study, an artificial neural network (ANN) approach was undertaken to assess retrospectively 230 human preimplantation embryos. After ICSI, embryos were subjected to time-lapse monitoring for 44 hours. For comparison as a standard embryo assessment methodology, a single senior embryologist assessed each embryo to predict development to blastocyst stage (BL) based on a single picture frame taken at 42 hours of development. In the experimental approach, in embryos that developed to blastocyst or destined to arrest (NoBL), cytoplasm movement velocity (CMV) was recorded by time-lapse monitoring during the first 44 hours of culture and analysed with a Particle Image Velocimetry (PIV) algorithm to extract quantitative information. Three main AI approaches, the k-Nearest Neighbor (k-NN), the Long-Short Term Memory Neural Network (LSTM-NN) and the hybrid ensemble classifier (HyEC) were employed to classify the two embryo classes.ResultsBlind operator assessment classified each embryo in terms of ability of development to blastocyst, reaching a 75.4% accuracy, 76.5% sensitivity, 74.3% specificity, 74.3% precision and 75.4% F1 score. After integration of results from AI models together with the blind operator classification, the performance metrics improved significantly, with a 82.6% accuracy, 79.4% sensitivity, 85.7% specificity, 84.4% precision and 81.8% F1 score.ConclusionsThe present study suggests the possibility to predict human blastocyst development at early cleavage stages by detection of CMV and AI analysis. This indicates the importance of the dynamics of the cytoplasm as a novel and valuable source of data to assess embryo viability.

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