Blastocyst Morphology Research Articles

Time of early cleavage affects the developmental potential of feline preimplantation embryos in vitro

The study compared developmental competence of embryos, based on the timing of the first cleavage and morula formation, with their subsequent ability to reach the blastocyst stage and the resulting blastocyst morphology and quality. Cleaved embryos were separated at 18 hours post insemination (hpi), 24 hpi, and 30 hpi and then cultured in droplets to follow the individual embryo development. The significantly higher percentage of the blastocyst formation was noted in the group I of embryos cleaving within less than 18 hpi (12.7%) compared with the group II cleaving within 18 to 24 hpi (10.7%), None of the late-cleaving embryos (group III >24–30 hpi) reached blastocyst stage in our experiment. Interestingly, the hatching ability was similar regardless the time of the first cleavage (I: <18 hpi; 6.4% and II: 18–24 hpi; 5.4%). The ability to hatch was correlated with the time of morula formation; only embryos that reached morula on Day 4 or Day 5 were able to develop into hatching blastocyst (8.4% and 3.3%, respectively). The differential cell staining revealed significantly more blastomeres in blastocysts obtained from embryos cleaving within 18 to 24 hpi than the blastocysts obtained from embryos cleaving less than 18 hpi (188.6 ± 21.9 vs. 129.3 ± 16). Embryos cleaving within 18 to 24 hpi also demonstrated the higher number and percentage of embryoblast cells (97.2 ± 12.6, 51.6 ± 2.9 vs. 46.6 ± 9, 36.3 ± 6.6). The presented results confirmed the association among the onset of the first cleavage, time reaching morula, and subsequent blastocyst formation and quality.

Does euploid embryo ranking by trophectoderm cell mitochondrial dna content correspond with ranking by blastocyst morphology within an individual patient’s cohort of blastocysts?

Does euploid embryo ranking by trophectoderm cell mitochondrial dna content correspond with ranking by blastocyst morphology within an individual patient’s cohort of blastocysts?

Blastocyst morphology is a poor indicator of euploid status

Blastocyst morphology is a poor indicator of euploid status

Are early morphokinectic parameters predictive of monozygotic splitting after single embryo transfer?

Extended in vitro culture has improved the ability to select the optimal embryo for transfer from both a genomic and morphokinetic perspective, though some have suggested that the delay in transferring embryos back to the uterus may be a risk factor for monozygotic twinning (MZT). Due to its rare incidence and IVF’s longstanding practice to return multiple embryos at transfer, a clear link between blastocyst morphology and MZT has yet to be demonstrated. Since MZT gestations confer additional perinatal morbidity and mortality, we undertook this study to assess whether early morphokinetic findings were correlated with monozygotic twinning.

Pregnancy and birth outcomes following fresh or vitrified embryo transfer according to blastocyst morphology and expansion stage, and culturing strategy for delayed development

How do live birth rates (LBRs), following fresh and vitrified/warmed embryo transfer, compare according to morphological grade, developmental stage and culturing strategy of human blastocysts in vitro? Equivalent LBRs were obtained after fresh embryo transfer and after vitrified/warmed embryo transfer of blastocysts of top or non-top quality, while vitrification after prolonged embryo culture of blastocysts with delayed development had a positive impact on LBR. Blastocyst morphology correlates with clinical outcome; however, few data are available on vitrified/warmed embryo transfer using non-top quality blastocysts. The aim of this study was to determine clinical outcomes of non-top quality blastocysts and blastocysts with delayed development that underwent vitrified/warmed embryo transfer. This retrospective, single-centre study (conducted January 2009 to June 2013) compared 1010 fresh embryo transfer and 1270 vitrified/warmed embryo transfer of blastocysts originating from the same stimulation cycle. Furthermore, 636 fresh embryo transfers and 304 vitrified/warmed embryo transfer after delayed expansion or blastulation in the same period were also analysed. Clinical outcomes after fresh and vitrified/warmed embryo transfer according to blastocyst morphology were compared in both groups. Similar LBRs after fresh embryo transfer or after vitrified/warmed embryo transfer of top or non-top quality blastocysts were observed. A statistically significant improvement in clinical outcomes was obtained after vitrified/warmed embryo transfer of Day 5 embryos with delayed expansion or blastulation when applying prolonged culture. Our study suggests that vitrification of non-top quality blastocysts as well as delayed cavitating and blastulating Day 5 embryos should be considered in autologous IVF cycles. Given that the present retrospective study used aseptic vitrification of blastocysts, the results, particularly the survival rates, may not be fully applicable to other vitrification protocols. The retrospective nature of the study has to be mentioned. Restriction of vitrification to top quality blastocysts may result in discarding potentially viable embryos. This study was not externally funded. There are no conflicts of interest to declare.

Total Cell Number and its Allocation to Trophectoderm and Inner Cell Mass in In Vitro Obtained Cats' Blastocysts.

The aim of this study was to evaluate the developmental kinetics of cats' blastocysts in connection with their morphology and blastomeres allocation to trophoblast or embryoblast cells. We examined gross blastocyst morphology and the total number of blastomeres together with its allocation to inner cell mass (ICM) or trophectoderm (TE) cells in pre-implantation feline embryos obtained from 6th to 9th day of invitro culture. From all the investigated embryos, 61.8% developed to early blastocyst, 37.4% to full and 7.6% to hatching blastocyst stage. The total cell number (TCN) varied form 58 cells in early day 6 to 245 in hatching day 8 blastocyst, with the mean 84.9 cells in early, 156.7 in full, and 204.4 in hatching ones. Day 8 blastocyst had the highest number of total cells, together with the highest mean number of ICM regardless of its morphological assessment. Early blastocyst (apart from day 6) had the highest number of arrested cells, while dead cells were the highest in full day 9 blastocyst. More data about the relationship between blastocyst development and morphology would facilitate the selection of optimal blastocysts for further procedures.

Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitrified/warmed single blastocyst transfer cycles

To evaluate the independent effects of the degree of blastocoele expansion and re-expansion and the inner cell mass (ICM) and trophectoderm (TE) grades on predicting live birth after fresh and vitrified/warmed single blastocyst transfer. Retrospective study. Reproductive medical center. Women undergoing 844 fresh and 370 vitrified/warmed single blastocyst transfer cycles. None. Live-birth rate correlated with blastocyst morphology parameters by logistic regression analysis and Spearman correlations analysis. The degree of blastocoele expansion and re-expansion was the only blastocyst morphology parameter that exhibited a significant ability to predict live birth in both fresh and vitrified/warmed single blastocyst transfer cycles respectively by multivariate logistic regression and Spearman correlations analysis. Although the ICM grade was significantly related to live birth in fresh cycles according to the univariate model, its effect was not maintained in the multivariate logistic analysis. In vitrified/warmed cycles, neither ICM nor TE grade was correlated with live birth by logistic regression analysis. This study is the first to confirm that the degree of blastocoele expansion and re-expansion is a better predictor of live birth after both fresh and vitrified/warmed single blastocyst transfer cycles than ICM or TE grade.

Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies.

STUDY QUESTIONIs blastocyst biopsy and quantitative real-time PCR based comprehensive chromosome screening a consistent and reproducible approach across different biopsy practitioners?SUMMARY ANSWERThe blastocyst biopsy approach provides highly consistent and reproducible laboratory and clinical outcomes across multiple practitioners from different IVF centres when all of the embryologists received identical training and use similar equipment.WHAT IS KNOWN ALREADYRecently there has been a trend towards trophectoderm (TE) biopsy in preimplantation genetic screening (PGS)/preimplantation genetic diagnosis (PGD) programmes. However, there is still a lack of knowledge about the reproducibility that can be obtained from multiple biopsy practitioners in different IVF centres in relation also to blastocysts of different morphology. Although it has been demonstrated that biopsy at the blastocyst stage has no impact on embryo viability, it remains a possibility that less experienced individual biopsy practitioners or laboratories performing TE biopsy may affect certain outcomes. We investigated whether TE biopsy practitioners can have an impact on the quality of the genetic test and the subsequent clinical outcomes.STUDY DESIGN, SIZE, DURATIONThis longitudinal cohort study, between April 2013 and December 2014, involved 2586 consecutive blastocyst biopsies performed at three different IVF centres and the analysis of 494 single frozen euploid embryo transfer cycles (FEET).PARTICIPANTS/MATERIALS, SETTING, METHODSSeven biopsy practitioners performed the blastocyst biopsies in the study period and quantitative PCR was used for comprehensive chromosome screening (CCS). The same practitioner performed both the biopsy and tubing procedures for each blastocyst they biopsied. To investigate the quality of the biopsied samples, the diagnostic rate, sample-specific concurrence and the cell number retrieved in the biopsy were evaluated for each biopsy operator. Clinical outcomes following FEET cycles were stratified by biopsy operator and compared. Cellularity of the biopsy sample was also correlated with clinical outcomes.MAIN RESULTS AND THE ROLE OF CHANCEThe seven practitioners performed 2586 biopsies, five in centre IVF-1 and one in each of the other two IVF centres (IVF-2 and IVF-3). Overall, 2437 out of 2586 (94.2%) blastocyst biopsies resulted in a conclusive diagnosis, 119 (4.6%) showed a nonconcurrent result and 30 (1.2%) failed to amplify, suggesting the absence of TE cells in the test tube or presence of degenerated/lysed cells only. Among the samples producing a conclusive diagnosis, a mean concurrence value of 0.253 (95% CI = 0.250–0.257) was observed. Logistic regression analysis adjusted for confounding factors showed no differences in the diagnosis rate and in the concurrence of the genetic analysis between different biopsy practitioners. An overall mean number of 7.32 cells (95% CI = 6.82–7.81; range 2–15) were predicted from all biopsies. Higher cellularity was significantly associated with a better quality of the CCS diagnosis (P < 0.01) and with the conclusive diagnosis rate, with nonconcurrent samples showing significantly lower numbers of cells (2.1; 95% CI=1.5–2.7) compared with samples resulting in a conclusive diagnosis (mean cells number 7.5; 95% CI = 7.1–7.9, P < 0.01). However, no differences were recorded between different biopsy practitioners regarding cellularity of the biopsy. Finally, logistic analysis showed no impact of the biopsy practitioners on the observed ongoing rates of implantation, biochemical pregnancy loss and miscarriage after the FEET cycles.LIMITATIONS, REASONS FOR CAUTIONThese data come from a restricted set of laboratories where all of the embryologists received identical training and use identical equipment. A single TE biopsy method and CCS technology was used and these data particularly apply to PGS programmes using blastocyst biopsy without zona opening at the cleavage stage and using qPCR-based CCS. To make firm conclusions on the potential impact of biopsy on biochemical pregnancy loss and miscarriages according to practitioner and biopsy cellularity, a larger sample size is needed.WIDER IMPLICATIONS OF THE FINDINGSWe reported a very high consistency and reproducibility of the blastocyst biopsy approach coupled with qPCR-based CSS for both genetic and clinical outcomes across different practitioners working in different IVF centres when appropriate training is provided and when the same laboratory setting is used. These data are important considering the trend towards the use of blastocyst biopsy worldwide for PGD/PGS applications.STUDY FUNDING/COMPETING INTEREST(S)None.

Frozen-thawed euploid blastocyst trasnsfer improves reproductive outcomes and blastocyst morphology is not critical on frozen-thawed embryo transfer

Frozen-thawed euploid blastocyst trasnsfer improves reproductive outcomes and blastocyst morphology is not critical on frozen-thawed embryo transfer

Morphokinetic analysis and embryonic prediction for blastocyst formation through an integrated time-lapse system

To describe the events associated with the blastocyst formation and implantation that occur in embryos during preimplantation development based on the largest sample size ever described with time-lapse monitoring. Observational, retrospective, single-center clinical study. University-affiliated private IVF center. A total of 7,483 zygotes from 990 first treatments of intracytoplasmic sperm injection (ICSI; 627 of oocyte donor vs. 363 autologous oocyte cycles), of which 832 blastocysts were transferred. No patient intervention. Embryos were cultured in a time-lapse monitoring system, and the embryos were transferred on day 5 after ICSI. Embryo selection was based on the multivariable model previously developed and on blastocyst morphology. Using a time-lapse system, embryo images were acquired every 15 minutes for 120 hours. Embryos cleavage time points up to the 9-cell stage (t2-t9) as well as to the morula stage (tM) and blastocyst formation (tB) were registered in hours after ICSI. Additionally, duration of the cell cycle and synchrony of the second and third cell cycles were defined. As a result, we have monitored the embryonic development of a total of 3,215 blastocysts, of which 832 were transferred. Finally, we analyzed the characteristics of embryonic development of blastocyst (phase 1) and of implanted and not implanted (phase 2) embryos as finally validated in an independent data set (phase 3). A detailed retrospective analysis of cleavage times was made for 7,483 zygotes. We analyzed 17 parameters and found several significantly correlated with subsequent blastocyst formation and implantation. The most predictive parameters for blastocyst formation were time of morula formation, tM (81.28-96.0 hours after ICSI), and t8-t5 (≤8.78 hours) or time of transition of 5-blastomere embryos to 8-blastomere embryos with a receiver operating characteristic curve (ROC) value = 0.849 (95% confidence interval [CI], 0.835-0.854; phase 1). These parameters were less predictive of implantation, with a ROC value = 0.546 (95% CI, 0.507-0.585). We also observed that time for expansion blastocyst, tEB (107.9-112.9 hours after ICSI), and t8-t5 (≤5.67 hours after ICSI) predict blastocyst implantation, with a ROC value = 0.591 (95% CI, 0.552-0.630; phase 2). The model was validated on an independent data set and gave a ROC of 0.596 (0.526-0.666; phase 3). The inclusion of kinetic parameters into score evaluation may improve blastocyst selection criteria and can predict blastocyst formation with high accuracy. We propose two multivariable models based on our findings to classify embryos according to their probabilities of blastocyst stage and implantation in the largest data set ever reported of human blastocysts.

Comparison of clinical pregnancy rates after single embryo transfer of day 5 versus day 6 euploid embryo

Comparison of clinical pregnancy rates after single embryo transfer of day 5 versus day 6 euploid embryo

Does blastocyst morphology predict pregnancy outcomes in euploid single embryo transfers?

Comprehensive chromosome screening (CCS) is a powerful tool which allows for selection of euploid embryos to improve pregnancy outcomes in IVF cycles. There is limited data suggesting a correlation between blastocyst morphology of euploid embryos and pregnancy outcomes. The objective of this study was to determine whether blastocyst morphology could be used to predict implantation potential of a euploid embryo.

Blastocyst morphology and chromosome status: correlation in women < age 35

Blastocyst morphology and chromosome status: correlation in women < age 35

The impact of comprehensive chromosome screening on embryo selection of day 5 blastocysts

Embryo selection is a critical component of successful infertility treatment. With the development of robust blastocyst culture, biopsy and vitrification techniques, comprehensive chromosome screening (CCS) is being applied for embryo selection with significant increases in live birth rates following a euploid blastocyst transfer. The aim of this study was to examine the relationship between D5 blastocyst morphology, chromosome constitution, and the impact on embryo selection.

Blastocyst Morphology Holds Clues Concerning The Chromosomal Status of The Embryo.

Embryo morphology has been proposed as an alternative marker of chro- mosomal status. The objective of this retrospective cohort study was to investigate the association between the chromosomal status on day 3 of embryo development and blas- tocyst morphology. A total of 596 embryos obtained from 106 cycles of intra- cytoplasmic sperm injection (ICSI) followed by preimplantation genetic aneuploidy screening (PGS) were included in this retrospective study. We evaluated the relation- ship between blastocyst morphological features and embryonic chromosomal altera- tion. Of the 564 embryos with fluorescent in situ hybridization (FISH) results, 200 reached the blastocyst stage on day 5 of development. There was a significantly high- er proportion of euploid embryos in those that achieved the blastocyst stage (59.0%) compared to embryos that did not develop to blastocysts (41.2%) on day 5 (P<0.001). Regarding blastocyst morphology, we observed that all embryos that had an abnormal inner cell mass (ICM) were aneuploid. Embryos with morphologically normal ICM had a significantly higher euploidy rate (62.1%, P<0.001). As regards to the trophectoderm (TE) morphology, an increased rate of euploidy was observed in embryos that had nor- mal TE (65.8%) compared to embryos with abnormal TE (37.5%, P<0.001). Finally, we observed a two-fold increase in the euploidy rate in high-quality blastocysts with both high-quality ICM and TE (70.4%) compared to that found in low-quality blastocysts (31.0%, P<0.001). Chromosomal abnormalities do not impair embryo development as ane- uploidy is frequently observed in embryos that reach the blastocyst stage. A high-quality blastocyst does not represent euploidy of chromosomes 13, 14, 15, 16, 18, 21, 22, X and Y. However, aneuploidy is associated with abnormalities in the ICM morphology. Further studies are necessary to confirm whether or not the transfer of blastocysts with low-quality ICM should be avoided.

Timing of First Cleavage of the Human Embryo Predicts a Successful Pregnancy by Assisted Reproductive Technologies: A Retrospective Study

Abstract: The absence of reliable markers to identify viable embryos for transfer at the early cleavage stage may contribute to the generally low implantation rates and high incidence of multiple births associated with IVF treatment. In the present study, we examined the relationships among the timing of the first cleavage, incidence of blastocyst formation in vitro, and pregnancy outcomes. All embryos (n=1,748) were examined for early cleavage at 25 h after insemination. Three groups were defined based on the persistence of two pronuclei (PN group), pronuclei breakdown (BD group), and early cleaved zygotes (EC group). Each embryo (n=1,120) was cultured until day 6 of development, and assessed for blastocyst formation. The rate of blastocysts showing good morphology was significantly higher in the EC group (44.7%) than in the BD (26.9%) or PN (13.9%) groups. The cryothawed good morphology blastocysts were transferred in 209 cycles. The clinical pregnancy rate was the highest and the abortion rate was the ...

The effect of macromolecule and growth factor combinations on in vitrodevelopment of bovine embryos

This study was conducted to determine the effects of different macromolecule sources added to synthetic oviduct fluid (SOF) culture medium supplemented with growth factors on the development of bovine embryos and blastocyst morphology. Zygotes were distributed into 5 treatment groups. Cleavage, morula, and blastocyst rates were evaluated under a stereomicroscope. Trophectoderm (TE) and inner cell mass (ICM) cells were determined by differential staining method. It was found that bovine serum albumin (BSA), either alone or in combination with growth factors, as compared to the control or polyvinyl-alcohol (PVA) resulted in higher embryo yield and faster development during early bovine embryo culture. The quality of bovine embryos, based on the number of blastocyst cells and the ratio of ICM to total blastomeres, was affected by the sources of macromolecules and their combinations with growth factors. Growth factors supplemented to SOFaa media with BSA and PVA significantly increased the number of ICM cells and the ratio of ICM cells to total number of cells. In conclusion, replacing BSA with PVA depressed the blastocyst rate and cell numbers, and the number of blastomeres and ICM and TE cell numbers were affected by both the type of macromolecule and the growth factor supplements.

Correlation between aneuploidy and blastocyst quality

Abstract Objective To carry out a detailed investigation of the impact of aneuploidy on blastocyst quality. Methods 1 257 cleavage-stage embryos from 203 patients underwent IVF treatment with aneuploidy screening were investigated. Comprehensive chromosome analysis involved the use of microarray comparative genomic hybridisation (aCGH) method on single blastomere biopsied on day 3 of embryo development. Embryo morphology was assessed and recorded on days 3 and/or 5–6 post-fertilisation. Morphologic grade A-C was assigned to the blastocysts with grade A being the highest grade and grade C the lowest. Results Among the 1 257 embryos that were analyzed, 474 (38%) were euploid, and 783 (62%) were aneuploid. 315/474 (66.5%) of euploid embyos developed into blastocysts, while 57/783 (32.8%) of aneuploid embryos grew to blastocysts ( P Conclusion Blastocyst morphology showed a significant link to aneuploidy ( P

The effects of fertilization mode, embryo morphology at day 3, and female age on blastocyst formation and the clinical outcomes

The aim of this study was to evaluate the influence of in vitro fertilization (IVF) versus intracytoplasmic sperm injection (ICSI), fertilization mode embryonic morphology at day 3, and female age on blastocyst development, on the clinical outcomes of pregnancy after blastocyst transfer. A total of 471 cycles were retrospectively investigated. The rates of blastocyst formation and of good blastocyst morphology were higher in IVF than in ICSI cycles but there were no significant differences in the clinical pregnancies or in the miscarriage rates. The rates of formation of blastocyst and of blastocysts with good morphology were significantly higher from good-morphology embryos than from poor-morphology embryos. Nevertheless, 16.9% of the poor-morphology embryos reached the blastocyst stage. The total rates of blastocyst formation, and rates of clinical pregnancy and implantation were statistically similar in the age <35, 35–39, and >39 year groups, although tending to decrease with increasing age. When equal numbers of embryos were transferred on day 3, the rates of clinical pregnancy and implantation after blastocyst transfer were significantly higher in the <35 year age group than in the 35–39 and >39 year age groups, which were not significantly different. The miscarriage rates after embryo or blastocyst transfers were not statistically different in groups of similar age. Therefore, extended embryo culture up to the blastocyst stage could be implemented for women aged younger than 35 years to increase the pregnancy rate. For older women, transfer and vitrification of available embryos at day 3 and extended culture of morphologically poor embryos to the blastocyst stage for cryopreservation may improve the clinical outcome.

Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome.

In assisted reproduction technology, embryo competence is routinely evaluated on morphological criteria. Over the last decade, efforts in improving non-invasive embryo assessment have looked into the secretome of embryos. Human embryos release genomic DNA (gDNA) and mitochondrial DNA (mtDNA) into the culture medium, and the mtDNA/gDNA ratio is significantly correlated with embryo fragmentation. Here, we investigate whether mtDNA/gDNA ratio in embryo spent medium is correlated with blastulation potential and implantation. The mtDNA/gDNA ratio was assessed in 699 Day 3 culture media by quantitative polymerase chain reaction (qPCR) to investigate its correlation with embryo morphology, blastocyst development and implantation. A logistic regression model evaluated whether mtDNA/gDNA ratio in the secretome may improve the prediction of blastulation. We found that embryos that successfully developed into blastocysts exhibited a significantly higher mtDNA/gDNA ratio in the culture medium compared with those that arrest (P = 0.0251), and mtDNA/gDNA, combined with morphological grading, has the potential to predict blastulation better than morphology alone (P = 0.02). Moreover, mtDNA/gDNA ratio was higher in the media from good-quality embryos that reached the full blastocyst stage on Day 5 compared with those that developed more slowly (P < 0.0001). With respect to blastocyst morphology, higher trophectoderm quality was associated with a higher mtDNA/gDNA ratio in the culture medium. Finally, a high mtDNA/gDNA ratio in spent medium was associated with successful implantation outcome (P = 0.0452) of good-quality embryos. In summary, the mtDNA/gDNA ratio in the Day 3 embryo secretome, in combination with morphological grading, may be a novel, non-invasive, early biomarker to improve identification of viable embryos with high developmental potential.