Traditional detection versus computer-controlled multilevel analysis of nuclear structures from donated human embryos

BACKGROUND: Multinuclearity is known to correlate with decreased implantation and pregnancy rates. Thus, a valid detection of nuclear structures especially among otherwise good quality embryos may be of great importance in order to improve clinical outcome. In this study, we have compared traditional manual microscopic analysis with computer-controlled multilevel morphological assessment for analysis of nuclear status in human embryos. METHODS: In total, 84 donated 2- and 4-cell embryos with < or = 20% fragmentation from patients referred for IVF or ICSI treatment were included. Mono- and multinuclearity was recorded using traditional analysis as well as computer-controlled multilevel analysis of each intact embryo. Subsequently, the embryos were separated into individual blastomeres to assess the number of nuclear structures. All nuclear structures were fixed and stained for DNA. RESULTS: There was no significant difference (P = 1.0) between embryonic nuclear status detected by computer-controlled analysis of the intact embryos and of the separated blastomeres. Additionally, 100% of the fixed nuclear structures contained DNA. However, using traditional morphological analysis, significantly more embryos (26%) had incorrect nuclear status detected (P = 0.002). Further, the presence of <10% embryonic fragmentation had no impact on the correct detection of nuclear structures using the multilevel analysis. For embryos with 11-20% fragmentation, 86% of the nuclear structures detected in the separated blastomeres were found in the intact embryos. The mean diameter of nuclear structures was significantly decreased from 22.1 microm in mononucleate 2-cell embryos to 18.7 microm in mononucleate 4-cell embryos (P < 0.001). CONCLUSION: The results of this study indicate that the use of computer-controlled multilevel morphological analysis can improve the detection of nuclear structures in human embryos.     

Caspase activity in preimplantation human embryos is not associated with apoptosis

BACKGROUND: Previous studies on mammalian preimplantation embryos have suggested an association between caspase activation, blastomere fragmentation and apoptosis. However, some reports on human embryos questioned the causal relationship between blastomere fragmentation and apoptosis, and information about the presence and activity of caspases in human embryos is lacking. METHODS: A fluorochrome-labelled universal caspase inhibitor was used to visualize active caspases in blastomeres and fragments of preimplantation human embryos. RESULTS: Caspase activity was detected only after fertilization, and was rare in blastomeres but frequent in fragments. The incidence of caspase activity in blastomeres and fragments was stable between the 2-cell and 12-cell stages. Caspase-positive blastomeres were only seen in poor-morphology embryos. The percentage of caspase-positive fragments was increased in embryos with multinucleated blastomeres but was unrelated to embryo morphology. Moreover, caspase-positive fragments detached from healthy blastomeres that were isolated by embryo biopsy and subsequently underwent mitotic division in culture. CONCLUSIONS: These data suggest that caspases in preimplantation human embryos are involved in developmental processes unrelated to cell death.     

Blastomere development after embryo biopsy: a new model to predict embryo development and to select for transfer

One of the most important and unsolved problems in in-vitro fertilization is to decide which embryos are more suitable to implant and therefore should be transferred. We analysed the in-vitro development of isolated biopsied blastomeres and compared it to the development of the original embryo, in order to find a relationship that could show the embryo's potential future development and so increase implantation rates. A total of 66 normally fertilized human embryos were biopsied at the 6- to 10-cell stages. At day 6, blastomeres were counted by nuclear labelling. A total of 33 embryos (50%) reached the blastocyst stage. Of the isolated blastomeres, 63% divided and 53% cavitated over 3 days in culture. Of the blastomeres taken from embryos that developed to the blastocyst stage, 88% divided, 79% cavitated, 76% divided and cavitated and 9% neither divided nor cavitated. In those from arrested embryos, 39% divided (P < 0.001), 21% cavitated (P < 0.001), 15% divided and cavitated (P < 0.001) and 55% neither divided nor cavitated (P < 0.001). Blastomeres biopsied from embryos that reached the blastocyst stage showed a significantly higher proportion of division and cavitation than those originated from arrested embryos. Culture of the isolated blastomeres can demonstrate those embryos more likely to develop to the blastocyst stage and that are probably more suitable to implant. Cryopreserving biopsed embryos and culturing blastomeres would increase implantation rates. Embryos can then be selected according to the blastomere development and thawed for transfer in a future cycle.     

Parameters guiding selection of best embryos for transfer after cryopreservation: a reappraisal

BACKGROUND: The purpose of this study was to evaluate the respective influences of blastomere survival and resumption of mitosis on the outcome of frozen-thawed embryos. METHODS: A retrospective analysis was performed in our centre on 363 thawing cycles, involving 4-cell day 2 grade 1 embryos with <10% fragmentation. RESULTS: A higher implantation rate per transferred embryo was observed when all transferred embryos were characterized by fully intact blastomeres (100% blastomere survival) as compared with damaged embryos (50 or 75% blastomere survival) (22.0 versus 7.2%; P < 0.0001). Moreover, the implantation rate per transferred embryo was significantly higher for cleaved embryos compared with uncleaved embryos (19.7 versus 3%; P < 0.0001). Transfer of fully intact, cleaved embryos resulted in the highest implantation rates compared with transfer of damaged and uncleaved embryos (27.4 versus 0%; P < 0.0001). Intermediate implantation rates were observed when only one of the two criteria was fulfilled (13 versus 11% respectively; P > 0.05). Multivariate analysis showed that the clinical pregnancy rate was influenced by both criteria (odds ratio = 3.4 for transfer of embryos with six or more cells versus embryos with less than six cells. CONCLUSION: The results of our study suggest that the most important factor to predict further embryo development is the total number of blastomeres in transferred embryos, however they are obtained (good survival and/or resumption of mitosis).     

Construction of an evidence-based integrated morphology cleavage embryo score for implantation potential of embryos scored and transferred on day 2 after oocyte retrieval

BACKGROUND: Evidence-based morphological embryo scoring models for ranking of implantation potential are still scarce, and the need for a precise model increases when aiming for singleton pregnancies. METHODS: Prospectively, 2266 IVF/ICSI double-embryo, day 2 transfers were studied. The five variables scored in 3- to 5-step scales for the embryos transferred are blastomere number (BL), fragmentation, blastomere size variation ('equality', EQ), symmetry of the cleavage and mononuclearity in the blastomeres (NU). The scoring results of embryos with an individual traceability from scoring to implantation, i.e. treatments resulting in either no implantation (n=1385) or twin implantation (n=228), were studied for prognostic potential. RESULTS: Although all five variables correlated highly with implantation potential, only BL, NU and EQ remained independently significant after regression analysis. The equation thus derived formed the basis for a 10-point integrated morphology cleavage (IMC) embryo score. A table with the scoring point for each possible combination of the embryo variables is presented. The scoring model was statistically validated on the singleton pregnancy group (n=653). CONCLUSIONS: We suggest that this IMC embryo scoring, incorporating cleavage stage and information on the variation in blastomere size and the number of mononucleated blastomeres, may optimize embryo ranking and selection for day 2 transfers.     

The frequency and developmental capability of human embryos containing multinucleated blastomeres

The frequency of multinucleated blastomeres (MNB) in 2- and 4-cell stage human embryos was recorded immediately before embryo transfer using a high-power inverted microscope. About 44% of patients (150/338) possessed embryos exhibiting MNB. The appearance of this nuclear abnormality was not correlated with maternal age. Overall, 15% of the otherwise good quality embryos (274/1885) that developed after monospermic fertilization contained several multinuclei (from two to seven) in at least one cell. Quite often MNB were found within all cells of the embryo (50% in 2-cell embryos). Blastomere multinucleation was significantly higher in 2-cell than 4-cell embryos (P <0.0001). This suggests that a considerable number of human embryos become abnormal during the first embryonic division. The embryos containing MNB were usually excluded for uterine transfers, with the exception of 19 cases when only such embryos could be replaced (6%; 19/338 patients). The results demonstrated that embryos with MNB may implant (4/19 cases; 21%) and they can lead to both spontaneous abortions and the successful birth of healthy infants (two cases). The fact that in the successful cases, 2-cell stage embryos with a mononucleated and a binucleated blastomere were transferred also suggests that due to the cell totipotency, development of a healthy baby is possible from one normal blastomere. Since multinucleation in early embryos may reflect gross chromosomal abnormalities or development of mosaic embryos, it is advisable not to replace embryos with MNB. Occasional transfers, however, can be considered because defective embryos may sometimes develop normally.      

Water and DMSO membrane permeability characteristics of in-vivo- and in- vitro-derived and cultured murine oocytes and embryos

Although embryo cryopreservation is routine for many mammalian species, it is important to know how the fundamental cryobiology of these cells changes with development. Progressive cleavage divisions result in a reduction in the blastomere surface area available for water and cryoprotectant mass transport. Therefore, the membrane permeability of murine oocytes, zygotes, 2-cell, 4-cell, and 8-cell embryos to water (Lp), and dimethylsulphoxide (PDMSO), and the reflection coefficient, sigma (sigma) were determined. Oocytes or zygotes were recovered, cumulus cells removed, then cultured until use. Oocytes and embryos were immobilized and perfused with treatment solutions at 24 degrees C. Osmotically induced cell volume changes over time were videotaped followed by image analysis. The Lp values in the presence of dimethylsulphoxide (DMSO) were 0.77, 0.81, 0.94, 0.86, and 1.10 microm/min/atm, and the PDMSO values were 1.85, 2.04, 2.41, 1.95, and 1.25x10(-3) cm/min for oocytes, zygotes, 2, 4, and 8-cell embryos respectively. The Lp values in the presence of DMSO were significantly (P < 0.05) higher than those in the absence of DMSO. Treating the whole embryo as a single osmotic entity leads to significantly (P < 0.05) elevated PDMSO estimates relative to those based upon measurements of individual blastomeres. These data indicate that both Lp and PDMSO estimates are lower when predicted on an individual blastomere basis. The data also show that neither Lp nor PDMSO differ among oocytes, zygotes, 2-cell and 4-cell embryos. However, the significantly higher Lp and lower PDMSO of the 8-cell stage support the hypothesis that fundamental cryobiological differences may require developmental stage- specific embryo cryopreservation protocols.      

Morphometric categorization of the human oocyte and early conceptus

Morphometrical procedures were used to quantitatively evaluate human oocytes and embryos in an IVF programme. The metaphase II oocyte was an irregular 3.5 x 10(6) microns 3 sphere of 1.05 coefficient of form. The ooplasmic volume of 1.4 x 10(6) microns 3 was reduced by 10% by fertilization. The zona pellucida behaved as a stable and almost spherical envelope of 1.8 x 10(6) microns 3 volume and 17 microns thickness. Through the first three cleavages, mean blastomere reduces 28.5% volume per division, evolving from an irregular spherical shape with 0.9 coefficient of form to an ellipsoid (0.8) at the 8-cell stage. The coefficient of diversity between sister blastomeres progressively moved from 1.4 to 1.6 during the first two (2n) cleavages. The coefficient of diversity also increased at 3-cell (2.2) and 6-cell (2.6) asynchronous divisions. Morphologically abnormal embryos showed some morphometrical differences. Embryos which successfully implanted and progressed to birth showed a higher coefficient of diversity between sister blastomeres.     

Human embryos with unevenly sized blastomeres have lower pregnancy and implantation rates: indications for aneuploidy and multinucleation

Uneven blastomere cleavage in human embryos of 'good morphology', i.e. those normally used for transfer, is a phenomenon which has been poorly investigated. The main objective in this study was to probe deeper into the aetiology behind previous findings that embryos with uneven cell cleavage have a lower developmental capacity in comparison with evenly cleaved embryos. Our hypothesis was that uneven cleavage may result in embryos with a higher degree of aneuploidy and/or multinuclear rate, which in turn might help to explain their low implantation rate. In the first part of the study, 378 embryo transfers performed over a 3-year period were analysed retrospectively, where all the transferred embryos in each cycle were of identical morphology score and cleavage stage. In the second part of the study, multicolour fluorescence in-situ hybridization (FISH) analyses on good quality embryos, representing the uneven (n = 11) and even (n = 13) study groups were performed. When comparing day 2 transfers between 4-cell embryos, it was found that unevenly cleaved embryos had significantly lower implantation (23.9 and 36.4%) and pregnancy rates (37.6 and 52.9%) compared with evenly cleaved embryos. A significantly higher degree of aneuploidy (29.4 and 8.5%) and multinuclear rate (21.1 and 2.1%) in blastomeres from uneven embryos was also found. It is concluded that uneven blastomere cleavage has a negative effect on both pregnancy and implantation rates in human IVF, and that this can partly be explained by a higher degree of aneuploidy/multinuclear rate. In the light of the results obtained, a new approach in the current embryo scoring system, placing more emphasis on blastomere size, is recommended.     

Morphological evaluation of human embryos and derivation of an embryo quality scoring system specific for day 3 embryos: a preliminary study

A scoring system specific for day 3 embryos has not been extensively explored. Most IVF laboratories continue to grade embryos solely on the basis of cell number and percentage fragmentation as was traditionally done for day 2 embryos. Additional morphological features, some unique to day 3 embryos, may be useful in selecting embryos most likely to blastulate and implant. The objective of this study was to derive an embryo scoring system for day 3 transfers which is predictive of positive pregnancy outcomes. A total of 316 transferred embryos from 93 patients was recorded on videotape and evaluated. The following parameters were used to grade the embryos: cell number, fragmentation pattern (FP), cytoplasmic pitting, compaction, equal sized blastomeres, blastomere expansion and absence of vacuoles. The clinical pregnancy rate was 41.9%, with an implantation rate of 18% per embryo transferred. The mean number of embryos transferred per patient was 3.4. Three formulae were derived to score embryo quality in each transfer based on the average score of individual embryos transferred. In the first scoring system, cell number alone was used to predict pregnancy outcome. The second scoring system was based on blastomere number and the observed FP. The third scoring system utilized both blastomere number and FP but also combined this with five morphological criteria to yield a final day 3 embryo quality (D3EQ) score. We found the D3EQ score to be prognostic of pregnancy outcome. This study suggests that although cell number and FP are certainly predictors of positive pregnancy outcomes, additional parameters specific to day 3 embryos should be used to stratify a cohort of embryos further.     

Spontaneous blastomere fusion after freezing and thawing of early human embryos leads to polyploidy and chromosomal mosaicism

The incidence of blastomere fusion after cryopreservation of early human embryos (day 2 and day 3) was investigated using the standard propanediol technique. The process of fusion was observed in all developmental stages (from 2 to 10 cells) and the frequency of this event was 4.6% in day 2 (41/889) and 1.5% in day 3 (10/646) embryos that survived the thawing (embryos with 50-100% intact cells). Fusion of two, and occasionally of several, blastomeres resulted in the formation of multinucleated hybrid cells, which clearly indicated that the ploidy of these newly created cells had been altered. This event, depending on the number of fused cells per embryo, transformed the embryos into either entirely polyploid embryos (complete fusion at 2- or 3-cell stage) or into mosaics being a mixture of polyploid and normal cells. Chromosomal preparations of embryos affected by blastomere fusion indicated the presence of tetraploid mitotic plates. Also, fluorescence in-situ hybridization (FISH) analysis using DNA probes targeting unique sequences on chromosomes 9, 15, 17 and 22 indicated the existence of tetraploid and diploid fluorescence signals in the interphase nuclei within mosaics. Therefore, observations on live and fixed embryos suggested that tetraploid (4n) or hexaploid (6n) and tetraploid-diploid or more complex aberrations of ploidy might be formed as a consequence of blastomere fusion. Furthermore, this demonstrates that freezing and thawing may induce numerical chromosomal changes in human embryos.     

Fertilization and early embryology: The presence of multinucleated blastomeres in human embryos is correlated with chromosomal abnormalities

The purpose of the present study was to determine whether thepresence of one or more multinucleated blastomeres during earlyembryonic development is associated with chromosomal abnormalitiesIn sibling blastomeres of that embryo. Embryos with multinucleatedcells (n = 47) detected on day 2 or 3 of development were comparedto dividing embryos without multinucleation. Arrested embryoswere excluded from this study. Chromosome abnormalities weredetected using fluorescent in-situ hybridization (FISH) withX, Y, 18 and 13/21 chromosome- specific probes. Of 47 embryosincluded in this study, 76.6% were chromosomafly abnormal, comparedto 50.9% in the control group (P < 0.001). Excluding aneuploidy,which is originated in the gametes and not the embryo, the differenceswere even higher, with 74.5% of multinucleated embryos beingchromosomafly abnormal compared to 32.3% of non-multinucleatedembryos (P < 0.001). Day of multinucleation appearance, numberof nuclei per cell, number of multinucleated cells per embryoand developmental quality of the embryos as well as the typeof fertilization (intracytoplasmic sperm injection versus standardinsemination) were not found to affect the rate of chromosomalabnormalities In embryos with multinucleated cells. These resultssuggest that embryos with multinucleated cells may not be suitablefor replacement and should be excluded unless no other embryosare available.     

The development of cytogenetically normal, abnormal and mosaic embryos: a theoretical model

Assisted reproduction and preimplantation genetic diagnosis (PGD) involve various complicated techniques, each of them with its own problems. However, the greatest problem with PGD for chromosome abnormalities is not of a technical nature but is a biological phenomenon: chromosomal mosaicism in the cleavage stage embryo. Here, we present a hypothetical, quantitative model for the development of chromosomally normal, abnormal and mosaic embryos. The arising of mosaicism in 2-8-cell embryos was described by a binomial probability model on the occurrence of mitotic events inducing chromosomal changes in the blastomeres. This model converted the 'mean' rate of mosaicism found in cross-sectional studies (60%) into an equal rate of mosaic embryos at arrival at the 8-cell stage (59.8%). The disappearance of > 90% of the mosaic embryos or the mosaicism itself from surviving embryos during the morula stage was explained by mitotic arrest of most of the mitotically changed cells under increasing cell cycle control. In our model, 25.9 and 14.3% of the embryos at the 8-cell stage are normal and abnormal respectively. The remaining 59.8% of the embryo shows mosaicism: 34.6% of abnormal/normal cells and 25.2% of abnormal/abnormal cells. The high proportion of abnormal and mosaic embryos together explains the high rate of abnormal laboratory findings in PGD for chromosomal abnormalities and aneuploidy screening. The poor representation of a 1- or 2-cell biopsy for the 7- or 6-cell post-biopsy embryo in the case of mosaicism explains the high rate of false-negative and false-positive results.     

Assessment of the viability and pregnancy potential of mouse embryos biopsied at different preimplantation stages of development

The developmental potential in vitro and in vivo of preimplantation mouse embryos biopsied at the 4-cell, 8-cell and morula stages were investigated. Biopsy had the least impact when performed at the 8-cell stage. There was no effect of biopsy on the development of 8-cells of blastocysts in vitro (95% compared with 99% of controls) or the implantation rate after transfers (82 versus 87%, P greater than 0.05); however, fewer embryos (52 versus 71%, P less than 0.05) resulted in viable fetuses. There was no effect of biopsy at the 8-cell stage on fetal weight on day 17. Blastocyst formation in vitro was significantly less for 4-cell biopsies compared with their controls (76 versus 90%, P less than 0.001) and biopsy also affected the implantation rate (44 versus 59%, P less than 0.01). Biopsy was most detrimental when performed on morulae, reducing the implantation rate from 65% for controls to 21% for biopsies (P less than 0.001). Fetal viability was also markedly affected with a reduction on day 17 from 42 to 26% accompanied by a significant reduction (24%, P = 0.02) of the mean fetal weight. Handling of embryos for biopsy at the morula stage, which involved removal of the zona pellucida, was a significant but not complete cause of the reduced implantation potential observed (sham-controls and intact-controls: 34 and 65%, P less than 0.001), while puncture of the zona during the biopsy of 4-cell and 8-cell embryos had no effect. Therefore, the 8-cell mouse embryo is the most suitable state for embryo biopsy.     

Influence of ovarian stimulation with HP-hMG or recombinant FSH on embryo quality parameters in patients undergoing IVF

BACKGROUND: There are limited data on the impact of different gonadotrophin preparations on embryo quality. METHODS: This evaluation was part of a randomized, assessor-blind, multinational trial, conducted in 731 women undergoing IVF after stimulation with highly purified human menopausal gonadotropin (HP-hMG; MENOPUR) (n = 363) or recombinant FSH (rFSH; GONAL-F) (n = 368). Ongoing pregnancy was the primary end-point [HP-hMG 27% and rFSH 22%; odds ratio (OR) (95% confidence interval, CI) 1.25 (0.89-1.75)]. All 7535 oocytes retrieved were evaluated daily until day 3 (embryo transfer) in a blinded manner both by local site embryologists and a central panel of three embryologists. RESULTS: The proportion of top-quality embryos per oocyte retrieved was higher with HP-hMG (11.3%) compared with rFSH (9.0%) (P = 0.044) in the local assessment, but comparable in the central assessment (9.5 and 8.0%, respectively). Significant differences in favour of HP-hMG were observed for number of blastomeres and degree of fragmentation, while uniformity of blastomere sizes, localization of fragments, frequency of multinucleation and homogeneous cytoplasm were comparable between HP-hMG and rFSH. The live birth, ongoing pregnancy and ongoing implantation rates for top-quality embryos were higher with HP-hMG than rFSH [48 versus 32% (P = 0.038), 48 versus 32% (P = 0.038), 41 versus 27% (P = 0.032)]. Both the proportion of embryos with at least 50% surviving blastomeres after cryopreservation and embryos resuming mitosis were more frequent with HP-hMG compared with rFSH. CONCLUSIONS: Composition of gonadotrophin preparations used during ovarian stimulation has an impact on some embryo quality parameters. The capacity to implant of the top-quality embryos derived from stimulation with HP-hMG appears to be improved, although the mechanism needs to be elucidated.     

Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development

BACKGROUND: To examine the relationship between the meiotic spindle, the first cleavage plane and any resulting influence on embryonic development parameters. METHODS: Sibling oocytes (n = 246) were allocated to either a control [polar body (PB)-aligned] or a treatment (spindle-aligned) microinjection group by use of a random numbers table. Spindles were identified by PolScope((R)) and the early embryo development parameters, and angle of first cleavage plane in relation to a defined animal-vegetal pole were analysed. RESULTS: Most oocytes (92.7%) had a visible spindle at the time of microinjection; however, 62.6% of first PBs (1PBs) were not located above the spindle (average deviation 37.3 +/- 33.2 degrees; range 0-176.6), with 6.9% of 1PBs in the opposite hemisphere to the spindle. The second PBs (2PBs) can also have an unpredictable deviation from the position of the meiotic spindle (12.5 +/- 16.7 degrees; range 0-91.8). This increased when the 1PB was above the spindle, forming a physical barrier to extrusion (average 24.7 +/- 16.1 degrees; range 7.9-91.8). Embryos developing from the spindle-aligned microinjection group had significantly more blastomeres per embryo (P = 0.044), a higher morphology score per embryo (P = 0.008) and a significantly higher average embryo score parameter (P = 0.003), with more embryos developing without any detectable fragmentation (P < 0.05) than the PB-aligned control group. Non-fragmented embryos undergo meridional cleavage, with a small angle between the spindle location and first cleavage plane (16.4 +/- 14.0 degrees ) compared with embryos with some degree of fragmentation (P = 0.002). This angle increased with the degree of fragmentation, with worst quality embryos having a spindle:cleavage angle of 45.1 +/- 17.7 degrees. CONCLUSIONS: The 1PB and, to a lesser degree, the 2PB can be unreliable predictors of the exact meiotic spindle location in human oocytes. Embryos from spindle-aligned oocytes have an increase in all measured development parameters over control siblings. When the animal pole is defined as the meiotic spindle location, non-fragmented embryos tend to develop from a meridional cleavage; with the most fragmented embryos developing from a more equatorial initial cleavage plane. This study proposes that the spindle accurately marks the animal pole in human oocytes, and provides evidence linking the meiotic spindle location to the first cleavage plane and resulting early embryo development parameters in human embryos.     

Embryo evaluation by analysing blastomere nuclei

BACKGROUND: To create a more effective selection standard for early embryos, we developed a new grading system consisting of conventional morphological evaluation in combination with analysis of blastomere nuclei. METHODS: A total of 744 embryos used during 459 cycles of embryo transfer on day 2 and blastocyst transfer were subjected to retrospective analysis. The overall implantation rate was 15.5% (115/744). Morphological evaluation of the embryos was performed on day 2 by referring to both the size of blastomere and fragmentation (conventional method) and the nucleic features of the blastomeres--either multinucleated or anucleic (nuclei counting method). The implantation rate for every transferred embryo and blastocyst was examined. RESULTS: Although a high implantation rate was observed with the highest quality embryos as judged by either the conventional method (24.1%; 57/237) or the nuclei counting method (26.1%; 104/399), the nuclei counting method predicted implantation rate better than the conventional method. The embryos that were considered to be high quality according to the conventional method, but low quality according to the nuclei counting method, had a limited implantation success rate of 6.3% (4/66). Also, after blastocyst transfer, implantation occurred most often when high quality embryos evaluated by the nuclei counting method were used (25.5%; 25/98), while the blastocysts from low quality embryos seldom implanted (3.2%; 2/63). CONCLUSIONS: When choosing which embryo to transfer, the normality of blastomere nuclei may be a more important index of quality than standard fragmentation features and/or blastomere uniformity analysis. When choosing among embryos, if nucleic status is identical, then embryos with the least fragmentation should be chosen. Moreover, in blastocyst transfer, a blastocyst whose nuclei were judged normal on day 2 should be selected on day 5 over any other blastocysts.     

Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres

Summary A detailed fate map of all the progeny derived from each of the blastomeres of the 4- and 8-cell stage South African clawed frog (Xenopus laevis) embryo is presented. Each “identified” blastomere that results from stereotypic cleavages has a characteristic set of progeny that distinguishes it from the other blastomeres of the embryo. The 4-cell dorsal (D) blastomere is the major progenitor of the stomodeum, cement gland, retina, notochord, head somite, pharynx and liver. The 4-cell ventral (V) blastomere is the major progenitor of the trunk and fin epidermis, ventral somite, nephrotome, lateral plate mesoderm and proctodeum. The other organs are derived from both blastomeres. At the next cell division, the animal hemisphere daughters of both blastomeres (D1 and V1, respectively) become the major progenitors for head ectodermal and mesodermal structures, and the vegetal hemisphere daughters become the major progenitors for trunk mesodermal (D2) or trunk endodermal (V2) structures. Semiquantitative lineage diagrams, using data from this and from previous studies demonstrate that as cleavage proceeds from the 2- to the 32-cell stage, the progenitors for particular organs or for specific regions of organs segregate into defined regions of the blastula. To determine whether this segregation is related to the position of the blastomere or to its geneological lineage, we compared the fates of radial 8-cell blastomeres to those of stereotypic 8-cell blastomeres. Radial blastomeres have fates nearly equivalent to the sum of the two 16-cell blastomeres that occupy the same position in the embryo, demonstrating that fate depends upon blastomere position rather than lineage.