Computer-controlled, multilevel, morphometric analysis of blastomere size as biomarker of fragmentation and multinuclearity in human embryos

BACKGROUND: Little is known about blastomere size at different cleavage stages and its correlation with embryo quality in human embryos. Using a computer system for multilevel embryo morphology analysis we have analysed blastomeres of human embryos and correlated mean blastomere size with embryonic fragmentation and multinuclearity. METHODS: A consecutive cohort of 232 human 2-, 3- and 4-cell embryos from patients referred for ICSI treatment were included. Sequences of digital images were taken by focusing at 5- micro m intervals through the embryo. Blastomere sizes and number of nuclear structures were evaluated based on these sequences. The degree of embryonic fragmentation was evaluated by normal morphological assessment prior to transfer and correlated to the blastomere sizes. RESULTS: As a result of normal cell cleavage, mean blastomere size decreased significantly from a volume of 0.28 x 10(6) microm(3) at the 2-cell stage to 0.15 x 10(6) microm(3) at the 4-cell stage (P < 0.001). Mean blastomere size decreased significantly (P < 0.001) with increasing degree of embryonic fragmentation, where highly fragmented embryos showed a 43-67% reduction in blastomere volume compared with embryos with no fragmentation. Multinucleated blastomeres were significantly larger than non-multinucleated blastomeres (P < 0.001). On average, multinucleated blastomeres were 51.5, 67.8 and 73.1% larger than their non-multinucleated sibling blastomeres at the 2-, 3- and 4-cell stage, respectively. Furthermore, the average volume of non-multinucleated blastomeres originating from multinucleated embryos was significantly smaller than the average volume of the blastomeres from mononucleated embryos (P < 0.001). CONCLUSIONS: The results of this study show that the average blastomere size is significantly affected by degree of fragmentation and multinuclearity, and that computer-assisted, multilevel analysis of blastomere size may function as a biomarker for embryo quality.     

The genetic constitution of multinuclear blastomeres and their derivative daughter blastomeres

The presence of multinuclear blastomeres (MNB) has been widely reported in in-vitro-cultured embryos. Multinucleation at the first mitotic division and affecting both blastomeres is considered abnormal and such embryos are not transferred. The objective of this study was to use fluorescent in-situ hybridization (FISH) and probes specific for chromosomes X, Y and 18 to examine the genetic constitution of embryos developing from the 2-cell stage in which both blastomeres were bi- or multinuclear. Initially, 2-cell embryos in which both blastomeres were bi- or multinuclear were cultured further. Of 101 embryos, 89 (88.1%) cleaved further and were analysed at the 3- to 8-cell stage on day 2 or 3. Among embryos analysed, 30.4% contained only mononuclear diploid blastomeres, 35.9% had a combination of mononuclear diploid and non- diploid blastomeres, and 33.7% had non-diploid blastomeres, indicative of chaotic division. Results obtained were similar with embryos derived from in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Also, no significant differences were found between 2-cell embryos with bi- or multinuclear blastomeres or between slowly or normally cleaved embryos. Twelve (11.9%) embryos arrested at the 2-cell stage on day 3; of these, one had diploid blastomeres and the others were abnormal and highly polyploid. Subsequently, 59 embryos were analysed at the 2-cell stage. Initial observations related to the high number of nuclei in metaphase at the moment of spreading, notably when multinuclear blastomeres were observed. Genetic analysis showed 44.7% of embryos to be susceptible to analysis; the genetic constitution corresponded in both blastomeres to a diploid status. A combined diploid blastomere and abnormal blastomere was found in 4.3% of embryos; both blastomeres were abnormal in 51%. These data show that the genetic constitution of bi- or multinuclear blastomeres, and the daughter cells developing from them, are not always abnormal.      

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.     

Multinucleation in cleavage stage embryos

BACKGROUND: The aim was to analyse multinucleation in relation to its incidence in time and in the population, and its correlation with clinical variables, with other morphological characteristics and with the implantation rate of cleavage stage embryos. METHODS: Retrospective analysis of 10 388 cleaved embryos from 1395 consecutive IVF/ICSI cycles in 700 patients between January 1, 1999 and June 30, 2002. RESULTS: Multinucleation was observed in 3491 (33.6%) embryos in 1107 cycles (79.4%) of 609 (87%) patients, more frequently on day 2 than on day 3: 2848 (27.4%) versus 1567 (15.1%) [relative risk (RR) = 1.82; 95% confidence interval (CI) = 1.72-1.92]. Its incidence increased with fragmentation: 31.0, 34.4 and 36.5% for fragmentation 相似文献   

Implantation: Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers

The purpose of this study was to devise an embryo score to predictthe likelihood of successful implantation after in-vitro fertilization(IVF). Unlike most studies dealing with the influence of embryostage and morphology on pregnancy, our study was based on singlerather than multiple embryo transfers. A total of 957 singleembryo transfers were carried out. No delivery was obtainedafter any of the 99 transfers using 1-cell embryos or embryosobtained after delayed fertilization. In the remaining 858 transfers,the embryos had cleaved. Higher pregnancy rates were obtainedwith embryos displaying no irregular cells (11.7 versus 6.9%;P < 0.01) and embryos displaying no fragmentation (11.5 versus8.1%; P < 0.05). The 4-cell embryos implanted 2-fold moreoften than embryos with more or less cells (15.6 versus 7.4%;P < 0.01). Based on these observations, we devised a 4-pointembryo score in which embryos are assigned 1 point each if they(i) are cleaved, (ii) present no fragmentation, (iii) displayno irregularities, and (iv) have four cells. Both pregnancyrate and take home baby rate were significantly correlated withembryo score. Each point of this score corresponds to a 4% increasein pregnancy rate. Interestingly, pregnancy rate was significantlylower in women aged >38 years (8.2 versus 11.4%; P < 0.05),even though embryo quality was similar regardless of age. Singleembryo transfer allowed us to define a simple and useful embryoscore to choose the best embryo for transfer to optimize IVFand embryo transfer outcome. The use of this embryo score coulddecrease multiple pregnancies after multiple embryo transfers.     

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).     

The window for embryo transfer in oocyte donation cycles depends on the duration of progesterone therapy

In 192 oocyte donation cycles performed between January 1993 and July 1996, we examined the width of 'the window for embryo transfer' using standard hormonal replacement methods. All transfers were performed within 48 h of insemination. We varied the day of embryo transfer with regard to the initiation of progesterone therapy and, thus, the duration of endometrial exposure to progesterone and analysed the resulting pregnancy rates. Patients were divided into five groups (I-V) and embryo transfers were performed 2, 3, 4, 5 or 6 days following initiation of progesterone therapy. The number of pregnancies per transfer cycle achieved in groups I-V were 0 (0%), 3 (12%), 16 (40%), 29 (48.3%), and 10 (20.4%) respectively. The increased pregnancy rate in group III in comparison to group II is statistically significant (P < 0.03). Furthermore, the pregnancy rate in group IV (5 days of progesterone administration before embryo transfer) was significantly higher than in group V (6 days of progesterone administration before embryo transfer; P < 0.005). We also noted that, when embryos were transferred 4 or 5 days after initiation of progesterone therapy, the pregnancy rates were not significantly different between menopausal and cycling recipients (50% vs 43.7%). Our results indicate that the window for embryo transfer is dependent on duration of treatment with progesterone; it begins approximately 48 h after starting progesterone administration and lasts for approximately 4 days. The optimum period for transferring embryos at the 4- to 8-cell stage corresponds to cycle days 18 and 19. Transfers performed on the 17th and 20th days of the cycle can result in successful implantation, although the rates of implantation are highest when transfers are done on days 18 and 19.      

Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization

This retrospective study of 1001 in-vitro fertilization (IVF) cycles included a consecutive series of single transfers (n = 341), dual transfers (n = 410) and triple transfers (n = 250) where all the transferred embryos in each cycle were of identical quality score and identical cleavage stage. In our 2 day culture system, transfer of 4- cell embryos resulted in a significantly higher implantation rate and pregnancy rate (23 and 49%) compared with 2-cell embryos (12 and 22%) and 3-cell embryos (7 and 15%). Furthermore, the transfer of 4-cell embryos resulted in a significantly higher pregnancy rate compared with embryos that had cleaved beyond the 4-cell stage (28%). The implantation rate (21%) and pregnancy rate (43%) after transfer of embryos of score 1.0 were significantly higher than after transfer of embryos of score 2.0 (14 and 32% respectively). Transferring embryos of score 2.1 resulted in significantly higher implantation rates (26%) and similar pregnancy rates compared with score 1.0. Transferring embryos of score 2.2-3.0 resulted in a significantly lower implantation rate (5%) and pregnancy rate (15%). A striking finding was that embryos of quality score 2.0 had a significantly lower implantation rate compared with embryos of quality score 1.0 and 2.1 and a significantly lower pregnancy rate compared to embryos of quality score 1.0. We also found a lower implantation rate and pregnancy rate when transferring 3-cell embryos. These findings may indicate periods of increased sensitivity to damage during the cell cycle. In conclusion, these results substantiate the idea of the superiority of 4-cell embryos and demonstrate that minor amounts of fragments in the embryo may not be of any importance. These findings may call for a shift when weighing the two main morphological components (quality score and cleavage stage) in the sense that reaching a 4-cell cleavage stage even with the presence of a minor amount of fragments should be preferred to a 2-cell embryo with no fragments.      

Resumption of mitosis during post-thaw culture: a key parameter in selecting the right embryos for transfer

This retrospective study of 701 thaw cycles analysed the clinical importance of whether or not embryos resumed mitosis during 24 h of post-thaw culture. A total of 3360 frozen embryos were thawed; 1922 embryos survived the freeze-thaw procedure with at least one intact blastomere and were then cultured for 24 h before transfer. All transfers were registered into either the 'cleaved embryo group' (n = 459), which was defined as transfers where at least one of the transferred embryos cleaved during the post-thaw culture period, or the 'non-cleaved embryo group' (n = 153), where none of the transferred embryos cleaved during the post-thaw culture period. A total of 1408 thawed embryos were transferred in 612 cycles; 459 embryo transfers were in the cleaved embryo group, resulting in an implantation rate of 10%, significantly higher than the 4% in the non-cleaved embryo group (P = 0.0003). A total of 130 pregnancies (28% per transfer) were obtained in the cleaved embryo group which was significantly higher than the 17 pregnancies (11% per transfer) obtained in the non-cleaved embryo group (P = 0.0001). However, the average number of transferred embryos was significantly higher in the cleaved embryo group (2.46 +/- 0.03) compared to the non-cleaved embryo group (1.82 +/- 0.07). No difference was found in the age of the women between the two groups. When analysing transfers where all transferred embryos had cleaved during the post-thaw culture period the clinical pregnancy rate increased significantly from 13% transferring two embryos to 36% transferring three embryos (P = 0.0136). In this latter subgroup an implantation rate as high as 17% was obtained. The overall multiple pregnancy rate was 16%. The multiple pregnancy rate was 19% in the cleaved embryo group. In conclusion, 24 h post-thaw culture may allow a better selection of the embryos and thereby we may be able to increase the implantation and pregnancy rates. This may enable us further to reduce the number of embryos transferred.      

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.      

GRIM-19在小鼠植入前胚胎中的表达及其作用

目的 通过研究干扰素/维甲酸联合应用诱导细胞凋亡相关基因 (GRIM-19)在小鼠植入前胚胎中的表达及其与胚胎质量之间的相关性,探讨GRIM-19在小鼠植入前胚胎发育中的作用。方法 采用实时定量PCR,检测小鼠植入前胚胎（2-细胞期、4-细胞期、8-细胞期、桑葚胚和囊胚）中GRIM-19 mRNA水平 (n =16);将小鼠8 细胞期胚胎按卵裂球大小、形态、透明带、胞质碎片分为优胚组和非优胚组,分别检测两组胚胎GRIM-19 mRNA水平,并分析其相关性 (n =13);制作假孕鼠（23只）,并随机分为A组（12只）和B组（11只）,采用移植器将优质和非优质8-细胞期胚胎移入假孕鼠的子宫内,观察两组雌鼠妊娠率及产仔率。结果 GRIM-19在小鼠植入前各期胚胎中均有表达,其mRNA水平从2-细胞期逐渐增高,至8-细胞期达高峰,随后呈下降趋势。优质8-细胞胚胎的GRIM-19 mRNA水平明显高于非优胚组（P <0.05）。两组8-细胞胚胎移植后,A组雌鼠妊娠率及产仔率显著高于B组雌鼠（P<0.05）。 结论 小鼠植入前胚胎中GRIM-19的表达量随发育时程的改变而变化,且与胚胎质量密切相关。     

Effect of using slush nitrogen (SN2) on development of microsurgically manipulated vitrified/warmed mouse embryos

BACKGROUND: This study evaluated the effect of vitrification using slush nitrogen (SN(2)) on cryopreservation of micromanipulated mouse embryos. METHODS: The zona pellucida of 4-cell embryos was either left intact or dissected or dissected with biopsy of an intact blastomere. In a second study, a blastomere was destroyed and either removed (removed group) or not removed (remained group) prior to vitrification/freezing. The micromanipulated embryos were equilibrated and loaded into an open pulled straw (OPS), and plunged into liquid nitrogen (LN(2)) or SN(2). RESULTS: When using LN(2) vitrification, recovery and blastocyst formation rates of embryos were lower for zona pellucida-opened and/or blastomere-biopsied embryos compared with zona pellucida-intact embryos. Using SN(2) for vitrification resulted in increased survival and development of vitrified/warmed embryos in both the zona pellucida-opened and blastomere-biopsy groups. Similar results were observed when using embryos with a destroyed blastomere either removed or left remaining before vitrification. However, the number of total and apoptotic cells were similar for both LN(2) and SN(2). In addition, using SN(2) increased the rate of intact recovery and blastocyst formation in warmed hemi-8-cell embryos derived from the same embryo. CONCLUSIONS: These results suggest that vitrification using SN(2) is useful in cryopreservation of micromanipulated embryos obtained from a variety of programs, including assisted hatching, preimplantation genetic diagnosis and nuclear transfer.     

Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage

Energy substrate requirements (pyruvate, lactate and amino acids) were determined for in-vitro development of hamster 1- and 2-cell embryos to blastocysts, using a chemically defined, protein free medium (hamster embryo culture medium, HECM). One-cell embryos were very sensitive to energy substrate type and concentration. Pyruvate alone could not support development of 1-cell embryos to greater than 4-cells, whereas lactate as sole energy substrate supported 14% development into morulae/blastocysts. Pyruvate, with lactate and 20 amino acids, inhibited 1-cell embryo development into blastocysts relative to lactate and 20 amino acids. The highest development of 1-cell embryos to blastocysts (up to 27%) occurred with reduced lactate concentration (less than 10 mM) and either 20 amino acids or 0.2 mM glutamine. Hamster 2-cell embryos were much less sensitive to energy substrates, requiring only lactate for development to blastocysts (53%). Lactate with 20 amino acids supported 70-75% of 2-cell embryos to blastocysts. Glutamine as sole energy and nitrogen source supported development to morulae and blastocysts of some 2-cell, but not 1-cell, embryos. Pyruvate did not enhance development of 2-cell embryos. We conclude that (i) altering the types and concentrations of available energy substrates drastically changes the developmental responses of 1-cell hamster embryos in vitro and (ii) energy substrate requirements for hamster embryo development in vitro are markedly different from those of mouse embryos, the standard model for studies on preimplantation development. This is the first report of successful in-vitro culture of hamster 1-cell embryos to the blastocyst stage.     

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.     

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.     

Block to development in cultured rat 1-cell embryos is overcome using medium HECM-1.

Rat pronuclear embryos were cultured in hamster embryo culture medium-1 (HECM-1) or a modified Krebs-Ringer bicarbonate solution (mKRB). Embryo cultures in HECM-1 were also challenged with low oxygen concentrations. In HECM-1, 57.9% (70/121) of the pronuclear embryos developed into the 4-cell stage after 48 h of culture. The rates of 8-cell, morula and blastocyst formation were 32.2% (39/121), 17.4% (21/121) and 9.9% (12/121), respectively. On the other hand, in mKRB, rat pronuclear embryos showed developmental blockages at the 2-cell and 4-cell stages, and never developed beyond the 4-cell stage. The rate of blastocyst formation under a low oxygen concentration was 20.1% (43/214), showing a significant difference from the value of 5.5% (11/201) obtained under a standard oxygen concentration (P less than 0.005). This is the first report of successful culture of rat pronuclear embryos to the blastocyst stage. Furthermore, it is suggested that protection from oxidation stress is a prerequisite for rat embryo development in vitro.     

Fertilization and early embryology: Timing of development is a critical parameter for predicting successful embryogenesis

Development of embryos from the 1-cell stage into blastocystsin vitro is generally slower than the time-course for developmentin vivo. It was the objective of this work to determine whetherembryos that reach the 8-cell stage within a normal time-framehave a developmental advantage (both in vitro and post-embryotransfer) over slower embryos. Hamster 1-cell embryos were collected10 h post-egg activation (PEA) and cultured for 48 h (58 h PEA= t⁵⁰ for 8-cell embryo development in vivo) in hamster embryoculture medium-6. Embryos were sorted according to stage reached,culture was continued in fresh medium and stage of developmentwas observed at 78, 82 and 86 h PEA. At 58 h PEA, embryos were<4-cell (4%), 4-cell (19%), 5- to 7-cell (16%) or 8-cell(61%). The 58 h 8-cell embryos had a significantly greater abilityto develop to the blastocyst stage than 58 h 4-cell embryosat 78, 82 and 86 h PEA (74 versus 13%, 69 versus 25% and 65versus 37% respectively). The percentages of 14-day-old fetusescollected after embryo transfer indicated that morulae and blastocystsderived from 58 h 4-cell embryos were, on average, less viable(26% fetuses) than morulae and blastocysts from 58 h 8-cellembryos (51% fetuses). Thus morulae and blastocysts developingin vitro from faster or slower cleaving embryos can be qualitativelyas well as quantitatively different. These data indicate thatthe timing of development in vitro, specifically the timingof completion of the third cell cycle, is a critically importantparameter for predicting successful embryogenesis in the hamster.     

A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos

The impact of cryopreservation on the implantation potential of early cleavage stage (day 2) embryos was assessed by analysing the outcome from > 5000 thawed embryos in relation to the outcome from a similar number of fresh embryos. Analysis of procedures in which all transferred embryos fulfilled equivalent defined criteria revealed no significant difference in the implantation rates (fetal hearts/100 embryos transferred) of fresh 4-cell embryos (16.6%) and fully intact thawed 4-cell embryos (16.9%). Although 2-cell embryos implanted at significantly lower rates, there was again no significant difference between fresh (6.5%) and fully intact thawed (7.2%) embryos. Similar analysis of all embryos (irrespective of cell number on day 2) demonstrated that the implantation potential of partially intact thawed embryos was related to the extent of blastomere loss with the implantation rate of embryos with 50% cell survival (5.4%) being approximately half the rate of fully intact embryos (11.3%). Combining the values obtained from 'pure' data for the implantation rates of embryos with defined levels of survival with their relative prevalence in the total population of thawed embryos gave a predicted number of implantations (441) which was similar to the observed outcome (463). This number was approximately 30% less than the number expected had the same embryos been transferred fresh (635). The results suggest that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. The observed frequency of blastomere loss results in a reduction of approximately 30% in the implantation potential of a population of embryos following cryopreservation.     

Towards defining parameters for a successful single embryo transfer in frozen cycles

BACKGROUND: Twin pregnancies in IVF should be avoided by transferring embryos one at a time, even for frozen cycles. In this study, we investigated the effect of blastomere lysis and cleavage in singleton frozen embryo transfer (sFET) cycles. Outcomes were compared with the transfer of two embryos in frozen transfer cycles (dFET). METHODS: A retrospective analysis was performed on 891 FET cycles, involving 404 sFET and 487 dFET cycles. RESULTS: Overall, in sFET cycles, the pregnancy and implantation rates were 8.9 and 8.7%. When blastomere lysis was more than 25% but no greater than 50%, the pregnancy and implantation rates were 3.2%. If blastomere lysis was greater than 50% there were no pregnancies. If blastomere lysis was less than 25%, but with no cleavage, the pregnancy and implantation rates were 4.1%. The results significantly improved (P = 0.007) in the group with less than 25% lysis, when cleavage occurred. The pregnancy and implantation rates for this group were 17.3 and 16.6%. This was not significantly different from unselected two embryo transfers (22 and 12.7%,P = 0.2 and 0.19, respectively). There were 21 twins with dFET (19.6% of pregnancies) and none in sFET. CONCLUSION: Both blastomere lysis and cleavage affect the outcome in sFET. To avoid the risk of twins, sFET should be considered when the embryo shows less than 25% blastomere lysis and at least one blastomere cleaves.