Assessment of embryo potential by visual and metabolic evaluation

Morphological evaluation of embryos is essential to the success of embryo transfer procedures and is presumed to reflect embryo metabolic activity. To investigate this assumption, correlations between morphological and metabolic parameters were determined for cultured murine morulae. After 18 h (n = 47) or 36 h (n = 48) of culture in M16, the developmental rate and quality (poor or good) of embryos were estimated, and, then, either their (14)C-glucose utilization or (35)s-methionine uptake and incorporation were measured. Retarded developing, or poor-quality embryos had lower mean glucose utilization, uptake and incorporation rates than normally developing or good-quality embryos (P < 0.05). After 18 h of culture, an association was found between developmental rate and metabolic activity, but this was not evident after 36 h of culture. Similarly, an association was found between embryo quality and metabolic activity. As expected, poor embryo quality was indicative of low metabolism throughout the culture period, but good quality did not necessarily indicate normal metabolic activity. Thus, morphological parameters do not always reflect metabolic competence, and some functional defects were not detectable by visual evaluation alone. Measuring metabolic parameters could complement visual evaluation for a better selection of embryos prior to transfer.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

Bovine embryo morphology and evaluation

The following paper briefly reviews the morphology of the bovine embryo and presents a retrospective analysis of bovine embryo transfer results accumulated from April to December of 1982 at a commercial embryo transfer center. Of particular interests were bovine embryo morphology, assessment of embryo quality, and recipient-donor, recipient-embryo synchrony requirements. Embryos were recovered from superovulated donors five to nine days after estrus (estrus = day O). All embryos were individually examined at 200X for cell stage of development and embryo quality. Embryos were nonsurgically transferred to recipients that were within two days of estrous cycle synchrony with the donor. Attempts were made to synchronize estimated developmental age of embryos to the day of the recipient cycle. A high degree of variability was observed in morphological development and embryo quality within and among donors. Embryo recovery in individual donors resulted in a wide range of embryonic cell stages, often differing in estimated developmental ages from 24 to 48 hours. A total of 783 embryos were transferred, resulting in 308 pregnancies. Stage of embryonic development (16-cell through hatched blastocyst) had little effect on pregnancy rates. Embryo quality was a more accurate predictor of success. Embryos of excellent, good, fair and poor categories resulted in 45%, 44%, 27% and 20% pregnancy rates, respectively. Recipient-donor estrous cycle synchrony of two days in either direction did not significantly alter pregnancy rates. However, 88% of 258 pregnancies (584 total transfers) occurred with a +/-1 day recipient-embryo synchrony compared to 74% based on +/-1 day recipient-donor cycle synchrony (P<0.001). Results suggest that transfer of bovine embryos based on synchrony between day of recipient cycle and state of embryonic development provides higher pregnancy rates than transfers based on recipient-donor cycle synchrony.     

Quantification of embryo quality by respirometry

It is generally accepted that assessment of embryo metabolism, in particular oxygen consumption, may improve embryo selection by identifying the embryos with higher developmental competence. Several methods have been employed to measure embryonic oxygen consumption, but most of them were detrimental to subsequent embryo development. Recently, we have introduced the Nanorespirometer system, which is a non-invasive and highly sensitive technology developed for the individual measurement of embryonic respiration rates. This technology is able to perform single measurements at a fixed time or stage of embryonic development without adversely influencing embryo viability. Concomitantly, and based on the same principles, a second technology -- the Embryo Respirometer -- has been developed. The Embryo Respirometer allows the continuous measurement of individual respiration rates with simultaneous acquisition of digital images of each embryo, during the entire culture period (6-7 days). In this review, both technologies are described and their potential use as diagnostic tools for improving embryo selection in bovine and human following IVF treatments is discussed. Correlations between respiration rates of individual embryos and other parameters such as morphological quality, sex, stage of development, kinetics, diameter, expression of key metabolic genes and subsequent viability following embryo transfer are also examined. On the basis of the results obtained, it is postulated that assessment of embryonic respiration rates in association with other viability parameters allows for a more accurate embryo evaluation, both under clinical and research conditions.     

Weedy adaptation in Setaria spp. IV. Changes in the germinative capacity of S. faberii (poaceae) embryos with development from anthesis to after abscission

Studies were conducted evaluating germinability states in giant foxtail (Setaria faberii) embryos, as well as surrounding tissues (hull, caryopsis), with germination assays. Further, seed age, fascicle arrangement, flowering patterns, and elongation in the inflorescence were evaluated. Both qualitatitive and quantitative morphological observations of the hull and the caryopsis were revealed by precisely determined fertilized spikelet age from anthesis until after seed abscission. Red coloration of the placental pad at ≈ 11 d after anthesis is probably a morphological indicator of physiological maturity. Germinability of giant foxtail embryos changed with development. Four qualitatively different types of embryo germination were observed during development of the seed: early disorganized callus growth at the basal, coleorhizal end of the embryo; germination of immature embryos with shortened and thickened axes; germination of the scutellum; and germination and growth of the coleoptile and coleorhiza in embryos aged 7 d after anthesis and older. Axis-specific embryo germinability was also observed. Inhibition of the embryo could be localized to the coleoptile, the coleorhiza, or both. These studies provide evidence for a complex model of germinability regulation based on the independent, asynchronous actions of the embryo, caryopsis, and hull compartments, as well as on their dependent, synchronous action. These studies provide evidence for a dynamic, developmental model of giant foxtail germinability regulation resulting in phenotypes with a wide range of germinability shed from an individual panicle. These diverse germinability phenotypes are found at all stages of development, but particularly when the seed is shed and the soil seed bank is replenished.     

Intra- and inter-observer analysis in the morphological assessment of early-stage embryos

Background  

The aim of this study was to determine the intra- and inter-observer variability in the evaluation of embryo quality. Multilevel images of embryos on day 1, day 2 and day 3, were analysed using different morphological parameters.     

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh,maturing cotyledonary zygotic embryos: biological,carbohydrate and proteomic analyses

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.     

New serum-free in vitro culture technique for midgestation mouse embryos

Current in vitro culture methods for mouse embryos are critically dependent on specially prepared rodent serum. Rodent serum requires careful preparation and stringent assessment of serum quality, while commercially available whole embryo culture serum is expensive and shows considerable lot variability. Thus, preparation and testing of suitable serum represents a considerable investment of time and resources, particularly for laboratories with only short-term embryo culture requirements. In addition, serum supplementation of culture medium may introduce unknown serum components that could interfere with interpretation of experimental results, especially where the study is geared towards analysis of a specific growth factor. Here we describe the composition of a standardized serum free culture medium comprised of commercially available stem cell media supplements. With this method, we have successfully cultured midgestation stage mouse embryos and demonstrated, using both morphological and gene expression criteria, that these embryos exhibited proper developmental progression. We believe this method to be a significant advance in whole embryo culture technology that will be of particular use to laboratories needing to utilize whole embryo culture to study midgestation organogenesis.     

油菜裂外壁小孢子胚在分子水平上具有胚体/胚柄分化

早期合子胚取材困难, 难以开展相关研究。前人的工作表明, 油菜(Brassica napus)裂外壁小孢子胚胎发生系统能够较好地模拟合子胚的分化模式, 因而可替代早期合子胚胎作为研究材料。但目前尚缺乏该胚胎发生系统中胚胎具有胚体/胚柄分化的分子水平的证据。该文首次证明了油菜WOX家族基因能够用于标记胚体/胚柄的分化过程, 利用胚柄标记基因BnWOX8的表达模式, 从分子水平上证明了带胚柄的裂外壁小孢子胚的确存在胚体/胚柄的分化。研究结果为充分利用油菜裂外壁小孢子胚胎发生系统, 解决早期胚胎取材困难的问题奠定了坚实的基础。同时, 建立了活体激光切割分离特定细胞的技术, 结合用于少量细胞RNA提取的活体特异细胞RNA提取技术, 为鉴定少量特异分化细胞的基因表达模式提供了一个可行且明确的解决方案。     

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.     

Effect of serum on the mitochondrial active area on developmental days 1 to 4 in in vitro-produced bovine embryos

Certain morphological changes at the subcellular level caused by the current techniques for in vitro embryo production seem to affect mitochondria. Many of these, including dysfunctional changes, have been associated with the presence of serum in the culture medium. Thus, the aim of the present work was to assess the mitochondrial dynamics occurring in embryos during the first 4 days of development, in order to analyze the most appropriate time for adding the serum. We used transmission electron microscopy (TEM) micrographs to calculate the embryo area occupied by the different morphological types of mitochondria, and analyzed them with Image Pro Plus analyzer. The results showed hooded mitochondria as the most representative type in 1- to 4-day-old embryos. Swollen, on-fusion, orthodox and vacuolated types were also present. When analyzed in embryos cultured without serum, the dynamics of the different mitochondrial types appeared to be similar, a fact that may provide evidence that the developmental changes control the mitochondrial dynamics, and that swollen mitochondria may not be completely inactive. In contrast, in culture medium supplemented with serum from estrous cows, we observed an increased area of hooded mitochondria by developmental day 4, a fact that may indicate an increased production of energy compared with previous days. According to these results, the bovine serum added to the culture medium seems not to be responsible for the functional changes in mitochondria.     

Relationship between timing of development,morula morphology,and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos

Noninvasive measurements of bovine embryo quality, such as timing of cleavage, morula morphology, blastocyst formation, and hatching ability, were linked with the number of inner cell mass (ICM) cells and trophectoderm (TE) cells of the resulting embryos. First, it was confirmed that fast-cleaving embryos proved to have significantly higher chances to reach advanced developmental stages vs. intermediate and slow cleavers (P = 0.01). They also showed significantly less fragmentation at the morula stage, implying the presence of more excellent morulae among fast-cleaving embryos (P < 0.05). Second, the quality of hatched blastocysts, resulting from morulae of different morphological grades, was examined by differential staining. The total cell and ICM cell numbers were significantly lower for hatched blastocysts developed from poor morulae compared to hatched blastocysts developed from excellent, good, or fair morulae. However, hatched blastocysts with <10 ICM cells were seen in embryos belonging to all four morphological scores. Finally, it was found that timing of first cleavage was not significantly correlated with timing of blastocyst formation or with cell number of blastocysts. Timing of blastocyst formation, however, was significantly correlated with cell number: day 8 blastocysts had significantly lower total cell and ICM cell numbers than day 6 and day 7 blastocysts (P < 0.001). These results suggest that the quality of in vitro-produced bovine embryos is very variable and cannot be linked with a single criterion such as embryo morphology and/or hatching ability. Timing of blastocyst formation was the most valuable criterion with regard to embryonic differentiation. Mol. Reprod. Dev. 47:47–56, 1997. © 1997 Wiley-Liss, Inc.     

Inactivation of a MAPK-like protein kinase and activation of a MBP kinase in germinating barley embryos

We provide evidence for involvement of two different 45 kDa protein kinases in rehydration and germination of barley embryos. In dry embryos, a myelin basic protein (MBP) phosphorylating kinase was detected, which could be immunoprecipitated with an anti-MAPK (mitogen-activated protein kinase) antibody. Rehydration of the embryo induced a decrease in activity of this 45 kDa MAPK-like protein kinase. In addition, activity of a MBP kinase of the same molecular weight was subsequently found to be induced. This second MBP kinase activity could not be immunoprecipitated with the anti-MAPK antibody and was induced only in germinating embryos, not in dormant embryos.     

THE CONTRIBUTIONS OF EMPIRICAL EVIDENCE TO SOCIO‐ETHICAL DEBATES ON FRESH EMBRYO DONATION FOR HUMAN EMBRYONIC STEM CELL RESEARCH

This article is a response to McLeod and Baylis (2007) who speculate on the dangers of requesting fresh ‘spare’ embryos from IVF patients for human embryonic stem cell (hESC) research, particularly when those embryos are good enough to be transferred back to the woman. They argue that these embryos should be frozen instead. We explore what is meant by ‘spare’ embryos. We then provide empirical evidence, from a study of embryo donation and of embryo donors' views, to substantiate some of their speculations about the problems associated with requesting fresh embryos. However, we also question whether such problems are resolved by embryo freezing, since further empirical evidence suggests that this raises other social and ethical problems for patients. There is little evidence that the request for embryos for research, in itself, causes patients distress. We suggest, however, that no requests for fresh embryos should be made in the first cycle of IVF treatment. Deferring the request to a later cycle ensures that potential donors are better informed (by experience and reflection) about the possible destinations of their embryos and about the definition of ‘spare embryos’. Both this article, and that by McLeod and Baylis, emphasize the need to consider the views and experiences of embryo donors when evaluating the ethics of embryo donation for hESC research.     

Metabolism of glucose by human embryos

Glucose turnover, as measured by CO2 production, lactate accumulation and carbon incorporation from [U-14C]glucose as sole energy substrate, was low on the 2nd day of culture of human embryos resulting from in-vitro fertilization but above that of unfertilized oocytes. In general, all parameters of metabolism increased substantially during the following 2 days of development but the rate of increase in lactate production was greater than that of CO2, especially between Days 3 and 4. Within developing embryos, no correlation was evident between the metabolic turnover of glucose and the method of patient stimulation, the morphological quality of embryos or the apparent rate of cleavage in culture. The results indicate that, before Day 3 of development, glucose is not effective as an energy source for the human embryo because of a blockade to glycolysis similar to that in mouse embryos.     

Loss of genomic imprinting in mouse embryos with fast rates of preimplantation development in culture

Currently, the stage of embryo development has been proposed as one of many criteria for identifying healthy embryos in infertility clinics with the fastest embryos being highlighted as the healthiest. However the validity of this as an accurate criterion with respect to genomic imprinting is unknown. Given that embryo development in culture generally requires an extra day compared to in vivo development, we hypothesized that loss of imprinting correlates with slower rates of embryonic development. To evaluate this, embryos were recovered at the 2-cell stage, separated into four groups based on morphological stage at two predetermined time points, and cultured to blastocysts. We examined cell number, embryo volume, embryo sex, imprinted Snrpn and H19 methylation, imprinted Snrpn, H19, and Cdkn1c expression, and expression of genes involved in embryo metabolism-Atp1a1, Slc2a1, and Mapk14-all within the same individual embryo. Contrary to our hypothesis, we observed that faster developing embryos exhibited greater cell numbers and embryo volumes as well as greater perturbations in genomic imprinting and metabolic marker expression. Embryos with slower rates of preimplantation development were most similar to in vivo derived embryos, displaying similar cell numbers, embryo volumes, Snrpn and H19 imprinted methylation, H19 imprinted expression, and Atp1a1 and Slc2a1 expression. We conclude that faster development rates in vitro are correlated with loss of genomic imprinting and aberrant metabolic marker expression. Importantly, we identified a subset of in vitro cultured embryos that, according to the parameters evaluated, are very similar to in vivo derived embryos and thus are likely most suitable for embryo transfer.     

Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study

The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I) blastocysts were compared for the occurrence of apoptosis in blastomeres. The total number of cells per embryo and the number of cells with damaged plasma membranes, fragmented DNA and fragmented nuclei per embryo were assessed in selected blastocysts by combining Ethidium homodimer (EthD-1), terminal dUTP nick end labeling (TUNEL) and Hoechst 33342 staining. In general, the level of blastomere apoptosis was low. A higher level of apoptosis was observed in in vitro produced equine, porcine and bovine blastocysts compared to their in vivo counterparts. Interestingly, 4 of the initially selected 29 bovine in vitro produced blastocysts exhibited extensive signs of apoptosis affecting the inner cell mass (ICM), which is not compatible with a viable conceptus. Repeated occurrence of this observation may explain the lower ET outcome of in vitro produced bovine embryos compared to in vivo produced embryos. It is concluded that, although in morphologically high quality blastocysts of several farm animal species a significant difference exists in the percentages of apoptotic cells between in vivo and in vitro produced embryos, the incidence of apoptosis at the blastocyst stage is at such a low level that it cannot reflect the substantial differences in embryo viability that have been described between in vivo and in vitro produced blastocysts following ET.     

Lung respiration, somatic activity and gas metabolism in embryonic chicks prevented from hatching by thiourea

Treating chick embryos with high doses of thiourea (32.8 mumol) on day 17 of incubation resulted in prevention of hatching and of active breathing. Furthermore, thiourea also prevented the increase of O2 consumption and the marked increase of somatic activity associated with the final hatching act. These findings provide evidence for the importance of active breathing in the prenatal period to initiate pipping and hatching of the avian embryo.     

Development in the Floating World: Defenses of Eggs and Embryos Against Damage from UV Radiation

Eggs and embryos of many aquatic organisms develop in the watercolumn and can experience ultraviolet radiation with potentiallydeleterious effects. This is especially vexing for floatingembryos that develop in the surface or neuston layer. Radiationdamage can be a particular problem for these embryos since thecell division cycle during the cleavage period is quite shortand often these cycles do not have mitotic checkpoints to insurefaithful transmission of DNA to the daughter cells. This couldresult in cell division with unrepaired DNA in the blastomeres,which could impact embryogenesis and the transmission of thegenome through the germ line. Described strategies to restrictradiation damage include mechanisms to limit oxidative damageand the use of sunscreens such as the mycosporines to curb radiationto sensitive targets. We describe a particularly ingenious useof sunscreens in the tunicate embryo, the use of extra-embryoniccells to shield the embryo from potentially harmful UV-A andUV-B radiation. We also raise questions regarding the natureof UV damage to embryos (is it DNA or also protein) and thecharacteristics of DNA repair in such embryos. It is likelythat unique mechanisms are present in floating embryos thatdevelop in this air-water interface to assure that cell andgenomic integrity are maintained in this challenging environment.