Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.     

Acid peptidase activity released from in vitro produced porcine embryos: a candidate marker to predict developmental competence

The ability to efficiently create high quality embryos, competent to produce normal viable offspring in vitro, facilitates diverse technological advancements in animal agriculture and assisted reproduction. Current methods for evaluation of embryos are predominantly based on morphological characteristics which are prone to potential bias of the scorer. Metabolic and genetic markers have also been explored for quality assessment, but they are cost prohibitive or require longer periods of time for evaluation. We hypothesized that secreted enzymes could provide another means of embryo quality assessment. In this report, we provide evidence that medium conditioned by porcine embryos often has proteolytic activity that operates in acidic conditions (acid peptidase activity or APA). The APA could be inhibited by pepstatin A, suggesting that the activity is derived from one or more aspartic peptidases. We also provide evidence that single embryos, incubated for as few as 24 hr, released enough APA that it was possible to measure it accurately at day 5 of culture. We also observed that such activity on day 6 could be positively correlated with advanced developmental stage and embryo quality. In addition, those embryos that were graded identically by morphological evaluations often differed in the amount of APA--with some being significantly higher than the experimental threshold value. Therefore, the APA of embryos might serve as an additional marker for evaluation of embryos.     

Relationship between development, metabolism, and mitochondrial organization in 2-cell hamster embryos in the presence of low levels of phosphate.

The effect of low concentrations of inorganic phosphate (P(i)) on development, metabolic activity, and mitochondrial organization in the same cohorts of cultured hamster embryos was evaluated. Two-cell embryos were collected from eCG-stimulated golden hamsters and cultured in HECM-10 with 0.0 (control), 1.25, 2.5, or 5.0 microM KH(2)PO(4). Glucose utilization through the Embden-Meyerhof pathway (EMP) and tricarboxylic acid (TCA)-cycle activity were determined following 5 h of culture. Mitochondrial organization in living embryos was evaluated using multiphoton microscopy at 6 h of culture. Development was assessed at 27 h (on-time 8-cell stage) and 51 h (on-time blastocyst stage) of culture. Total cell numbers, as well as cell allocation to the trophectoderm and inner cell mass were determined for morula- and blastocyst-stage embryos. Culture with P(i) did not alter TCA-cycle activity. However, culture with > or 2.5 microM P(i) significantly increased (P < 0.01) EMP activity compared to control. Mitochondrial organization was significantly (P < 0.01) disrupted by P(i) in a dose-dependent manner. Development to the 8-cell, morula/blastocyst, and blastocyst stages was significantly reduced (P < 0.05) in the presence of > or =2.5 microM P(i) compared to both control and 1.25 microM P(i). This study clearly demonstrates that, for hamster embryos, inclusion of even exceptionally low concentrations of P(i) in culture medium dramatically alters embryo physiology. Additionally, although 2-cell embryos can tolerate some structural disruption without concomitant, detrimental effects on development or metabolic activity, metabolic disturbance is associated with decreased developmental competence.     

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.     

Oxygen regulates amino acid turnover and carbohydrate uptake during the preimplantation period of mouse embryo development

Oxygen is a powerful regulator of preimplantation embryo development, affecting gene expression, the proteome, and energy metabolism. Even a transient exposure to atmospheric oxygen can have a negative impact on embryo development, which is greatest prior to compaction, and subsequent postcompaction culture at low oxygen cannot alleviate this damage. In spite of this evidence, the majority of human in vitro fertilization is still performed at atmospheric oxygen. One of the physiological parameters shown to be affected by the relative oxygen concentration, carbohydrate metabolism, is linked to the ability of the mammalian embryo to develop in culture and remain viable after transfer. The aim of this study was, therefore, to determine the effect of oxygen concentration on the ability of mouse embryos to utilize both amino acids and carbohydrates both before and after compaction. Metabolomic and fluorometric analysis of embryo culture media revealed that when embryos were exposed to atmospheric oxygen during the cleavage stages, they exhibited significantly greater amino acid utilization and pyruvate uptake than when cultured under 5% oxygen. In contrast, postcompaction embryos cultured in atmospheric oxygen showed significantly lower mean amino acid utilization and glucose uptake. These metabolic changes correlated with developmental compromise because embryos grown in atmospheric oxygen at all stages showed significantly lower blastocyst formation and proliferation. These findings confirm the need to consider both embryo development and metabolism in establishing optimal human embryo growth conditions and prognostic markers of viability, and further highlight the impact of oxygen on such vital parameters.     

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.     

Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo

Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.     

The role of the visceral yolk sac in hyperglycemia-induced embryopathies in mouse embryos in vitro.

The adverse developmental effects of hyperglycemia to rodent embryos have been shown using whole embryo culture. Although, a mechanism by which hyperglycemia-induced effects occur is unknown, recent work has focused on the visceral yolk sac as a potential target tissue. Therefore, we have evaluated the developmental effects of hyperglycemia in early head fold stage mouse embryos in vitro and assessed the histiotrophic function of the visceral yolk sac. As has been previously shown in rodents, hyperglycemia produced neural tube closure defects in a concentration dependent manner at 33, 50, and 67 mM glucose using a 44 h exposure period. However, exposure times between 6 and 12 h were sufficient to alter embryonic development when the glucose concentration was 50 or 67 mM. In contrast, early somite stage embryos (4-6 somite stage) appear to be less sensitive to dysmorphogenesis and a 48 h exposure to 67 mM glucose but not 33 or 50 mM also produced neural tube defects. Hyperglycemia (67 mM) did not alter the uptake of 35S-methionine and 35S-cysteine-labeled hemoglobin (35S-Hb) in the visceral yolk sac (VYS) in early headfold staged embryos. However, the accumulation of 35S in the embryo was reduced by 16-18% at glucose concentrations of 50 or 67 mM during the last 12 h of a 44 h exposure period. No effect on VYS uptake or embryonic accumulation of 35S-labeled products was observed at shorter exposure periods (12-24 and 24-36 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.     

Determination of pentose phosphate and Embden-Meyerhof pathway activities in bovine embryos

Quantitative determination was made of the activity of pentose phosphate pathway (PPP) and Embden-Meyerhof pathway (EMP) in individual bovine embryos from the six-cell to the hatched blastocyst stage. Embryos were collected from superovulated cross-bred heifers and classified into good and poor categories. A single embryo in 1 ul of medium was mixed with 2 ul of medium containing 3 to 30 nCi radiolabeled glucose previously placed on a detached lid of the 1.5-ml polypropylene microcentrifugé vial. The lid was then fitted to its vial which had been loaded in advance with 1.5 ml of 0.1 N NaOH. Vials were then incubated at 37 degrees C for 3 h. At the end of the incubation period, a 1.5-ml NaOH trap was inverted and placed into a 20-ml scintillation vial containing 10 ml of aqueous counting solution and counted in a liquid scintillation spectrophotometer. The PPP activity in good-quality embryos was greatest at the six-cell stage and decreased with increasing embryo development. The EMP activity showed the reverse trend. Poor- quality embryos had a lower glucose metabolism and higher PPP activity. Similar measurements were made on embryos following 24 h of culture, and total glucose metabolism and percentage of PPP activity were increased. In conclusion, these data suggest that in good quality bovine embryos total glucose utilization is low until 16-cell stage, with PPP being the predominant pathway. Total glucose utilization increases significantly at the morula stage; EMP activity increases with increasing embryo development; and PPP activity increases significantly in poor quality embryos and in embryos 24 h in culture.     

Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat ( Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose-family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 m M galactose-containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated ¹⁴C from ¹⁴C-fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no ¹⁴C incorporation into stachyose was observed in embryos from afterripened caryopses. No ¹⁴C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose-family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening-induced germination.     

Development of phenobarbital-sensitive control mechanisms for uridine diphosphate glucuronyltransferase activity in chick embryo liver

Uridine diphosphate (UDP) glucuronyltransferase activity in chick liver rises at hatching from near zero to adult levels. This rise will occur prematurely in embryo liver during organ culture. Increase in enzyme activity during organ culture differs with embryo age: in liver from 11-day old embryos it ceases at adult values; in liver from 5-day old embryos it continues to much higher-than-adult levels. Phenobarbital added to culture medium accelerates these rises in enzyme activity and elevates the plateau reached in 11-day embryo liver to that observed in 5-day embryo liver. Kinetic analysis of the changes in enzyme activity induced by phenobarbital during culture suggests that the regulatory mechanisms for enzyme activity are different in 5- and 11-day embryo liver and that these differences reflect developmental changes occurring in ovo.     

A dysmorphology score system for assessing embryo abnormalities in rat whole embryo culture

BACKGROUND: The rodent whole embryo culture (WEC) system is a well‐established model for characterizing developmental toxicity of test compounds and conducting mechanistic studies. Laboratories have taken various approaches in describing type and severity of developmental findings of organogenesis‐stage rodent embryos, but the Brown and Fabro morphological score system is commonly used as a quantitative approach. The associated score criteria is based upon developmental stage and growth parameters, where a series of embryonic structures are assessed and assigned respective scores relative to their gestational stage, with a Total Morphological Score (TMS) assigned to the embryo. This score system is beneficial because it assesses a series of stage‐specific anatomical landmarks, facilitating harmonized evaluation across laboratories. Although the TMS provides a quantitative approach to assess growth and determine developmental delay, it is limited to its ability to identify and/or delineate subtle or structure‐specific abnormalities. Because of this, the TMS may not be sufficiently sensitive for identifying compounds that induce structure or organ‐selective effects. METHOD: This study describes a distinct morphological score system called the “Dysmorphology Score System (DMS system)” that has been developed for assessing gestation day 11 (approximately 20–26 somite stage) rat embryos using numerical scores to differentiate normal from abnormal morphology and define the respective severity of dysmorphology of specific embryonic structures and organ systems. This method can also be used in scoring mouse embryos of the equivalent developmental stage. RESULT AND CONCLUSION: The DMS system enhances capabilities to rank‐order compounds based upon teratogenic potency, conduct structure‐ relationships of chemicals, and develop statistical prediction models to support abbreviated developmental toxicity screens. Birth Defects Res (Part B) 89:485–492, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence of paternal nutrient provisioning to embryos in broad-nosed pipefish Syngnathus typhle

In two experiments, radioactively labelled nutrients (either (3)H-labelled amino-acid mixture or (14)C-labelled glucose) were tube-fed to brooding male Syngnathus typhle. Both nutrients were taken up by the males and radioactivity generally increased in the brood pouch tissue with time. Furthermore, a low but significant increase of (3)H-labelled amino acids in embryos was found over the experimental interval (48 h), whereas in the (14)C-glucose experiment the radioactivity was taken up by the embryos but did not increase over the experimental time (320 min). Uptake of radioisotopes per embryo did not differ with embryo size. A higher uptake mg(-1) tissue of both (3)H-labelled amino acids and (14)C-labelled glucose was found in smaller embryos, possibly due to a higher relative metabolic rate or to a higher surface-area-to-volume ratio compared to larger embryos. Uptake in embryos was not influenced by male size, embryonic developmental advancement or position in the brood pouch. It is concluded that brooding males provide amino acids, and probably also glucose, to the developing embryos in the brood pouch.     

In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.     

Expression of Bcl-2 and Bax proteins in relation to quality of bovine oocytes and embryos produced in vitro

The mechanisms underlying the visual assessment and selection of immature oocytes resulting in optimum embryonic development following in vitro maturation, fertilization and culture (in vitro maturation (IVM)/in vitro fertilization (IVF)/in vitro embryo culture (IVC)) are unknown. Also, the reasons for the more frequent occurrence of cytoplasmic fragmentation in in vitro produced bovine embryos, resulting in poor survival following cryopreservation and decreased pregnancy rates following embryo transfer are not clear. The objectives of this study are: (1) to investigate whether differences in the quality of immature oocytes and embryo fragmentation are associated with apoptosis; and (2) to study the pattern of Bcl-2 and Bax expression in oocytes and embryos to help elucidate their potential roles in the regulation of apoptosis during development. Bovine oocytes were obtained from slaughterhouse ovaries and divided into four grades (grades I–IV) based on their morphology. Oocytes of different grades were cultured in serum-free medium for 48 h. Embryos were produced only from grade I oocytes (highest quality) via IVM, IVF and IVC procedures. The morphological analysis of apoptosis in oocytes and embryos was carried out using propidium iodide staining and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling. The expression of Bcl-2 and Bax in oocytes and embryos of different qualities and stages was determined using western blotting. The results showed that the number of morphologically abnormal oocytes with shrinkage and/or fragmentation of the ooplasm, which are typical features of apoptosis, was significantly higher in grade IV oocytes (denuded oocytes, the lowest quality) than in grade I oocytes after 48 h in vitro culture (P<0.05). DNA fragmentation, a hallmark of the biochemical changes seen in apoptotic cell death, was observed in morphologically fragmented oocytes and embryos. The expression of Bcl-2 was high in good quality oocytes and embryos, low in fragmented embryos, and hardly detectable in denuded oocytes. In contrast, the expression of Bax was found in all types of oocytes and embryos with the highest expression in the denuded oocytes. This implies that the ratio of Bcl-2 to Bax may be used to gauge the tendency of oocytes and embryos towards either survival or apoptosis. Overall, our results show that apoptosis appears to be an underlying mechanism of bovine oocyte degeneration and embryo fragmentation. Interactions between the Bcl-2 family of proteins may play a critical role in pre-implantation embryo development. These findings could have important implications for improving IVF and related techniques.     

Development retardation in cultured preimplantation rabbit embryos

Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine. In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P less than 0.001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.     

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.