Time Of Blastocyst Formation Research Articles

The destinies of human embryos reaching blastocyst stage between Day 4 and Day 7 diverge as early as fertilization.

What clinical and laboratory differences emerge from parallel direct comparison of embryos reaching the blastocyst stage between Days 4, 5, 6, and 7 (Days 4-7)? Increasing times to blastocyst formation are associated with a worse clinical outcome and perturbations in developmental patterns appear as early as the fertilization stage. Previous evidence indicates that later times to blastocyst development are associated with a worse clinical outcome. However, the vast majority of these data concern Day 5 and Day 6 blastocysts, while Day 4 and Day 7 blastocysts remain less thoroughly investigated. In addition, studies comparing in parallel the developmental patterns and trajectories of Day 4-7 blastocysts are lacking. This leaves unanswered the question of when and how differences among such embryos emerge. Acquisition of such knowledge would significantly contribute to understanding the relative impact of intrinsic and extrinsic causes of embryo developmental kinetics and competence. This retrospective study involved time-lapse technology (TLT) monitoring of Day 4 (N = 70), Day 5 (N = 6147), Day 6 (N = 3243), and Day 7 (N = 149) blastocysts generated in 9450 ICSI cycles. Oocyte retrievals were carried out after clomiphene citrate-based minimal ovarian stimulation, between January 2020 and April 2021. Couples included in the study presented with different diagnoses, mainly male factor and unexplained infertility. Cases involving cryopreserved gametes or surgically retrieved sperm were excluded. Microinjected oocytes were assessed by a combined TLT-culture system. Day 4-7 blastocyst groups were compared in terms of morphokinetics (pronuclear dynamics, cleavage patterns and timings, and embryo quality) and clinical outcome. Clinically usable blastocysts were cryopreserved and transferred in single vitrified-warmed blastocyst transfers (SVBT). From 19846 microinjected oocytes, 17144 zygotes (86.4%) were obtained. Overall, the blastocyst development rate was 56.0%. Rates of blastocysts formation on Days 4, 5, 6, and 7 were 0.7%, 64.0%, 33.8%, and 1.6%, respectively. The average expanded blastocyst development times were 98.4 ± 0.4, 112.4 ± 0.1, 131.6 ± 0.1, and 151.2 ± 0.5 h in the Day 4-7 groups, respectively. Female age was positively associated with longer times to blastocyst development. Rates of both inner cell mass (ICM) and trophectoderm (TE) morphological grade A blastocysts were negatively associated with the day of blastocyst development (P < 0.0001). The differences in development times and intervals increased progressively until blastocyst expansion (P < 0.0001 for all development times). Strikingly, such differences were already markedly evident as early as the time of pronuclear fading (tPNf) (20.6 ± 0.3, 22.5 ± 0.0, 24.0 ± 0.0, 25.5 ± 0.3; Days 4-7, respectively; P < 0.0001). Rates of cleavage anomalies (tri-/multi-chotomous mitosis or rapid cleavage) occurring at the first or second/third division cycles were also positively associated with longer times to blastocyst development. Implantation, ongoing pregnancy, and live birth rates were progressively reduced with increasing blastocyst development times (P < 0.0001), even after stratification for maternal age. When controlled for female age, male age, number of previous embryo transfer cycles, morphological grade of the ICM and TE, and progesterone supplementation, the probabilities of implantation, clinical, and ongoing pregnancy and live birth were significantly decreased in Day 6 blastocysts in comparison to Day 5 blastocysts. Follow-up data on birth length, weight, and malformations were comparable among the four blastocyst groups. The study is limited by its retrospective design. Having been obtained from a single centre, the data require independent validation. This study extends previous data on the relation between time of blastocyst formation and clinical outcome. It also indicates that differences in developmental times and patterns of Day 4-7 blastocysts occur as early as the fertilization stage, possibly dictated by intrinsic gamete-derived factors. This study was supported by the participating institutions. The authors have no conflict of interest to declare. N/A.

Morphokinetic changes in vitrified and non-vitrified in vitro-derived ovine embryos

The present experiment employed time-lapse (TL) imaging to assess the effects of vitrification on the development of ovine blastocysts and to see if the timing of blastocyst formation and expansion was correlated with the numbers of embryo- and trophoblasts determined through differential staining of the expanded blastocysts. Ovaries were obtained after slaughter from cycling (October–March) Polish Longwool ewes aged 1–3 years and cumulus-oocyte complexes were collected by ovarian scarification. In vitro maturation was performed in TCM 199 medium supplemented with Earle's Salt, 10% of FBS, and 5 μg/mL of LH/FSH at 38 °C for 24 h. After maturation, the oocytes were incubated with thawed ram semen (IVF) for 19 h and all presumptive zygotes were transferred to a 16-well dish containing Cult medium for monitoring with the Primo Vision TL system. A portion of ovine embryos were vitrified (Cryotop system) at the early cavitation stage and TL observations of warmed (n = 30) and non-vitrified (n = 32) blastocysts continued until the attainment of the expanded blastocyst stage, at which point they were differentially stained with bisbenzimide and propidium iodide for microscopic enumeration of embryoblasts (inner cell mass blastomeres) and trophoblast-cells. There were no statistically significant differences in the timing of blastulation (tB) and formation of expanded blastocysts (tEB) between vitrified and non-vitrified ovine embryos, but non-vitrified blastocysts exceeded (P < 0.05) their vitrified and warmed counterparts in the mean number of embryo- and trophoblasts. In addition, the number of trophoblasts was negatively and moderately correlated with tB and tEB, for both vitrified and non-vitrified embryos. It can be concluded that even though vitrification of ovine embryos is associated with a significant reduction in the number of blastomeres, the rate of blastocyst development remains closely linked to the numbers of trophoblastic cells.

The higher the score, the better the clinical outcome: retrospective evaluation of automatic embryo grading as a support tool for embryo selection in IVF laboratories.

Is the automatic embryo grading function of specific time-lapse systems clinically useful as a decision support tool for IVF laboratories? Blastocyst grading according to the automatic scoring system is directly associated with the likelihood of implantation and live birth, at least in treatments without preimplantation genetic testing for aneuploidy (PGT-A). Several embryo selection algorithms have been described since the introduction of time-lapse technology in IVF laboratories, but no one algorithm has yet been sufficiently consolidated for universal use. Multicentric models based on automated grading systems offer promise for standardization of embryo selection. A retrospective cohort study was performed including 1678 patients who underwent IVF treatments between 2018 and 2020 and whose embryos (n = 12468) were cultured in time-lapse systems. After obtaining the required parameters (division time to 2, 3, 4 and 5 cells; time of blastocyst formation; inner cell mass quality; and trophectoderm quality), the automatic embryo score was calculated using the software included in the appropriate workstation. First, embryo score was compared with conventional morphological quality and the subsequent clinical outcomes of 1952 single blastocyst transfers. Second, we quantified the contribution of the automatic embryo score and conventional morphological grade to implantation and live birth outcome with multivariate logistic regression analysis in different patient populations. A higher embryo score was associated with a better clinical outcome of IVF treatment. The mean of the automatic embryo score varied significantly (P < 0.001) among embryos with different morphological categories, between euploid and aneuploid embryos, between embryos resulting in positive versus negative pregnancy, between implanted and non-implanted embryos, and between embryos resulting in positive and negative live birth. Embryo score was related to the odds of implantation and live birth in the oocyte donation program (odds ratio (OR)=1.29; 95% CI [1.19-1.39]; P < 0.001 for implantation and OR = 1.26; 95% CI [1.16-1.36]; P < 0.001 for live birth) and in conventional treatments with autologous oocytes (OR = 1.38; 95% CI [1.24-1.54]; P < 0.001 for implantation and OR = 1.47; 95% CI [1.30-1.65]; P < 0.001 for live birth). There was no significant association of embryo score with implantation or live birth in treatments involving PGT-A. This study is limited by its retrospective nature. Further prospective randomized trials are required to confirm the clinical impact of these findings. The single-center design should be taken into account when considering the universal application of the model. Evidence of the clinical efficiency of automated embryo scoring for ranking embryos with different morphological grade and potential in order to achieve higher implantation and live birth rates may make it a decision support tool for embryologists when selecting blastocysts for embryo transfer. This research has been funded by a grant from the Ministry of Science, Innovation and Universities FIS (PI21/00283) awarded to M.M. There are no competing interests to declare. N/A.

Randomized study of G-TL and global media for blastocyst culture in the EmbryoScope: morphokinetics, pregnancy, and live births after single-embryo transfer

To evaluate the efficacy of two different invitro fertilization culture media for blastocyst development, pregnancy, and live birth rate. Global (GB) medium (used without refreshment) and G-TL medium (designed specifically for culture in time-lapse incubators) were compared. Prospective randomized study of sibling embryo culture in two culture media. Invitro fertilization clinic. Women undergoing fresh or frozen cycles using autologous or donor oocytes. None. The primary endpoints were implantation, pregnancy, and live birth rate (LBR) after single blastocyst transfer. Secondary endpoints included embryo morphokinetics, development of good-quality blastocysts, and euploidy rate. Kinetic data from 10,768 sibling pronucleate embryos cultured in the EmbryoScope were compared. GB embryos initiated compaction earlier and formed morula sooner than their G-TL counterparts. The mean timing for start of blastulation did not differ. The interval between start of blastulation and time of blastocyst formation was observed to be <12 hours for proportionately more GB compared with G-TL-cultured embryos. Despite a higher rate of observed dysmorphisms in GB embryos, the euploidy rate among biopsied blastocysts did not differ between media. A total of 820 single-embryo transfer cycles were performed. Implantation rates were similar between media, independent of whether the embryo transferred was fresh (GB 58.7% vs. G-TL 61.7%) or frozen (GB 64.1% vs. G-TL 60.5%). Live birth rates were also not different. With GB medium, the LBR for fresh and frozen transfers was 54.2% and 53.1%, respectively, as compared with 51.1% and 50%, respectively, with G-TL. Uninterrupted culture in a time-lapse incubator without medium refreshment was well supported by both media tested. Differences in morphokinetics did not necessarily dictate the superiority of one media over the other. Both pregnancy and LBR were not significantly influenced by choice of culture medium. The euploidy rate was also independent of culture medium.

62 Sequential nutrient restriction and provision during bovine in vitro embryo culture differentially affect blastocyst development and quality with oocytes from varied sources

We have demonstrated that bovine blastocyst development was improved after culture in medium with only 6.25% of standard carbohydrate and amino acid concentrations, supplemented with fatty acids. However, these blastocysts had lower cell numbers. We hypothesised that this was due to deficiencies in embryo metabolism at the time of blastocyst formation. Thus, our objectives were to (1) determine whether using a sequential combination of nutrient concentrations could rescue blastocyst cell number; and (2) investigate the efficacy of reduced nutrient medium in 2 sources of oocytes. Oocytes were in vitro matured in identical medium either in our laboratory or during shipment from a commercial supplier. Oocytes in our laboratory were derived from feedlot heifers while purchased oocytes were obtained from culled cows. Zygotes were cultured using sequential medium with fraction V BSA. In step 1/step 2, embryos were cultured using 100% (glucose 0.5 mM/fructose 3.0mM, pyruvate 0.3/0.1mM, lactate 10.0/6.0mM, NEEA 1×/1× MEM, EAA 0.25×/0.5× MEM), 25% or 6.25% of standard nutrient concentrations. On Day 3, embryos were moved to step 2 as follows: 100% to 100%, 25% to 25%, 25% to 100%, 6.25% to 25%, or 6.25% to 100%. Lipid content of single mature oocytes from both sources was determined using gas chromatography coupled to an ISQ-LT MS/MS (GC-MS; Thermo Scientific, Waltham, MA, USA). Data (mean±s.e.m.) were analysed using ANOVA (P&amp;lt;0.05). When oocytes from feedlot heifers were used, blastocyst development and cell number did not differ between treatments. When oocytes from culled cows were used, blastocyst development was improved after embryo culture in 25-25% (45.1±3.3%) and 6.25-25% (46.6±3.2%) compared with 100-100% (34.2±3.2%). However, inner cell mass number of blastocysts cultured in 25-25% (25.6±2.5) and 6.25-25% (26.0±2.6) was reduced compared with 100-100% (41.4±4.5); TE and total cell number did not differ. Embryos cultured in 100-100%, 25-100%, and 6.25-100% were equivalent. Metabolomics revealed that 10 lipid compounds (polyunsaturated fatty acids, glycosyldiacylglycerols, and glycerophospholipids) differed in abundance between the two sources of oocytes. These results show that oocytes from different sources lead to different experimental outcomes, likely due to a combination of age, body condition, diet, and hormone treatment of the female. Oocytes from culled cows result in embryos that develop to blastocysts better in a reduced nutrient environment, although these embryos have fewer inner cell masses, suggesting that quality may be reduced. Embryos from feedlot heifer oocytes are relatively immune to nutrient fluctuations. Different endogenous fatty acid reserves in the oocyte may lead to differing metabolic strategies in the subsequent embryo, altering their response to substrate availability during in vitro culture. These results also demonstrate that reduction of nutrients during culture has no detrimental effect on blastocyst development or total cell number in either oocyte source, but that inner cell mass formation requires increased nutrient provision.

Localisation of stem cell factor, stanniocalcin-1, connective tissue growth factor and heparin-binding epidermal growth factor in the bovine uterus at the time of blastocyst formation.

Early embryonic losses before implantation account for the highest rates of reproductive failure in mammals, in particular when in vitro-produced embryos are transferred. In the present study, we used molecular biology techniques (real-time quantitative polymerase chain reaction), classical immunohistochemical staining coupled with confocal microscopy and proteomic analysis (multiple reaction monitoring and western blot analysis) to investigate the role of four growth factors in embryo-uterine interactions during blastocyst development. Supported by a validated embryo transfer model, the study investigated: (1) the expression of stem cell factor (SCF), stanniocalcin-1 (STC1), connective tissue growth factor (CTGF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bovine uterine fluid; (2) the presence of SCF, STC1, CTGF and HB-EGF mRNA and protein in the bovine endometrium and embryos; and (3) the existence of reciprocal regulation between endometrial and embryonic expression of SCF, STC1, CTGF and HB-EGF. The results suggest that these growth factors most likely play an important role during preimplantation embryo development in cattle. The information obtained from the present study can contribute to improving the performance of in vitro culture technology in cattle and other species.

The relationship between blastocyst grade, time of blastocyst formation, maternal age and chromosomal aneuploidy

To evaluate the correlation between blastocyst grade, time of blastocyst formation, maternal age and chromosomal aneuploidy. Retrospective analysis. 377 patients who underwent IVF at our center from Jan 2013 to Dec 2013 were included. A total of 1403 blastocysts underwent trophectoderm biopsy and comparative genomic hybridization (CGH). The association of morphologic score, time of blastocyst formation, maternal age and the rate of aneuploidy were analyzed. Embryos that progressed to blastocysts on day5 were less likely to be aneuploid than day6 blastocysts (Table1 A). A surprisingly high percentage of grade AA blastocysts were aneuploid. More than half of the grade AB and BB blastocysts were aneuploid (Table 1B). The aneuploidy rate increased significantly with maternal age (Table 1C). Our data suggest a correlation between aneuploidy and the time of blastocyst formation, blastocyst quality and maternal age. A significant proportion of grade AA blastocysts were aneuploid suggesting morphologic analysis alone is insufficient to ensure transfer of normal embryos. Younger age groups had a high percentage of aneuploidy suggesting that PGS testing may be considered even in younger patients. Very few D6 blastocysts from patients >40 years were euploid. PGS-testing should be strongly considered in D6 embryos in this age group.Tabled 1THE ASSOCIATION BETWEEN TIME OF BLASTOCYST FORMATION, EMBRYO GRADE, MATERNAL AGE AND RATE OF ANEUPLOIDYTable1 (A) Association between time of blastocyst formation and aneuploidyAneuploidy Rate (%)Age GroupsD5 BlastocystD6 Blastocystp valuedonor29 (n=217)38 (n=88)0.17<3524 (n=229)46 (n=128)0.00*35-3737 (n=139)53 (n=100)0.02*38-4055 (n=160)68 (n=124)0.04*41-4276 (n=80)84 (n=45)0.36>4276 (n=42)93 (n=51)0.04*Table1 (B) Association between embryo grade and aneuploidyAneuploidy Rate (%)p valueAAAB/BABBAA vs AB/BAAA vs BBAB/BA vs BBD5 Blastocyst31 (n=527)55 (n=197)58 (n=118)0.00*0.00*0.64D6 Blatocyst42 (n=177)58 (n=118)69 (n=224)0.120.00*0.04*Table1 (C) Association between maternal age and aneuploidyAge Groupsdonor<3535-3738-4041-42>42Aneuploidy Rate (%)323344617985p valuedonor vs <35 p=0.87; donor vs 35-37 p=0.00*; donor vs 38-40 p=0.00*; donor vs 41-42 p=0.00*; donor vs >42 p=0.00*; <35 vs 35-37 p=0.00*, <35 vs 38-40 p=0.00*; <35 vs 41-42 p=0.00*; <35 vs >42 p=0.00*; 35-37 vs 38-40 p=0.00*; 35-37 vs 41-42 p=0.00*; 35-37 vs >42 p=0.00*; 38-40 vs 41-42 p=0.00*; 38-40 vs >42 p=0.00*; 41-42 vs >42 p=0.29* p<0.05 statistical significant difference Open table in a new tab

Metabolic Induction and Early Responses of Mouse Blastocyst Developmental Programming following Maternal Low Protein Diet Affecting Life-Long Health

Previously, we have shown that a maternal low protein diet, fed exclusively during the preimplantation period of mouse development (Emb-LPD), is sufficient to induce by the blastocyst stage a compensatory growth phenotype in late gestation and postnatally, correlating with increased risk of adult onset cardiovascular disease and behavioural dysfunction. Here, we examine mechanisms of induction of maternal Emb-LPD programming and early compensatory responses by the embryo. Emb-LPD induced changes in maternal serum metabolites at the time of blastocyst formation (E3.5), notably reduced insulin and increased glucose, together with reduced levels of free amino acids (AAs) including branched chain AAs leucine, isoleucine and valine. Emb-LPD also caused reduction in the branched chain AAs within uterine fluid at the blastocyst stage. These maternal changes coincided with an altered content of blastocyst AAs and reduced mTORC1 signalling within blastocysts evident in reduced phosphorylation of effector S6 ribosomal protein and its ratio to total S6 protein but no change in effector 4E-BP1 phosphorylated and total pools. These changes were accompanied by increased proliferation of blastocyst trophectoderm and total cells and subsequent increased spreading of trophoblast cells in blastocyst outgrowths. We propose that induction of metabolic programming following Emb-LPD is achieved through mTORC1signalling which acts as a sensor for preimplantation embryos to detect maternal nutrient levels via branched chain AAs and/or insulin availability. Moreover, this induction step associates with changes in extra-embryonic trophectoderm behaviour occurring as early compensatory responses leading to later nutrient recovery.

The effect of timing of embryonic progression on chromosomal abnormality

To evaluate the relationship between aneuploidy and timing of blastocyst formation. Historical cohort study. Private IVF clinic. Ninety-four couples undergoing IVF treatment in combination with chromosomal screening of embryos. The mean maternal age was 39.2 years and average number of embryos per patient 5.3. A total of 530 embryos were biopsied on day 3 and underwent chromosome screening with microarray-based comparative genomic hybridization. Effect of day of embryo blastulation and morphologic grade on aneuploidy rate. Day 5 morulas that progressed to blastocysts on day 6 were significantly less likely to be aneuploid (79.8%) than day 5 morulas that did not progress to blastocysts (92.9%). However, there was no significant difference in aneuploidy rates when embryos that became blastocysts on day 5 were directly compared with embryos that became blastocysts on day 6. Delayed blastulation is not associated with increased aneuploidy rates, but absence of blastulation is associated with increased aneuploidy. Therefore, we conclude that when choosing a morula for transfer on day 5, there may be a benefit in waiting an extra day for the possibility of blastulation to occur.

Mechanisms of trophectoderm fate specification in preimplantation mouse development

During preimplantation mouse development, embryos establish two distinct cell lineages by the time of blastocyst formation: trophectoderm (TE) and inner cell mass (ICM). To explain the mechanism of this cell fate specification, two classical models, namely the inside-outside model and polarity model have been proposed based on experimental manipulation studies on embryos. This review summarizes recent findings on the molecular mechanisms of fate specification, and discusses how these findings fit into the classical models. TE development is regulated by a transcription factor cascade, the core transcription factors of which are Tead4 and Cdx2. The transcriptional activity of Tead4 is regulated by the position-dependent Hippo signaling pathway, thus supporting the inside-outside model. In contrast, several findings support the polarity model; some other findings suggest different mechanisms. We also discuss how the two classical models could be further developed in the light of recent molecular findings.

Culture of early stage ovine embryos to blastocyst enhances survival rate after cryopreservation

The current study assessed both the effects of in vitro culture and developmental stage of early stage in vivo produced ovine embryos on their ability to survive cryopreservation. Early stage embryos ( n = 226) were recovered from the oviduct, at different days of the early luteal phase, at three different developmental stages: 2- to 4-cell, 5- to 8-cell and 9- to 12-cell. For each stage, half of the embryos were cultured to the blastocyst stage and frozen thereafter (CF), while the remainder was frozen just after recovery (EF). A third experimental group (BF; n = 43) included blastocysts obtained from the uterus and frozen immediately after recovery. Embryo viability post-thawing was determined by assessing their rate of development to the hatched blastocyst stage following in vitro culture. Culture negatively affected embryo viability, since survival rate was higher in blastocysts obtained from the uterus than in those from culture (83.7% versus 66.1%; P < 0.05); also the cryosurvival of cultured embryos was lower when the timing of blastocyst formation was extended ( P < 0.01). However, survival following freezing–thawing of early developmental stages was significantly lower when compared to viability of their counterparts cultured to the blastocyst stage (23.1% versus 66.1%, P < 0.0001). In conclusion, our results indicate that, despite the deleterious effects of culture per se, the culture of early in vivo produced ovine embryos to the blastocyst stage prior to be frozen improves their survival after thawing.

Assessment of mammalian embryo quality: what can we learn from embryo morphology?

Embryo morphology assessment, however imperfect it may be, is at present the most popular method for embryo selection prior to transfer, both in human and bovine assisted reproduction. A major difference between human and bovine embryos is the fact that in the latter, assessment of morphology is jeopardized by the opacity of the blastomeres, which is caused by lipid droplet accumulation. This opacity makes it difficult to assess nuclear and nucleolar morphology, aspects which can easily be evaluated in human zygotes or early cleaving embryos. However, recent research which focused on correlation between bovine embryo morphology and embryonic ultrastructure, gene expression and cryoresistance, has provided evidence that much more can be deduced from mere embryo morphology than previously thought. Morphological features such as colour of the blastomeres, the extent of compaction, timing of blastocyst formation and expansion and diameter of the embryo at hatching can be linked with embryo quality. On the other hand, cattle embryos of deviant chromosomal constitution or with aberrant genetic make-up cannot be selected against by means of the current morphological techniques. Possible solutions include the visualization of bovine pronuclei at the zygote stage by means of ultracentrifugation or multiphoton laser scanning microscopy, and adjustment of genetic analysis in order to reconstruct embryo genetic make-up starting from the biopsy material.

Protein Kinase C Regulates the Phosphorylation and Cellular Localization of Occludin

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.

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.

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.

Culture of sheep zygotes in synthetic oviduct fluid medium with different concentrations of sodium bicarbonate and HEPES

An investigation was conducted of the viability of sheep zygotes in vivo subsequent to culture in synthetic oviduct fluid medium (SOFM) in which sodium bicarbonate was partly or wholly replaced with HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid); concentrations of 0, 6.25, 12.5, 18.75 and 25 mM HEPES were studied. The addition of HEPES lowered the pH during the culture period, and was associated with a reduction in the percentage of zygotes that developed to blastocysts (range 60.4 to 85.2%); this reduction was significant (P < 0.05) at concentrations of 18.75 and 25 mM. There was also a significant (P < 0.05) reduction in the percentage of zygotes that commenced hatching (range 47.2 to 74.1%) and a significant (P < 0.05) delay in the time of blastocyst formation (range 4 to 7 d) in medium containing 25 mM HEPES. Viability, as assessed by elongation of the trophoblast after transfer to recipient ewes, was similar for zygotes cultured for 3 d either in medium with 12.5 mM HEPES or in medium without HEPES (90.3 and 93.9%, respectively). Extending the culture period to 5 d was associated with a general decline in viability (P < 0.05), with 67.7% of zygotes cultured with HEPES elongating compared with 45.5% for those cultured without HEPES. This study confirms that sheep zygotes can be successfully cultured in SOFM for up to 3 d without loss of viability. The addition of HEPES provides additional buffering capacity but zygote development may be compromised.

Nuclear reprogramming in nuclear transplant rabbit embryos.

The first six genetically verified nuclear transplant rabbits have been produced in this study. Individual eight-cell stage embryo blastomeres were transferred and fused with enucleated mature oocytes of which six full-term offspring were produced out of 164 manipulated eggs. The following efficiency rates were determined for the nuclear transplantation procedure: chromosomal removal from oocytes, 92%; fusion rate, 84%; activation rate, 46%; embryo transfer rate, 27%. Additional reasons for the low efficiency rate of nuclear transplant embryos may include limited development due to aging in recipient oocytes and asynchronous transfers of manipulated embryos to recipient females. The successful development to term may have been due to the ability of the mature oocyte to reprogram the eight-cell stage nuclei. The number of cells in blastocysts derived from isolated eight-cell blastomeres (18 +/- .08) was lower than that of nonmanipulated pronuclear (106 +/- 5.1) and nuclear transplant embryos derived from eight-cell stage nuclei (91 +/- 10.2) (p less than 0.001). This evidence along with the significant amount of nuclear swelling in nuclear transplant embryos and a delay in the time of blastocyst formation indicate that nuclear reprogramming had taken place in these embryos. Successful nuclear reprogramming indicates that serial transfers could result in the expanded multiplication of mammalian embryos.

The developmental potential of mouse 16-cell blastomeres.

Based on the criteria of relative size and cell surface polarization, subpopulations of outer (larger, polar) and inner (smaller, apolar) blastomeres have been isolated from mouse 16-cell morulae and reaggregated in groups of 16 cells, and the developmental potential of the aggregates has been assessed both in vitro and in vivo. Aggregates of outer, inner, or mixed groups of cells all formed blastocysts which outgrew and contained both characteristic trophectoderm and alkaline phosphatase positive inner cell masses in vitro. However, significant differences were observed in the timing of blastocyst formation, depending on cell type. Aggregates of outer cells recompacted more slowly, commenced fluid accumulation earlier, and contained more cells at the blastocyst stage than did inner cell aggregates. Aggregates containing both outer and inner cells were intermediate. Postimplantation development of blastocysts derived from aggregates of outer or inner cells, after transfer to pseudopregnant recipients, was normal and comparable to zona-intact control embryos. This relationship between the expression of different morphological and behavioural properties by the precursor cells of the trophectoderm and inner cell mass and the existence of totipotent cells in both outer and inner subpopulations is discussed.

Protein content of porcine embryos during the first nine days of development

Little is known about the physiochemical aspects of porcine embryos prior to implantation. The purpose of this study was to quantitate the total protein content of porcine embryos from fertilization through day 9 of development. Thirty-seven gilts and two sows were hand mated and the reproductive tracts collected at slaughter 1 to 9 days after the onset of estrus. The uteri were flushed with phosphate buffered saline (PBS), and embryos were collected, washed with PBS and transferred directly to test tubes containing distilled water. Only embryos which appeared morphologically normal were used. Protein content was determined by the Bio-Rad microassay. Standard curves were constructed for each assay using. 8 to 19 μg gamma globulin (Bio-Rad assay). Protein standards were run in triplicate. Regression lines were calculated for protein standards, and the resulting line was used to determine total protein in the unknown samples. Protein content of unfertilized eggs and embryos increased steadily through days 3, 4 and 5 of development, from 273 to 334, 491 and 620 ng, respectively. This result suggests that the protein content of porcine embryos increases only slightly from fertilization through day 5 of development. A dramatic increase in embryo protein content was observed between days 6 and 9 of development, which is the time of blastocyst formation and hatching of the embryo from the zona pellucida.

Interruption of pregnancy in the rat and hamster by administration of PMS or HCG.

Intraperitoneal injection of 50 IU PMS on Days 1–3 after mating resulted in the early transport of ova from the rat tube into the uterus. This effect was less markedly shown in the hamster but retardation of cleavage was observed in a few cases. When 100 IU HCG was injected, tubal transport was not markedly accelerated in either the rat or the hamster. Following treatment with 50 IU PMS on Days 1–3, no living embryos were found in rats and hamsters killed on Days 16 and 13, respectively. Implantation was inhibited in the rat, though not in the hamster. It also failed when normal blastocysts were transferred synchronously into the PMS-treated rats. Implantation did occur in rats treated with various doses of PMS on Days 5–7, but fetal mortality (54–100%) increased proportionately with the dose. A single dose of 100 IU PMS on one of Days 5, 7, 9 and 11 in the rat induced 67, 92, 75 and 56% fetal mortality, respectively. Administration of 100 IU HCG on Days 1–3 had a slight effect on preand post-implantation losses in the rat. The same treatment in the hamster reduced the number of implantation sites and provoked considerable embryonic mortality (73%). Although rats proved relatively more sensitive to PMS than HCG given during implantation, the effects of HCG given at this time are seen more readily in the hamster than in the rat. A single dose of 100 IU HCG given to the hamster on one of Days 4, 6, 8 and 10 induced 13, 92, 39 and 24% embryonic mortality, respectively. Supplemental treatment with progesterone in the rat and hamster did not reduce the adverse effect on pregnancy in the PMS-treated females, but was beneficial in animals ovariectomized at the time of blastocyst formation. It thus seems likely that antifertility effects of PMS may have been due to abnormal steroid production in the ovary. (Endocrinology83: 217, 1968)