Human Morula Research Articles

Treatment with Laevo (L)-carnitine reverses the mitochondrial function of human embryos.

Laevo (l)-carnitine plays important roles in reducing the cytotoxic effects of free fatty acids by forming acyl-carnitine and promoting beta-oxidation, leading to alleviation of cell damage. Recently, the mitochondrial functions in morula has been shown to decrease with the maternal age. Here, we assessed the effect of l-carnitine on mitochondrial function in human embryos and embryo development. To examine the effect of L-carnitine on mitochondrial function in morulae, 38 vitrified-thawed embryos at the 6-11-cell stage on day 3 after ICSI were donated from 19 couples. Each couple donated two embryos. Two siblings from each couple were divided randomly into two groups and were cultured in medium with or without 1 mM L-carnitine. The oxygen consumption rates (OCRs) were measured at morula stage. The development of 1029 zygotes cultured in medium with or without L-carnitine was prospectively analyzed. Addition of L-carnitine to the culture medium significantly increased the OCRs of morulae and improved the morphologically-good blastocyst formation rate per zygote compared with sibling embryos. Twenty healthy babies were born from embryos cultured in L-carnitine-supplemented medium after single embryo transfers. L-carnitine is a promising culture medium supplement that might be able to counteract the decreased mitochondrial function in human morula stage embryos.

Maternal-biased H3K27me3 correlates with paternal-specific gene expression in the human morula.

Genomic imprinting is an epigenetic mechanism by which genes are expressed in a parental origin-dependent manner. We recently discovered that, like DNA methylation, oocyte-inherited H3K27me3 can also serve as an imprinting mark in mouse preimplantation embryos. In this study, we found H3K27me3 is strongly biased toward the maternal allele with some associated with DNA methylation-independent paternally expressed genes (PEGs) in human morulae. The H3K27me3 domains largely overlap with DNA partially methylated domains (PMDs) and occupy developmental gene promoters. Thus, our study not only reveals the H3K27me3 landscape but also establishes a correlation between maternal-biased H3K27me3 and PEGs in human morulae.

The Usefulness of the Measurement of Zona Pellucida Thickness in the Morphological Evaluation of Human Morula

The relationship between morphologic criteria including zona pellucida thickness (ZPT) and the ET outcomes was studied for day-4 fresh morula transfer (n=306). Pregnancy rate (PR) was significantly correlated with the proportion of compaction (P<0.0001), the morphology (P<0.0001) and the amount of fragmentation (P=0.0004) of the best morula. In cycles with single morula transfer (n=121), only the morphology was significantly correlated with PR (P=0.03). The average ZPT of the best morula was significantly thinner (P<0.0001) in conception cycles (7.9 ± 2.1 μm) than in non-conception cycles (9.3 ± 1.8 μm). Also in cycles with single morula transfer, the average ZPT was significantly thinner (P=0.004) in conception cycles (8.5 ± 1.9 μm) than in non-conception cycles (9.7 ± 2.0 μm). Factors influencing conception were assessed by logistic-regression analysis. The average ZPT (P<0.0001), the morphology (P=0.0003) and the amount of fragmentation (P=0.02) of the best morula were significantly related to the conception, whereas only the average ZPT (P=0.01) was related to the conception in single morula transfer cycles. We concluded that the average ZPT and the morphology of embryo seem to be strong predictors of pregnancy in day-4 morula transfer and thus important indicators of good embryo quality.

Cryopreservation of human embryos at the morula stage and outcomes after transfer

Objective To evaluate the survival rate of human morula embryo freezing and the morphological alterations during freezing, during and after thawing, and their applications in embryo selection. Design Retrospective observational study. Setting Private infertility clinic. Patient(s) Consecutive patients under age 39 undergoing frozen morula embryo transfers from December 1999 to May 2003. Intervention(s) Embryo freezing was performed at the morula stage. Embryo thaw and post-thaw ETs were conducted on the same day, which is equivalent to a day 4 ET. Main outcome measure(s) Morphological alterations during freezing and thawing and after thawing. Post-thaw embryo survival rates, transferable rates, pregnancy rates, and implantation rates. Result(s) Morula embryos showed reversed morphological alterations during the freezing process; these alterations were recovered during thawing or shortly after the thawing. Post-thaw survival rates showed no significant difference between any of the morula substages. However, embryos scored as grade 3, which represented good quality, had significantly higher post-thaw survival and transferable rates than grade 2 and 1 embryos. Patients who received at least one grade 3 embryo had significantly higher pregnancy rates, implantation rates, and ongoing/live birth rates than other groups. Conclusion(s) An acceptable survival rate can be achieved after cryopreservation of human morula embryos, and morphological alterations that occur during and shortly after an embryo thaw can be a feasible index for determining viable embryos.

Morula-derived human embryonic stem cells

Human embryonic stem (ES) cells are known to derive from the inner cell mass of blastocyst. Although the embryos of other developmental stages have also been used as a source for ES cells in animal models, the feasibility of obtaining ES cell lines from human morula is not known, despite being an obvious source available through assisted reproduction and preimplantation genetic diagnosis programmes. This study describes an original technique for derivation of ES cells from human morula, which enabled the establishment of eight morula-derived ES cell lines. These ES cell lines were shown to have no morphological differences from the ES cells derived from blastocysts, and expressed the same ES cell specific markers, including Oct-4, tumour-resistance antigens TRA-2–39, stage-specific embryonic antigens SSEA-3 and SSEA-4, and high molecular weight glycoproteins TRA-1–60 and TRA-1–81, detected in the same colony of morula-derived ES cells showing specific alkaline phosphatase expression. No differences were observed in these marker expressions in the morula-derived ES cells cultured in the feeder layer free medium. Similar to ES cell originating from blastocyst, the morula-derived ES cells were shown to spontaneously differentiate in vitro into a variety of cell types, including the neuron-like and contracting primitive cardiocyte-like cells.

Developmental capacity of different morphological types of day 5 human morulae and blastocysts

After prolonged culture of human embryos, the expanded blastocysts with oval inner cell mass (ICM) and cohesive trophectoderm (TE) are preferably selected for transfer. In cycles with poor embryonic development, the selection has to be done from among suboptimal blastocysts for which no grading system exists. In this study, 1396 transferred blastocysts and morulae were classified into eight morphologic categories. The B1 category constituted the optimal blastocysts. The other categories were characterized by different deviations from optimal blastocysts: cytoplasmic fragments and necrosis in TE (B2), unexpanded blastocoele (B3), non-compact or small ICM (B4), fragments in TE and ICM (B5), up to 20% excluded blastomeres (B6), necrotic TE and ICM (B7), and more than 20% excluded cells from blastocysts (B8). The live birth rate was calculated from blastocysts with known outcome after transfer (88.9% transferred blastocysts). The birth rate declined from B1 to B8 by the same order and was: 45.2, 32.8, 26.9, 23, 17.7, 16.7, 7.7 and 1.2% respectively. Normal ICM was recognized as the most important parameter for implantation. There was a strong relation between such ordered morphology categories and implantation capacity ( P < 0.0001). Such a grading system is helpful in selecting the best of all available day-5 embryos for transfer.

Expression of cystic fibrosis transmembrane conductance regulator during early human embryo development.

Formation of the blastocyst is one of the first morphological changes in early embryonic development. Ion transport has been shown to be crucial for blastocoele cavity formation and expansion, although the mechanisms that underlie this process are presently unknown. As a transmembrane Cl(-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR) may participate in ion transport and early blastocoele formation. CFTR mRNA was detected throughout preimplantation embryo development and in the unfertilized oocyte. Immunocytochemistry disclosed the presence of CFTR protein from the 8-cell stage, reaching maximum immunoreactivity at early blastocyst stage embryos. Patch clamp electrophysiology of morulae and blastocysts demonstrated typical CFTR Cl(-) channel activities in the apical membrane of trophectoderm cells. Thus CFTR is expressed both at mRNA and protein levels in human morulae and blastocysts, and functions as a cAMP-regulated apical membrane Cl(-) channel. These data suggest that CFTR may contribute to blastocoele formation in the early human embryo.

Glucose metabolism of human morula and blastocyst-stage embryos and its relationship to viability after transfer

The pregnancy rate and implantation rate following blastocyst transfer in the human have been reported to be high; however, it has remained necessary to transfer 2–3 blastocysts to achieve these rates. Morphological criteria are currently used to select blastocysts for transfer and have some limited correlation with ongoing viability. Glucose metabolism of 189 human morula to blastocyst stage embryos was analysed using a non-invasive ultramicrofluorescence technique to determine if this could be used to predict viability. There was a linear trend to increased glucose uptake with progression from the morula to the hatching/hatched blastocyst stage of development, whereas glycolytic activity did not vary. There was no consistent difference in glucose uptake or glycolytic activity for embryos at the various morphological stages on day 5 compared to day 6 in vitro. Glucose uptake and glycolytic activity of the nine embryos positively identified as having implanted following transfer varied and were apparently not different from the values for embryos that failed to implant. In addition, viability was demonstrated to be compatible with high glycolytic activity, with four of nine implanted embryos having a glycolytic activity in the highest 15% of the population of embryos studied. Glucose uptake and glycolytic activity of male and female embryos did not appear to be different. Glucose metabolism cannot be used prospectively to select viable human morula or blastocyst stage embryos for transfer and it is also unlikely to be a useful tool to predict the sex of the embryo.

Assessment of polyploidy in human morulae and blastocysts using co-culture and fluorescent in-situ hybridization.

Fluorescence in-situ hybridization with DNA probes for X, Y and no. 18 chromosomes was used to analyse human morulae (n = 13) and blastocysts (n = 41), obtained after co-culture on Vero cells. On the basis of the number of hybridization signals, the proportion of embryos with more than five polyploid cells was 30.8% for morulae and 29.3% for blastocysts. These values are similar to those for mixoploidy (mosaicism of diploid and polyploid cells) observed in blastocysts of animal species. The results were confirmed by scanning electron microscopy, which showed a wide variation in the size of blastocyst nuclei, and by classical cytogenetic analysis. Mixoploidy seems to be a normal feature in preimplantation embryos and to occur very early in human embryo development. This lays open to doubt the preimplantation diagnosis of genetic errors at these stages, since results obtained from single cell analysis may not be representative of the whole embryo.

Tight junctions and cavitation in the human pre-embryo.

In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intracellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome-like bodies. Since the vacuoles and intracellular and extracellular cavities contain electron-dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocytes, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed.

An investigation by mathematical modelling of whether mouse and human preimplantation embryos in static culture can satisfy their demands for oxygen by diffusion

Mammalian preimplantation embryos are conventionally grown in small, static droplets of medium. By contrast, embryos within the oviduct are subject to mixing forces arising from the action of cilia and the contractions of the myosalpinx. Such forces will minimize the buildup of unstirred layers around the embryos and facilitate the exchange of gases and metabolites. We have devised a mathematical model to investigate whether preimplantation mouse and human embryos grown in static culture can satisfy their requirement for oxygen solely by diffusion i.e. in the absence of stirring. The model incorporates the diffusion coefficients for oxygen in the medium outside and within the embryo, the oxygen tension of the culture medium, the size of the embryo and its oxygen uptake. Solutions of the model are provided for mouse oocytes and 2-cell embryos, mouse blastocysts and human morulae. In each case, the model is solved for two different values of the diffusion coefficient of oxygen within the cell and of the oxygen content of the medium (5 and 20%). The conclusion is that mouse embryos in static culture are likely to be able to satisfy their demands for oxygen by diffusion alone, but that human embryos may become marginally hypoxic, especially at lower oxygen levels.

Ultrastructural studies of the implantation process.

The human ovum arrives at the uterine cavity by day 4 of development. At that time the ovum is a morula or an early blastocyst. Hertig et al. (1954) have described a human morula of about 96 h developmental age found within the uterine cavity and consisting of 58 cells: 5 embryonic and 53 trophoblastic. All the trophoblastic cells were of a fairly uniform cuboidal nature. The zona pellucida was still present (Carnegie No. 8794). In Hertig’s paper is a description of a human blastocyst of 4½ days of developmental age and consisting of 107 cells: 8 embryonic and 99 trophoblastic. An embryonic disc was formed at this stage. The trophoblastic cells were thinned out, except those placed at the embryonic pole. These cells (the “polar trophoblasts”) make the first contact with the uterine epithelium at the implantation site.