1 Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Toronto, Ontario, Canada and 2 Toronto Center for Advanced Reproductive Technology, Toronto, Ontario, Canada
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Abstract |
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Key words: blastocyst/ß-human chorionic gonadotrophin/human leukocyte antigen-G/implantation/pregnancy specific ß-1 glycoprotein
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Introduction |
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Development of spare embryos to the blastocyst stage in vitro ranges between 3 and 62% depending on the medium and culture conditions used (for overview see Olivennes et al., 1994). Rates of blastocyst formation for non-preselected embryos suggest that ~50% are able to reach this developmental stage in vitro, but only a subset of these (2535%) are able to establish a viable pregnancy (Alves da Motta et al., 1998
; Jones et al., 1998
; Rijnders and Jansen, 1998
). Thus, even among groups of human embryos capable of reaching the blastocyst stage, some embryos are not able to initiate implantation and/or proceed further in development. This observation is further supported by observations of morphological (Dokras et al., 1991
), molecular (Woodward et al., 1994
; Jurisicova et al., 1996a
; Saith et al., 1996
) and metabolic (Magnusson et al., 1986
; Gardner and Lane, 1997
) variability in the quality of human blastocysts. Furthermore, cellular abnormalities such as lack of inner cell mass (ICM) cells (Winston et al., 1991
; Desai et al., 1997
), a high rate of cell death (for review see Hardy, 1997
) and abnormalities in thinning of the zona pellucida or inability to initiate hatching also contribute to lower than expected pregnancy rates observed after blastocyst transfer. These results prompted us to analyse the quality of human blastocysts with respect to their transcriptional activity. Assessment of cell number, cell death and expression of three markers of embryonic quality, human chorionic gonadotrophin beta (ß-HCG), pregnancy specific ß-1 glycoprotein (SP-1) and human leukocyte antigen G (HLA-G), was performed.
ß-HCG and SP-1 are placental hormones secreted by syncytiotrophoblast with concentrations detectable in the maternal blood around day 14 post-fertilization (Grudzinkas et al., 1977). Both of these markers are used for assessment of functionality as well as chromosomal normality of the developing placenta and embryo (Bogart et al., 1987
; Graham et al., 1992
). Moreover, both these markers have been detected at variable levels in culture media of human embryos (Dimitriadou et al., 1992
; Woodward et al., 1993
).
HLA-G is a non-classical class I antigen with low polymorphism, found on the surface of invasive cytotrophoblast cells that are in direct contact with maternal decidua (Kovats et al., 1990a). Expression of HLA-G by some cleavage stage human embryos (Jurisicova et al., 1996a) and the possibility that HLA-G may be a candidate for a human homologue of the mouse preimplantation embryo development (Ped) gene (Jurisicova et al., 1996b
) have previously been reported. Both SP-1 and HLA-G are thought to play a role in protecting the fetus from maternal immune system recognition and rejection, while ß-HCG is involved in paracrine interaction with the corpus luteum and endometrium.
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Materials and methods |
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Spare embryos of variable quality that appeared to arise from normally fertilized oocytes with two pronuclei (2PN), as well as abnormally fertilized (3PN) or activated oocytes (1PN), were used in this study. IVF or intracytoplasmic sperm injection (ICSI) was performed according to standard techniques as previously described (Segal and Casper, 1992; Greenblatt et al., 1995
). At day 2 (48 h post-insemination), single embryos were moved from human tubal fluid (HTF) medium into either a new 1 ml aliquot of HTF (Irvine Scientific, CA, USA) supplemented with 10% synthetic serum supplement (SSS) (Irvine Scientific) or synthetic oviductal medium enriched with potassium (KSOM) (Erbach et al., 1994
) medium containing 0.5xnon-essential amino acids (Gibco BRL, Grand Island, NY, USA) supplemented with 10% SSS and were maintained at 37°C in 5% CO2 and 95% air. Assessment of embryo quality and developmental stage were recorded daily until the embryos reached the expanded blastocyst stage. From the subset of embryos, the culture media was collected and stored at 70°C for further analysis.
Analysis of cell number and cell death
In order to analyse the status of chromatin in all obtained blastocysts, we used nuclear staining with the fluorochrome 4,6-diamidino-2-phenylindole (DAPI, Sigma, St Louis, MO, USA). The assessment of cell death was based on DNA condensation and nuclear morphology (Jurisicova et al., 1998). The cell death index (CDI) was calculated as the percentage of total cells which exhibited nuclear abnormalities.
Analysis of gene expression in human blastocysts
A subset of human blastocysts (n = 37) obtained from normally fertilized oocytes was stained by adding DAPI to the culture medium. After 30 min incubation, the zona pellucida was removed and embryos were viewed briefly under fluorescence. The numbers of normal cells and cells with condensed DNA were recorded. Subsequently, these randomly chosen blastocysts were used for analysis of gene expression. All embryos analysed in this group were cultured in HTF medium. ß-HCG and HLA-G expression were determined by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) based assay (Rambhatla et al., 1995). The amplified material was dot blotted and analysed by hybridization with radiolabelled cDNA probes, followed by quantification of signals on a phosphorimager. Results are presented in counts per minute (c.p.m.) generated by a bound probe. Estimates of mRNA copy number were calculated as described (Rambhatla et al., 1995
) with some modification regarding size differences between human (120 µm) and mouse (80 µm) oocytes as well as differences in mRNA turnover due to the delayed activation of the zygotic genome in humans.
The cDNA probes used for analysis recognized the 3' untranslated region of the genes, and were either donated for this study (see Acknowledgements) or were cloned in this laboratory. cDNA products of single embryos were prescreened for the quality of cDNA by hybridization with elongation factor 1 (EF-1
). Using this control, it was determined that 5/37 samples failed to produce good quality hybridization signals, and these cDNA samples were excluded from further analysis.
Determination of SP-1 concentrations in culture medium
A double antibody enzyme-linked immunosorbent assay (ELISA) was used to measure SP1 in embryo culture media. High binding capacity 96-well plates were coated with 17 µg/ml of rabbit polyclonal anti-SP1 immunoglobulin G (IgG) (Dako, Copenhagen, Denmark). Excess sites were blocked with phosphate buffered saline (PBS), 0.5% Tween 20 (PBST), 5% non-fat milk powder for 2 h at room temperature. A 100 µl aliquot of embryo culture media was added per well and incubated overnight at 4°C. After washing with PBST, 10 µg/ml of biotinylated anti-SP1 was added per well and incubated for 2 h at room temperature. Streptavidin-horseradish peroxidase (Calbiochem, La Jolla, CA, USA) diluted 1:200 in PBST was applied and reactions were developed with o-phenylenediamine in citrate buffer containing 0.04% H2O2. Pooled cord blood serum (n = 50) from term vaginal deliveries diluted in PBS were used as assay standards (Grudzinkas et al., 1977).
Statistical analysis
Differences between the proportions of embryos developing to the blastocyst stage were compared by 2 analysis with Yates' correction. The effect of culture conditions, abnormal nuclear morphology and ploidy on total cell number and the cell death index were analysed by the MannWhitney rank sum test and by KruskalWallis one-way analysis of variance on ranks respectively. Analysis of cell number and the level of expression of studied genes and protein product were performed by Pearson's rank correlation using the SigmaStat statistical package (Version 1.0).
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Results |
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The type of culture medium used affected the rate of blastocyst formation of 2PN embryos (Table I). A significantly higher proportion of normally fertilized embryos developed to blastocyst stage in KSOM medium (25 versus 11.8% in HTF). In contrast, no effect of medium on the rate of blastocyst formation was observed in 1PN (7.8 versus 7.4%) or 3PN (14.8 versus 11.2%) embryos, suggesting that these embryos were developmentally compromised and that improved culture medium, such as KSOM, was unable to increase their developmental potential (see Table I
). The rate of blastocyst formation for embryos with multinucleated blastomeres was 10% (12/122). Interestingly, none of the embryos containing blastomeres with fractured nuclei developed beyond the 16-cell stage. The remaining embryos arrested and/or further fragmented at various points during in-vitro culture.
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Discussion |
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In-vitro development past the four-cell stage is believed to be one of the factors allowing selection of embryos that may reach the blastocyst stage. In the experiments in this study, spare embryos were used which were of poor quality and contained either excessive fragmentation (<30%), polynucleated blastomeres or cells with fractured nuclei and thus were not suitable for transfer or cryopreservation. Even though this pool represents a group of developmentally compromised embryos, gene expression analysis of this subset can offer at least some information regarding mechanisms governing molecular events occurring during human preimplantation embryo development.
The rates of blastocyst formation observed in this study are within the reported range of development for spare embryos. Cell numbers and CDI, recorded at day 6 and 7, were comparable with those previously reported (Hardy et al., 1989) and no significant effect of culture media on cell number or cell death rates was observed. Interestingly, a positive effect of KSOM media on the frequency of blastulation in the group of normally fertilized embryos was observed. In addition, KSOM supplemented with non-essential amino acids stimulated the ability of embryos to secrete SP-1, since concentrations almost twice as high were recorded in this medium when compared to HTF. Thus, KSOM not only improves the developmental potential of human embryos, but its composition also appears to have a positive influence on the rate of metabolism. This observation is in agreement with that of a previous study (Ho et al., 1995
), which reported that mouse blastocysts cultured in KSOM supplemented with amino acids displayed similar rates of gene transcription to those that developed in vivo.
Cell death appears to be an integral component of development of mammalian embryos at the blastocyst stage (for review see Hardy, 1997). Results of several experiments published recently identified some triggers of apoptosis in mouse and rat blastocysts, such as hyperglycaemia (Moley et al., 1998
), tumour necrosis factor (TNF)-
(Pampfer et al., 1997
) and glutamine (Devreker and Hardy, 1997
). Unfortunately, the molecular triggers of this process in the human remain unknown. It would be interesting to assess whether removal of glutamine from KSOM medium or addition of transforming growth factor (TGF)-
(Brison and Schultz, 1997
) can decrease cell death rates in human embryos, as was previously observed for mouse embryos.
One of the most widely studied markers of embryonic development is ß-HCG. Initiation of ß-HCG transcription was previously observed between the six- to eight-cell stage using in-situ hybridization (Bonduelle et al., 1988). In the current study, expression was detected as early as at the two-cell stage, which could be attributed to the more sensitive RT-PCR technique employed. Previously, it was proposed that the trophoectodermal (TE) lineage in humans is established by a single cell formed after the second cleavage division (Edwards and Beard, 1997
). Moreover, based on experiments performed with X inactivation in the mouse (Sheardown et al., 1997
), it appears that the default differentiation state of the blastomeres is the TE state. In a subset of cells in eight-cell embryos, Oct-4, a transcriptional repressor of placental markers (Liu and Roberts, 1996
), appears not to localize to the nucleus and thus fails to repress the TE state (reviewed in Edwards and Beard, 1997
). Oct-4 transcript also could be detected in human oocytes and early cleavage-stage embryos (Abdel-Rahman et al., 1995
), but whether Oct-4 product is able to function at such an early stage remains unknown. Consistent with this hypothesis is our observation that HLA-G antigen could be found on the surface of all blastomeres of two- to eight-cell human embryos, but at the blastocyst stage only TE cells maintain this expression (Jurisicova et al., 1996a
). At present, it is not known whether a subset of blastomere(s) at the 8-cell stage also expresses ß-HCG.
Secretion of ß-HCG protein by blastocysts commences around day 7 with peak concentrations observed around day 10 (Dokras et al., 1991; Woodward et al., 1993
). The biochemical profile of ß-HCG molecules changes with the differentiation of trophectoderm, which in turn may influence the biological activity of this molecule (Lopata et al., 1997
). Interestingly, variability in the secretion of ß-HCG among human blastocysts cultured in vitro was observed, with only a subset of embryos being able to produce detectable concentrations (48% reported by Woodward et al., 1994
, 55% reported by Dimitriadiou et al., 1992). Moreover, embryos maintained in favourable culture conditions, such as on co-culture with Vero cells, secrete higher concentrations of HCG (Turner and Lenton, 1996
). At the present time, it is not clear whether lack of transcription, translation or secretion is responsible for such variability. The fact that in this study detectable, but variable, amounts of ß-HCG transcript were observed suggest that lack of transcription is an unlikely cause of HCG variability. Attempts were also made to measure ß-HCG in culture medium from these embryos using a radioimmunoassay with a sensitivity of 5 IU/l. ß-HCG was undetectable in the range of the standard curve. This could be explained either by the dilution by the larger volume of media used to culture embryos (1 ml) or insufficient sensitivity of the assay. Previously, no association could be found between ability to secrete ß-HCG, cleavage rates and morphological assessment of early embryos (Woodward et al., 1994
). In addition, no correlation was found between the rate of ß-HCG transcription, and cell numbers or cell death rates at the blastocyst stage.
Similarly, amounts of HLA-G mRNA proved to be variable in the population of embryos studied. This observation is consistent with our previous report (Jurisicova et al., 1996b) that only a proportion (47%) of human blastocysts are able to express HLA-G. In the current study group, the proportion of HLA-G positive blastocysts was higher (90%) than previously reported, but a different RT-PCR approach was used and the embryos were pre-selected based on expression of elongation factor EF-1
, which eliminates those with low transcriptional activity. As in the case of ß-HCG, no association could be found between the amount of HLA-G transcript and cell numbers. However, a very strong positive correlation was observed between amounts of HLA-G and ß-HCG transcript. It is not clear whether this relationship reflects the fact that both genes are regulated by the same transcription factors or whether a direct relationship exists between expression of ß-HCG and HLA-G. Originally, it was proposed that HLA-G may be a human homologue of the murine Ped gene that appears to influence the rate of mouse embryo cleavage. More blastomeres were observed at 48 and 72 h post-fertilization in HLA-G positive embryos then in those negative for HLA-G (Jurisicova et al., 1996a
). However, some recent studies revealed biochemical differences between HLA-G and the Qa-2 antigen, a protein product of the Ped gene, and failed to show an association between cleavage rate and HLA-G mRNA expression (Warner et al., 1998
).
SP-1 is another marker that could be detected in the culture media of a subset (55%) of early cleavage stage embryos from day 3 onwards. However, no relationship could be found between the ability to secrete SP-1 and ß-HCG, nor did there appear to be a connection between the developmental potential of embryos and the concentrations of these two hormones (Dimitriadou et al., 1992). The lack of correlation between concentrations of SP-1 protein and the rate of ß-HCG transcription also supports this observation and confirms the hypothesis that SP-1 and ß-HCG do not share the same regulatory pathways.
In a recent study (Saith et al., 1996), cumulative SP-1 concentrations were decreased in vacuolated morulae, but failed to distinguish blastocysts of different morphological grades. In this study, a significant positive correlation was found between cell number and concentrations of SP-1. This observation may have clinical relevance, since this molecule can be used as a marker for selecting blastocysts with higher cell numbers. However, the real predictive value of SP-1 with respect to developmental potential needs to be evaluated by transfer of such blastocysts and prospective analysis of pregnancy outcome.
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Acknowledgments |
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Notes |
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References |
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Submitted on November 25, 1998; accepted on April 8, 1999.