Variability in the expression of trophectodermal markers ß-human chorionic gonadotrophin, human leukocyte antigen-G and pregnancy specific ß-1 glycoprotein by the human blastocyst

Andrea Jurisicova1, Monica Antenos1, Kubra Kapasi1, James Meriano2 and Robert F. Casper1,2,3

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Improved culture conditions that support the development of human embryos to the blastocyst stage in vitro led to the prospect of blastocyst transfer to increase pregnancy rates. Thus, there is a need for characterization of possible biochemical markers able to predict the implantation potential of human blastocysts. In this study, the expression of three placental markers that are expressed prior to implantation, ß-human chorionic gonadotrophin (HCG), human leukocyte antigen (HLA)-G and pregnancy specific ß-1 glycoprotein (SP-1), was investigated. ß-HCG transcript could be detected as early as the two-cell stage, which is one to two cleavage divisions earlier than previously reported. Both ß-HCG and HLA-G transcripts could be detected in the majority of blastocysts, but their levels were highly variable. No association could be found between the amount of transcript for these genes, total cell number or cell death rate. Interestingly, there was a highly positive correlation between accumulation of ß-HCG and HLA-G transcripts. SP-1 protein concentrations were assessed in the culture medium of blastocysts using enzyme-linked immunosorbent assay. There was a significant positive correlation between SP-1 concentrations and blastocyst cell numbers. Moreover, synthetic oviductal medium enriched with potassium resulted in an SP-1 concentration twice as high as that observed using human tubal fluid medium. These data suggest that SP-1 may be used to select blastocysts with higher cell number, possibly resulting in higher pregnancy rates.

Key words: blastocyst/ß-human chorionic gonadotrophin/human leukocyte antigen-G/implantation/pregnancy specific ß-1 glycoprotein


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite several improvements implemented in the in-vitro fertilization (IVF) procedure, pregnancy rates have not changed dramatically in the past decade. New knowledge on metabolic requirements of the preimplantation embryo and improvement of culture media allowed extended in-vitro embryo culture up to the blastocyst stage. A series of preliminary trials of blastocyst transfer in selected groups of patients appeared to result in excellent pregnancy rates (for reviews, see Bavister and Boatman, 1997Go; Desai, 1998Go). Implantation rates up to 50% were reported when embryo transfer was performed at day 5 (Gardner et al., 1998Go), suggesting that blastocyst transfers are a desirable addition to new reproductive technologies. However, as with any change applied to a routine clinical setting, the transfer of embryos at this later developmental stage has several pitfalls and benefits that should be considered (Gardner and Schoolcraft, 1998Go; Tsirigotis, 1998Go).

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., 1994Go). 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 (25–35%) are able to establish a viable pregnancy (Alves da Motta et al., 1998Go; Jones et al., 1998Go; Rijnders and Jansen, 1998Go). 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., 1991Go), molecular (Woodward et al., 1994Go; Jurisicova et al., 1996aGo; Saith et al., 1996Go) and metabolic (Magnusson et al., 1986Go; Gardner and Lane, 1997Go) variability in the quality of human blastocysts. Furthermore, cellular abnormalities such as lack of inner cell mass (ICM) cells (Winston et al., 1991Go; Desai et al., 1997Go), a high rate of cell death (for review see Hardy, 1997Go) 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., 1977Go). Both of these markers are used for assessment of functionality as well as chromosomal normality of the developing placenta and embryo (Bogart et al., 1987Go; Graham et al., 1992Go). Moreover, both these markers have been detected at variable levels in culture media of human embryos (Dimitriadou et al., 1992Go; Woodward et al., 1993Go).

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., 1996aGo) 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., 1996bGo) 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryo culture
Spare human preimplantation embryos were obtained from the IVF Program, Division of Reproductive Sciences, at the Toronto Hospital and from the Toronto Center for Advanced Reproductive Technologies. Patients who elected not to freeze their spare embryos for future transfers either by choice or because the embryos were not suitable for cryopreservation due to extensive fragmentation or multinucleation of blastomeres, were asked to donate these embryos for research. This project was approved by the human ethics committee of the Toronto Hospital and by the University of Toronto.

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, 1992Go; Greenblatt et al., 1995Go). 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., 1994Go) 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., 1998Go). 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., 1995Go). 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., 1995Go) 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{alpha} (EF-1{alpha}). 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., 1977Go).

Statistical analysis
Differences between the proportions of embryos developing to the blastocyst stage were compared by {chi}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 Mann–Whitney rank sum test and by Kruskal–Wallis 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).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryo culture
A total of 1040 spare human preimplantation embryos was obtained after either IVF or ICSI at day 2 post-insemination. The major proportion of embryos (n = 611) developed from zygotes with two pronuclei (2PN) and ~70% of these embryos exhibited at least 30% of cellular fragmentation (grade 3). Of these 611 embryos, 122 contained at least one multinucleated blastomere and 16 contained blastomeres with fractured nuclei. All the abnormally fertilized or activated oocytes were obtained by ICSI. One hundred and seventy of them appeared to have only a single pronucleus (1PN) with two polar bodies, while 259 had three visible pronuclei (3PN).

The type of culture medium used affected the rate of blastocyst formation of 2PN embryos (Table IGo). 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 IGo). 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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Table I. Developmental potential of human embryos cultured in human tubal fluid (HTF) or synthetic oviductal medium supplemented with potassium (KSOM) in vitro
 
All blastocysts were subjected to analysis of cell number, but five were lost during this procedure. The mean (±SEM) number of cells per blastocyst on day 6 was 54 ± 3, with only a few cells (6 ± 1) displaying signs of cell death. By day 7, after further development to the fully expanded or hatching blastocyst stage, the mean cell number had increased significantly to 83 ± 7 (Table IIGo). Interestingly, the number of cells with typical signs of apoptosis doubled to 14 ± 2. Occasionally necrotic cells (0.8 ± 0.6 per blastocyst) were noticed, which most likely represented arrested blastomeres from earlier cleavage divisions.


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Table II. Comparison of cell number and cell death in the blastocysts developed from normally fertilized (2PN) and abnormally fertilized (1PN and 3PN) embryos. Total cell no. and dead cell no. values are mean ± SE. Cells were classified as dead based on 4,6-diamidino-2-phenylindole staining (DAPI) when chromatin condensation and change in nuclear shape was observed. Cell death index (CDI) was calculated as the proportion of cells showing apoptotic changes per embryo based on DAPI classification
 
Some differences in cell number and cell death index were observed among blastocysts developed from normally (2PN) and abnormally (1PN or 3PN) fertilized embryos. Blastocysts that originated from activated oocytes (1PN) appeared to have a higher incidence of cell death than normally fertilized embryos. However, due to the small sample size, these differences did not reach statistical significance (see Table IIGo). When cell number and CDI were compared between 2PN blastocysts with respect to culture medium, no effect was observed either at day 6 or at day 7 (Table IIIGo.).


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Table III. Comparison of cell number and cell death in normally fertilized embryos that developed to the blastocyst stage in two different culture media. Numbers shown in the third, fourth and fifth columns are mean number of cells or percentage ± SE. Cells were classified as apoptotic when chromatin condensation and change in nuclear shape was observed. CDI was calculated as a proportion of cells showing apoptotic changes per embryo based on the DAPI classification
 
Analysis of ß-HCG and HLA-G expression in human blastocysts
In order to establish the exact initiation of ß-HCG transcription in human embryos, mRNA accumulation was evaluated during human preimplantation embryo development (Figure 1Go). ß-HCG expression was detected at the two-cell stage with steadily increasing expression towards the blastocyst stage. No detectable expression was found in oocytes, suggesting that ß-HCG is solely expressed from the embryonic genome before the major wave of zygotic genome activation.



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Figure 1. Expression of ß-human chorionic gonadotrophin (HCG) in human oocytes and normal embryos. This developmental profile of HCG expression was based on a previously described technique (Rambhatla et al., 1995Go). The upper panel shows the data expressed in units of bound counts per minute (c.p.m.). The lower panel shows the same data converted to estimated copies per embryo. Bars denote mean ± SEM of expression. Oo = MII oocytes, c = cell stage, M = morula, B = blastocyst.

 
Amounts of ß-HCG and HLA-G in the population of studied blastocysts (n = 32) varied significantly. All blastocysts contained detectable amounts of ß-HCG transcript with mean ± SD value of 9383 ± 6588 c.p.m. (range 596–25 968 c.p.m.). Even though all blastocysts expressed ß-HCG transcript, one-third of the embryos (n = 11) had expression levels within the lowest 20th percentile. As expected, a great variability in expression of HLA-G was also observed. The mean ± SD value of HLA-G expression was 11 704 ± 10 085 c.p.m., with three blastocysts completely lacking any signal and an additional five having amounts of expression within the bottom 20% (range 0–40 589 c.p.m.). This is consistent with our previous observation that only a subset of human blastocysts expressed detectable amounts of HLA-G mRNA using semi-nested RT-PCR (Jurisicova et al., 1996aGo). There was no relationship between transcript accumulation for either of these genes and cell number or CDI. However, a very strong positive correlation was observed between accumulation of HLA-G and ß-HCG transcripts (Figure 2Go, r = 0.66, P < 0.0001).



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Figure 2. Correlation between amounts of ß-HCG and human leukocyte antigen (HLA)-G transcript levels as observed in 32 blastocysts developed from 2PN embryos cultured in HTF medium. Amounts are expressed in units of bound probes measured as counts per minute (c.p.m.).

 
Analysis of SP-1 concentrations in culture medium
In order to investigate a possible association between cell number and cumulative secretion of embryonic markers such as SP-1, an ELISA assay was performed using an SP-1 specific antibody. Medium from 92 blastocysts was analysed, out of which 80 originated from normally fertilized oocytes and 12 from 3PN zygotes. The mean cumulative concentration ± SD of SP-1 in culture medium was 2.1 ± 1.7 mg/l per embryo and there was no difference between concentrations secreted by normally and abnormally fertilized embryos. Interestingly, in 14 blastocysts SP-1 was undetectable. When SP-1 concentrations were compared between the two different culture media used in this study, significantly higher mean ± SEM concentrations were found in KSOM (3.1 ± 0.3 mg/l) in comparison with HTF (1.5 ± 0.2 mg/l; P = 0.0001) medium. Moreover, there was a positive correlation between the levels of SP-1 production and the total number of cells per blastocyst (see Figure 3Go, r = 0.27, P = 0.008). No association could be found between SP-1 and transcript levels for either HLA-G or ß-HCG.



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Figure 3. Relationship between pregnancy specific ß-1 glycoprotein (SP-1) concentrations expressed in mg/l and total cell number per blastocyst assessed by nuclear staining with DAPI.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The developmental potential of early cleavage stage human embryos obtained by IVF is variable as later reflected in the quality of obtained blastocysts. In order to increase the chance of a successful pregnancy, the transfer of embryos at the blastocyst stage coupled with selection of the most viable embryos is desirable. However, at the present time, our knowledge of suitable biochemical markers that could be used to predict viability of blastocysts is extremely limited.

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., 1989Go) 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., 1995Go), 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, 1997Go). Results of several experiments published recently identified some triggers of apoptosis in mouse and rat blastocysts, such as hyperglycaemia (Moley et al., 1998Go), tumour necrosis factor (TNF)-{alpha} (Pampfer et al., 1997Go) and glutamine (Devreker and Hardy, 1997Go). 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)-{alpha} (Brison and Schultz, 1997Go) 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., 1988Go). 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, 1997Go). Moreover, based on experiments performed with X inactivation in the mouse (Sheardown et al., 1997Go), 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, 1996Go), appears not to localize to the nucleus and thus fails to repress the TE state (reviewed in Edwards and Beard, 1997Go). Oct-4 transcript also could be detected in human oocytes and early cleavage-stage embryos (Abdel-Rahman et al., 1995Go), 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., 1996aGo). 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., 1991Go; Woodward et al., 1993Go). 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., 1997Go). 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., 1994Go, 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, 1996Go). 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., 1994Go). 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., 1996bGo) 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{alpha}, 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., 1996aGo). 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., 1998Go).

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., 1992Go). 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., 1996Go), 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.


    Acknowledgments
 
The authors would like to thank Drs Ted Brown and Neil MacLusky for their help with statistical analysis and for valuable suggestions in the preparation of this manuscript. We are in debt to the investigators who donated cDNA probes for genes used in these studies: Dr Cindy Schmidt for HLA-G and Dr Goodman for ß-HCG. This study was supported by a grant from Medical Research Council of Canada (MRC-MT-14059) to R.F.C.


    Notes
 
3 To whom correspondence should be addressed at: 6–246 EN, The Toronto General Hospital, 200 Elizabeth Street, Toronto, ON, Canada M5G 2C4 Back


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 Abstract
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 Results
 Discussion
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Submitted on November 25, 1998; accepted on April 8, 1999.