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Embryo |
a Fertilitetscentrum AB, 402 29 Göteborg, Sweden
b Department of Obstetrics and Gynaecology and Reproductive Medicine Unit, Adelaide University, Adelaide 5005, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was detected in embryos from the first-cleavage through blastocyst stages of development, but the GM-CSF-receptor beta common subunit (ßc) could not be detected at any stage. When neutralizing antibodies reactive with GM-R were added to embryo culture experiments, the development-promoting effect of GM-CSF was ablated. In contrast, GM-CSF activity in embryos was not inhibited either by antibodies to ßc or by E21R, a synthetic GM-CSF analogue that acts to antagonize ßc-mediated GM-CSF signaling. Unexpectedly, E21R was found to mimic native GM-CSF in promoting blastulation. When embryos were assessed for apoptosis and cell number by confocal microscopy after TUNEL and propidium iodine staining, it was found that blastocysts cultured in GM-CSF contained 50% fewer apoptotic nuclei and 30% more viable inner cell mass cells. Together, these data indicate that GM-CSF regulates cell viability in human embryos and that this potentially occurs through a novel receptor mechanism that is independent of ßc.
apoptosis, cytokines, embryo, growth factors, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine originally identified as a regulator of the proliferation, differentiation, and activation of myeloid hemopoietic cells [11]. In hemopoietic precursors, GM-CSF acts as a survival factor through suppressing apoptosis [12–14]. It is synthesized by estrogen-primed epithelial cells in the oviduct and uterus in mice [15], sheep [16], cows [17], and humans [18, 19]. Several recent findings implicate a physiological role for GM-CSF in regulating preimplantation embryo development. Exposure of in vitro-produced cow embryos to GM-CSF improves blastocyst development [20], and in ovine embryos, GM-CSF increases implantation potential through upregulating expression of interferon-
[16]. In preimplantation mouse embryos, in vitro culture in GM-CSF enhances blastocyst formation, hatching, and attachment [21] and also stimulates glucose metabolism and promotes the proliferation and/or viability of inner cell mass (ICM) cells. In genetically GM-CSF-deficient mice, the rate of blastocyst development is retarded, and on the day of implantation, blastocysts have a smaller number of blastomeres. This is associated with perturbations in subsequent development, with decreased fetal size and increased fetal resorption evident late in gestation, and with greater mortality during postnatal life [22].
In recent experiments, we have shown that addition of recombinant human (rh) GM-CSF to culture medium dramatically increases the blastulation rate in human embryos. Human blastocysts exposed to rhGM-CSF blastulate earlier, have significantly more blastomeres both in the ICM and trophectoderm (TE), and show improved ability to hatch from the zona pellucida and to attach to the culture dish [10].
In hemopoietic cells, the GM-CSF receptor is comprised of two subunits that belong to the superfamily of cytokine receptors typified by the growth hormone receptor. The GM-CSF-receptor alpha subunit (GM-R or CD116) confers low-affinity binding, whereas the GM-CSF-receptor beta common subunit (ßc) does not bind to GM-CSF itself but, rather, forms a high-affinity complex when associated with ligand-coupled GM-R [23, 24]. In Xenopus oocytes and melanoma cells, GM-R has been reported to act independently of ßc, with ligation stimulating glucose uptake through a kinase-independent pathway [25, 26]. In fact, GM-R mRNA, but not ßc mRNA, expression has been detected in mouse embryos by reverse transcription-polymerase chain reaction (RT-PCR) throughout preimplantation development [21], suggesting that the effects of GM-CSF in mouse embryos are elicited independently of ßc.
Dissecting the roles of individual receptor components is now facilitated by the availability of several subunit-specific neutralizing antibodies and antagonists. The mouse monoclonal antibody 2B7 targets epitopes within the GM-CSF-binding site and completely abrogates both high- and low-affinity binding of GM-CSF to native and recombinant receptors [27], potently inhibiting the actions of GM-CSF in several different bioassays. BION-1 is a mouse monoclonal antibody that is reactive with the membrane proximal domain of ßc [28], which on binding blocks dimerization between ßc and GM-R, thereby inhibiting ßc-dependent signaling. In the synthetic GM-CSF analogue E21R, glutamic acid at amino acid residue 21 is substituted with arginine. Because this residue is essential for binding of GM-CSF to ßc, the mutation does not affect binding to GM-R but, rather, generates a molecule incapable of forming high-affinity complexes with ßc and subsequent induction of ßc-mediated signaling [29]. In hemopoietic cells, E21R is demonstrated to act as a potent antagonist of native GM-CSF.
In the present study, we further investigate the mechanisms mediating the embryotrophic actions of GM-CSF. The expression of GM-CSF-receptor mRNA in human preimplantation embryos is assessed by RT-PCR and immunocytochemistry. Using in vitro culture experiments including E21R or GM-R- and ßc-specific antibodies, we demonstrate that GM-CSF mediates effects in human embryos through GM-R independently of ßc. Furthermore, with TUNEL and confocal microscopy, we identify suppression of apoptosis as a likely mechanism through which GM-CSF increases the abundance of ICM cells in blastocyst-stage embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryos were donated with consent by couples undergoing in vitro fertilization (IVF) at Fertilitetscentrum AB. Ethics approval (700-96) was obtained from the University of Göteborg. Patients underwent ovarian hyperstimulation as previously described [10, 30]. Oocytes were retrieved 36–38 h after hCG administration, assessed morphologically, and fertilized in vitro. Embryos were cultured in IVF-50 (Vitrolife AB, Göteborg, Sweden) and frozen on Day 2 using a three-step propanediol cryopreservation kit (Freeze Kit 1; Vitrolife). For culture experiments, embryos frozen at the 2- to 4-cell stage were thawed at or beyond their storage limit in liquid nitrogen. Blastocysts used for immunocytochemistry and RT-PCR were cultured from excess embryos, surplus to treatment and freezing requirements.
Reagents and GM-CSF Bioassay
Recombinant human GM-CSF was from R&D Systems (Abingdon, U.K.), mouse anti-human GM-R (anti-CD116) was from Serotec (Oxford, U.K.), mouse anti-ßc (BION-1) was kindly provided by Angel Lopez (Hanson Centre for Cancer Research, Adelaide, Australia), and ßc-antagonist E21R was kindly provided by Bresagen Pty. Ltd. (Adelaide, Australia). The neutralizing activities of the GM-R- and ßc-reactive antibodies anti-CD116 and BION-1, respectively, and antagonist activity of E21R were confirmed in a bioassay employing GM-CSF-responsive human myeloid TF-1 cells, essentially as described previously [31]. Briefly, duplicate serial 1:2 dilutions of rhGM-CSF or of GM-CSF plus E21R or neutralizing antibodies were incubated with 1 x 104 TF-1 cells, starved of GM-CSF for the previous 24 h, in 200 µl of RPMI-1640 supplemented with 10% (v/v) fetal calf serum. After 48 h, cultures were pulsed with 1 µCi of [3H]thymidine for 4 h and harvested onto glass-fiber paper for liquid scintillation counting.
Reverse Transcription-Polymerase Chain Reaction
Embryos were cultured in vitro and harvested at the 2- to 4-cell stage on Day 2, the 6- to 8-cell stage on Day 3, the morula stage on Day 4, or the blastocyst stage on Day 5. Culture was performed in 20-µl droplets of IVF-50 overlaid with Ovoil-150 (Vitrolife) until Day 3 (72 h postinsemination) when embryos were transferred into CCM (Vitrolife). Embryos were washed in Ca2+- and Mg2+-free phosphate-buffered saline (PBS), snap-frozen in liquid nitrogen, and stored at -70°C before extraction. Total cellular RNA was extracted from TF-1 cells, from two cohorts each of 20 blastocysts, and from groups of twenty to thirty 2- to 4-cell embryos, 6- to 8-cell embryos, morulae, or blastocysts using a modification of the method described by Braude and Pelham [32] as described previously [21]. Residual chromosomal DNA was removed by treatment with RNase-free DNase (Boehringer Mannheim GmbH, Mannheim, Germany) for 60 min at 37°C. First-strand cDNA synthesis was achieved by RT of RNA primed with random hexamers (Bresatec, Adelaide, Australia) using a Superscript RNase H-reverse transcriptase kit (Gibco, Grand Island, NY). Detection of mRNA by RT-PCR was performed using primer pairs specific for GM-R and ßc as well as ß-actin (detailed in Table 1) and reagents supplied in a Taq DNA polymerase kit (Biotech International Ltd., Perth, Australia). The PCR products were analyzed by electrophoresis through 2% (w/v) agarose gels stained with ethidium bromide in parallel with molecular weight markers (1-kilobase DNA ladder; Bresatec).
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Immunocytochemistry
On Day 5 of culture (120–124 h postinsemination), the zona pellucida was removed from blastocysts in Acid Tyrode solution (ZD-10; Vitrolife) containing 4 mg/ml of polyvinyl-pyrolidone (PVP; Mr 360 000) and washed four times in PBS containing 4 mg/ml of PVP (PBS-PVP). Blastocysts were incubated in anti-CD116, BION-1, or irrelevant mouse immunoglobulin (Ig) G2 (Dakopatts AB, Älvsjö, Sweden; all 10 µg/ml) in PBS containing 1% (w/v) human serum albumin, 10% (v/v) normal goat serum, and 10 mM sodium azide (PBS-NGS) for 4°C for 60 min. Embryos were washed in PBS-NGS and incubated in biotinylated goat anti-mouse antibody (Dakopatts, 1:200 in PBS-NGS, 4°C for 45 min), then washed again in PBS-NGS and incubated in streptavidin-fluorescein isothiocyanate (Dakopatts, 1:50 in PBS-NGS, 4°C for 30 min). After mounting under cover-slips in 20% (v/v) glycerol in PBS, stained embryos were examined by laser confocal microscopy (Leica TCS 4D, Wetzlar, Germany).
Embryo Culture Experiments
Frozen 2- to 4-cell embryos were thawed in four steps using a propanediol method for embryo thawing (Thaw Kit 1; Vitrolife). In the first experiment, embryos were graded according to morphological criteria and randomized by conventional means, with equal numbers of embryos of each grade allocated into each treatment group. In the latter two experiments, embryos were separated into groups using optimal allocation according to the Pancock sequential method of randomization [33] balanced for prognostic factors including number of oocytes retrieved, successful pregnancy in the cycle, nature of treatment (standard IVF or intracytoplasmic sperm injection), and grade of embryo morphology after thawing. The embryos were cultured at a density of five embryos per 20-µl drop of Sydney IVF Cleavage Medium (Cook IVF, Brisbane, Australia) covered by Ovoil-150. At the 6- to 8-cell stage, embryos were transferred into 1 ml of Sydney IVF Blastocyst Medium (Cook IVF). In all three experiments, the culture media were renewed every 48 h. Blastocysts were scored on Day 5 at 120 h postinsemination according to criteria described previously [34]. During the course of three experiments, embryos were cultured in media containing carrier BSA (2 ng/ml), rhGM-CSF (2 ng/ml), or rhGM-CSF in combination with E21R (1 µg/ml), anti-CD116 (250 µg/ml), BION-1 (10 µg/ml), E21R alone (1 µg/ml or 2 ng/ml), or anti-CD116 alone (250 µg/ml).
TUNEL and Cell Allocation Analysis
Blastocysts formed in culture experiments were washed four times in PBS-PVP, fixed in 3.7% (w/v) paraformaldehyde in PBS (pH 7.3) overnight at 4°C, then washed again in PBS-PVP and stored at 4°C until analysis. The TUNEL was performed using an in situ cell-death detection kit (Boehringer Mannheim) with minor modifications to a protocol described previously [35, 36]. Blastocysts were treated in 0.5% (v/v) Triton X-100 in PBS for 60 min at room temperature, washed four times in PBS-PVP, and then incubated in fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase (TdT) under oil for 1 h at 37°C in the dark. A TUNEL-negative control was obtained by omitting TdT from the labeling mix. Embryos were then washed in PBS-PVP and counterstained with propidium iodide (PI; 50 µg/ml in PBS) for 60 min at room temperature in the dark. To avoid background staining from cytoplasmic RNA, RNase A (50 µg/ml) was added to the PI labeling mix. After further washing in PBS-PVP, embryos were mounted under cover-slips in 20% (v/v) glycerol in PBS and examined by laser confocal microscopy. Twenty optical sections were taken through each blastocyst at 1.5- to 3-µm intervals. For cell number analysis, nuclei were scored as associated with ICM cells or TE cells based on location and morphology [36]. Cells with TUNEL-positive (yellow-stained), fragmented nuclei were scored as apoptotic.
Statistical Analysis
Statistical analysis was performed using the Fisher exact test, chi-square test, and Mann-Whitney U-Test (StatSoft, Inc., Tulsa, OK). Differences in data were considered to be significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In hemopoietic cells, GM-CSF acts via binding to receptors comprised of heterodimeric complexes of GM-R and ßc. To investigate whether preimplantation embryos express mRNA for either component of the GM-CSF receptor, mRNA prepared from pools of 20 blastocysts was reverse transcribed and analyzed by PCR with primers specific for GM-R, ßc, and ß-actin cDNAs. The GM-R mRNA was clearly evident in each of four blastocyst preparations (representative data, Fig. 1A). In contrast, mRNA encoding ßc was not detected even when highly sensitive, nested PCR was employed. Identity between the GM-R and ßc fragments amplified from TF-1 cell cDNA and their cognate templates was confirmed by sequencing.
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To examine the expression of GM-R during the course of development in preimplantation embryos, cDNA was prepared from embryos at the 2- to 4-cell, 6- to 8-cell, morula, and blastocyst stages. The GM-R mRNA was expressed at each stage throughout preimplantation development (Fig. 1B).
Expression of GM-CSF-Receptor Protein> in Preimplantation Embryos
To examine the presence of GM-CSF-receptor proteins in embryos, immunocytochemistry was performed in human 8-cell embryos and blastocysts using the GM-R- and ßc-reactive antibodies anti-CD116 and BION-1, respectively. Initially, the reactivity of both reagents and the efficacy of the labeling protocol were confirmed by flow cytometric analysis in TF-1 cells (data not shown). Embryos probed with anti-CD116 or BION-1 were analyzed by laser-scanning confocal microscopy. In three individual experiments, all of eight blastocysts and eight 8-cell embryos clearly demonstrated binding of anti-CD116 to the surface of TE cells, confirming the presence of GM-R protein (Fig. 2). Binding of BION-1 could not be detected in any of six blastocysts examined, even at maximal sensitivity settings, at which the staining was negligible and indistinguishable from that seen in negative-control embryos probed with an irrelevant IgG2a antibody. These data indicate that GM-R protein, but not ßc protein, is present in the blastomere cell membranes of preimplantation embryos.
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Inhibition of GM-CSF Bioactivity> with Receptor-Neutralizing Antibodies and Antagonists
To further examine the significance of GM-R and ßc in mediating the effect of GM-CSF in embryo development, 2- to 4-cell embryos were cultured to the blastocyst stage in the presence of GM-CSF alone or in the presence of receptor-neutralizing antibodies and the ßc-antagonist E21R. Initially, TF-1 cell proliferation assays were used to confirm the biological activities of E21R and antibodies BION-1 and anti-CD116 and to determine effective concentrations for neutralization studies. Each reagent inhibited TF-1 cell proliferation elicited by rhGM-CSF in a dose-dependent manner, with the potency of each reagent being similar to those in previous reports [27, 28, 37] (data not shown).
As expected, the rate and extent of development of 2- to 4-cell embryos to blastocysts was significantly increased by the addition of 2 ng/ml of rhGM-CSF in each of three experiments to an extent (71% in rhGM-CSF vs. 36% in medium-only control, a total of n = 82 and n = 80 embryos, respectively) (Table 2) comparable to that reported previously [10]. The effect was exerted in embryos irrespective of the presence in culture medium of ßc-neutralizing antibody BION-1 (71% blastocyst development, n = 17) or ßc-antagonist E21R (71%, n = 17), at concentrations shown to be inhibitory in TF-1 assays. In contrast, the development-promoting effect of GM-CSF in embryos was neutralized by addition of 250 µg/ml of the GM-R-blocking antibody anti-CD116 (43% blastocyst development, n = 35). The effect of anti-CD116 was the consequence of its receptor-neutralizing action, because addition of a comparable concentration of anti-CD116 in the absence of rhGM-CSF had no detrimental effect on embryo development. Together, these data indicate that GM-R is essential to mediation of the effect of rhGM-CSF in human embryos.
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Interestingly, culture of embryos in the GM-CSF-analogue E21R elicited an effect comparable to that of rhGM-CSF, with 76% development seen in high concentrations (1 µg/ml) of E21R added to culture media in the absence of rhGM-CSF. The potency of E21R was comparable to that of native GM-CSF, with no significant difference between the proportion of blastocysts developing in rhGM-CSF or E21R when both were used at 2 ng/ml. This result is surprising, because E21R has not previously been observed to duplicate the actions of native GM-CSF in hemopoietic cells. As expected, culture of TF-1 cells in E21R did not drive their proliferation (data not shown). However, the antagonistic action of E21R in hemopoietic cells is the result of aberrant binding to ßc and consequent failure to elicit the conformational change in tertiary structure on which ßc signaling is contingent. In contrast, the region of E21R that binds to GM-R is indistinguishable from that in the native cytokine, such that E21R binding to GM-R could reasonably be expected to mimic binding of rhGM-CSF. Thus, our finding of an agonistic, as opposed to antagonistic, effect of E21R in embryos is consistent with the interpretation that, in blastomeres, GM-CSF signaling is contingent on binding to GM-R but is independent of ßc.
Effect of GM-CSF on Apoptosis in Blastocysts
To investigate the effect of GM-CSF on the incidence of cell death, groups of blastocysts cultured in the presence or absence of rhGM-CSF were subjected to TUNEL and examined by laser-scanning confocal microscopy. Within each group, the proportions of embryos at the blastocyst and late-blastocyst stages of development were similar (data not shown). Nuclei labeled by TUNEL showed the fragmented nuclear morphology characteristic of apoptosis, indicating that the observed cell death likely was apoptotic rather than necrotic (Fig. 3). The incidence of cell death was significantly higher in blastocysts formed in media alone compared to blastocysts formed in the presence of GM-CSF, principally because of increased cell death in the ICM. The results confirm previous findings that blastocysts cultured in the presence of GM-CSF have a significantly greater total cell number compared with blastocysts cultured in media alone (mean ± SD; 111 ± 17 vs. 134 ± 20, n = 29 and n = 32, respectively) (Table 3). Increases in the number of TE cells and, particularly, in the number of ICM cells contributed to the greater cell number. The cell death index was significantly higher in embryos cultured in media alone as compared to embryos exposed to GM-CSF (4.9% and 2.1%, n = 29 and n = 32, respectively). No effect of culture in GM-CSF was observed on the mitotic index.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that may occur independently of ßc.
The data show that, similar to mouse embryos [21], human preimplantation embryos express GM-R mRNA, but not ßc mRNA, throughout preimplantation development. Corroborating data were obtained using immunocytochemistry, whereby we detected GM-R protein, but not ßc protein, associated with the cell membrane of both 8-cell embryos and blastocysts. The suggestion of moderately diminished GM-R mRNA expression at third cleavage is consistent with synthesis of GM-R from maternal transcripts at first cleavage and induction of embryonic expression from the morula stage onward.
The present experiments confirm previous findings that addition of rhGM-CSF to the culture media dramatically increases the survival and developmental potential of human embryos in vitro. The beneficial effect was exerted in the presence of ßc-neutralizing antibody but was inhibited by GM-R-neutralizing antibody. The conclusion that GM-CSF signaling in embryos is mediated by an unconventional pathway is supported by culture experiments with the GM-CSF-analogue E21R. A glutamic acid residue at amino acid residue 21 in the GM-CSF molecule is essential for high-affinity binding and signal transduction through ßc, such that substitution of this amino acid with arginine in E21R prevents normal interaction with ßc but does not interfere with binding to GM-R [24]. In human embryos, E21R failed to antagonize the bioactivity of GM-CSF and, when added alone to culture media, stimulated blastocyst development to the same extent as GM-CSF. This agonistic, as opposed to antagonistic, activity in embryos is consistent with normal binding of E21R to GM-R and the interpretation that GM-CSF signaling in embryos does not require ßc.
Ligation of GM-R in the absence of ßc is reported to elicit effects in other nonhemopoietic cell lineages [25, 26]. We have recently demonstrated in mouse embryos that GM-CSF acts independently of ßc to promote blastomere viability. As in Xenopus oocytes and melanoma cells [25, 26], ligation of GM-R was observed to stimulate glucose transport by a phosphorylation-independent signaling mechanism [21]. Whether GM-CSF signaling in any or all of these cell lineages is truly mediated by ligation of GM-R in isolation or, alternatively, whether it requires association between GM-R and other ßc homologues or entirely distinct coreceptors remains to be explored. A recently described variant of ßc with a truncated cytoplasmic tail [39] is unlikely to fulfil this role in embryos, given that our ßc primer sets target sequences encoding membrane-distal regions of the molecule, which are conserved in the variant. Also, heparan sulfate proteoglycans such as syndecan-2, which are present in preimplantation embryos [40] and implicated in mediating GM-CSF signaling in osteoblasts [41], may be involved.
As in previous experiments [10], embryo culture in the presence of GM-CSF caused an increase in the total number of blastomeres, primarily because of a relatively larger ICM. In the present study, we show this likely is largely the consequence of a lower incidence of cell death by apoptosis. Although we did not observe an increase in mitotic figures in ICM cells of blastocysts cultured in GM-CSF, it certainly remains possible that GM-CSF also provides a cell-replication stimulus and that ICM cells are more abundant as the net result of both antiapoptotic and proliferative effects. The means by which GM-CSF protects against apoptosis in embryos is not clear and, given the distinct receptor configuration in embryos, likely is dissimilar to mechanisms in other cell lineages. Withdrawal of GM-CSF causes apoptosis in several types of hemopoietic cells [12, 42–44], whereas addition of exogenous GM-CSF can rescue myeloid leukemic cells from apoptosis induced by antiproliferative compounds [43]. However, these effects appear to be contingent on the expression of ßc, because experiments showing that E21R causes apoptosis through inducing ßc dephosphorylation [45] suggest that maintenance of a phosphorylated state in ßc is the prime protective consequence of GM-CSF binding in hemopoietic cells.
Several experiments in mice support the conclusion that cytokines and growth factors other than GM-CSF can alleviate the effect of suboptimal culture conditions on induction of apoptosis in blastocysts [35, 36]. In the case of insulin-like growth factor (IGF)-1, addition to culture can increase the rate of blastulation, increase the number of blastomeres [8], and provide protection from apoptosis [46] in both mice and human embryos. The 41% improvement in the rate of apoptosis elicited by IGF-1 in human embryos [46] is comparable to that seen in the present study with GM-CSF, despite the difference in background incidence of cell death, which is presumably a function of differences in embryo culture conditions. In other nonhemopoietic cells, GM-CSF stimulates cell viability and proliferation through promoting glucose uptake and metabolism, and our experiments in mouse embryos implicate this pathway in blastomeres [21]. The lower incidence of apoptosis in human embryos cultured in GM-CSF is consistent with their having a higher state of metabolic activity, and metabolic measurements in single human blastocysts might eventually lead to proof of a similar explanation in humans. Whether the mechanisms underlying the ability of different growth factors to regulate blastomere survival are related is not known, but it could reasonably be proposed that each has an influence on the metabolic activity of ICM and/or TE cells and that the diminished survival rate of embryos cultured in growth factor-free medium is caused by apoptosis resulting from metabolic starvation.
Importantly, the rate of apoptosis in blastocysts appears to be physiologically significant. In mouse experiments, high rates of apoptosis correlate with diminished reproductive outcome, specifically giving rise to smaller fetuses later in gestation [36, 47]. Thus, the incidence of apoptotic cell death likely is a key indicator of the viability and developmental competence of human preimplantation embryos. Indeed, it is has been speculated on the basis of animal studies [48, 49] that smaller birth weights in human IVF progeny are at least partially the consequence of compromised preimplantation embryo development [50, 51]. The discovery of growth factors that improve cell number and inhibit apoptosis in human embryos may thus have clinical significance, but the prospects for using growth factors such as GM-CSF in in vitro culture media need to be tempered by caution in relation to the consequences for fetal viability and health.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Cecilia Sjöblom, Cell Therapeutics Scandinavia AB, Medicinaregatan 8A, 413 46 Göteborg, Sweden. FAX: 46 31 741 17 50; cecilia.sjoblom{at}celltherapeutics.se
Received: 15 November 2001.
First decision: 6 December 2001.
Accepted: 24 May 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Biol Reprod 1997 56:1088-1096[Abstract]-deficient mouse blastocysts. Biol Reprod 1998 59:136-144 decreases the viability of mouse blastocysts in vitro and in vivo. Biol Reprod 1999 60:479-483This article has been cited by other articles:
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1 and B
