Murine Embryo Development Research Articles

Expression of cell adhesion molecules during human preimplantation embryo development.

Formation of a fully differentiated, implantation competent blastocyst requires the expression of a complex repertoire of molecules. However, the events that drive morphogenesis are poorly elucidated in the human embryo. In this work, we describe the amplification of representative cDNAs from morphologically and developmentally normal, individual human embryos at all stages from pronucleate to blastocyst. These cDNAs were probed to reveal the temporal expression pattern of cell adhesion molecules thought to play a key role in murine preimplantation embryo development. We demonstrated constitutive expression of beta actin, beta 1 and alpha 6 integrins, ZO-1 and E-cadherin, as shown previously in mouse embryos. No expression of beta 3, alpha 2, alpha 3 or alpha 7 integrins nor of L or P selectin was detected at any stage of preimplantation development. beta 5 integrin showed a regulated pattern of expression and was not expressed in blastocysts, while desmocollin-2 could only be detected at the blastocyst stage. Expression and localization of beta 1, beta 5 and alpha 6 integrins and ZO-1 and E-cadherin proteins was confirmed in blastocyst stage embryos by immunocytochemistry. We have identified differences in the expression of integrin molecules between mouse and human embryos, and propose a role for alpha v beta 5 and alpha 6 beta 1 integrin dimers in the human embryo at implantation.

The significance of cytokines, chemical composition, and murine embryo development in hydrosalpinx fluid for predicting the IVF outcome in women with hydrosalpinx.

The aim of this study was to determine whether differences in cytokine concentrations, chemical composition, and murine embryogenesis in hydrosalpinx fluid could be observed between women with hydrosalpinx who achieved and did not achieve pregnancy. Hydrosalpinx fluids were collected from 39 infertile women participating in an IVF-embryo transfer programme at a university teaching hospital. The fluids were analysed for concentrations of cytokine concentration (epidermal growth factor, interferon-gamma, leukaemia inhibitory factor, and tumour necrosis factor-alpha) and chemical composition. Mouse embryos were cultured in medium with various concentrations (0, 10, 50, 100%) of hydrosalpinx fluid. Logistic regression demonstrated that a murine blastocyst formation rate >or=53.3% in 50% hydrosalpinx fluid (OR = 16.6, 95% CI 2.4-116.1, P = 0.005) and patient age (OR = 0.778, 95% CI 0.61-0.99, P = 0.045) were independent predictors of IVF outcome. The diagnostic accuracy did not significantly improve when age and number of good quality embryos transferred was taken into account. The chemical composition and cytokine concentrations of hydrosalpinx fluid were not predictive of subsequent IVF outcome. The mouse embryo assay of hydrosalpinx fluid might potentially serve as a predictor of subsequent IVF outcome in women with hydrosalpinx. This technique may be useful in selecting appropriate treatment options for patients with hydrosalpinx undergoing IVF treatment.

Hydrosalpinx fluid affects murine embryonic development in a coculture system with epithelial endometrial cells

Objective: The aim of the present investigation was to assess whether a coculture system protects from the effect of hydrosalpinx fluid (HF) on murine embryo development, evaluated through blastocyst cell number. Design: Controlled prospective study. Setting: Academic research center. Patient(s): Endometrium and HF from six patients and endometrium from six normal patients. Intervention(s): Murine embryos were exposed to the absence or presence of different concentrations of human HF: 0% HF (control), 50% HF, 70% HF in human tubal fluid, and 100% HF, in a simple culture system (SCS), epithelial coculture system (ECS), and hydrosalpinx epithelial coculture system (HECS). Main Outcome Measure(s): Embryonic development at 72 hours and blastocyst cell number determined by the Tarcowsky method. Result(s): In SCS, 91.9% of the embryos reached the blastocyst stage, and no significant differences were shown in the presence of HF. However, significant differences were observed in the blastocyst cell number. Of the embryos cultured in ECS, 97.1% reached the blastocyst stage, and high concentrations of HF caused a decrease in embryonic development. A significant difference was observed between ECS and HECS in embryo development without HF. When HF was added, a significant decrease in blastocyst cell number was seen in embryos exposed to HECS compared with ECS. Conclusion(s): Our data suggest that normal and hydrosalpinx endometria do not protect from the deleterious effect of HF on embryo development at the concentrations evaluated. This effect is dose dependent and was determined through the blastocyst cell number.

The effects of 1.5T magnetic resonance imaging on early murine in-vitro embryo development.

Although no ionizing radiation is involved, patients undergoing magnetic resonance imaging (MRI) are exposed to powerful static magnetic fields, magnetic field gradients, and radio-frequency fields that may be potentially damaging. Our study aims to document the effect of MRI imaging sequences on early murine embryo development (two-cell to blastocyst stage) in vitro. Two-cell murine embryos were exposed to various lengths of MRI using pulse sequences employed in present day clinical imaging. Early murine embryo development was documented in vitro, and blastocyst development rates were computed for both the control and exposed groups. There were no significant differences detected in the rate of blastocyst formation between the control groups and the embryos exposed to MRI.

Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro.

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N-omega-nitro-L-arginine methyl ester, L-NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10(-7) M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10(-3) and 10(-5) M L-NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four-cell stage was observed by the addition of high concentration of SNP (10(-3) M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10(-5) and 10(-7) M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L-NAME reversed the inhibitory effect by each SNP or L-NAME treatment. Furthermore, low concentration of SNP (10(-7) M) but not high concentration of SNP (10(-3) M) significantly stimulated trophoblast outgrowth, whereas the addition of L-NAME suppressed the spreading of blastocysts in a dose-dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner.

Hydrosalpinx Fluid Effect on the Murine Embryonic Development in a Coculture System With Epithelial Endometrial Cells

Objective: The aim of the present investigation was to assess whether a coculture system protects the effect of the hydrosalpinx fluid (HF) on murine embryo development evaluated though the number of blastocyst cells.

Effects of hydrosalpingeal fluid on murine embryo development and implantation.

Our purpose was to evaluate the effects of various concentrations of hydrosalpingeal fluid (HSF) on the preimplantation development and implantation of murine embryos. One-cell mouse embryos were cultured in KSOM culture medium with 0.1, 1.0, 10, or 50% HSF, without and with lactate supplementation. Late-stage embryos were transferred into the uteri of pseudopregnant CD-1 females to determine implantation rates. The embryo transfer technique used was developed by our group and its effectiveness was evaluated during this experiment. Blastocyst development in the 0.1, 1.0, 10, and 50% group was 45, 55.0, 12.5, and 17.5%, respectively, with lactate supplementation, and 35.0, 52.5, 12.5, and 5.0%, respectively, without lactate supplementation, while in the KSOM (control) group it was 63.8%. Blastocyst development was reduced compared to controls in the 10% HSF and 50% HSF groups. Implantation rates for the 0.1 and 1.0% groups with lactate supplementation were 43.0 and 25.0%, respectively, and those with lactate supplementation were 50.6 and 61.8%, respectively, while in the KSOM group the implantation rate was 65.5%. None of the implantation rates were significantly different. Hydrosalpingeal fluid has a concentration-dependent inhibitory effect on in vitro murine embryo development, but it has minimal effects on implantation rates.

Expression and regulation of genes associated with cell death during murine preimplantation embryo development.

The newly fertilized preimplantation embryo depends entirely on maternal mRNAs and proteins deposited and stored in the oocyte prior to its ovulation. If the oocyte is not sufficiently equipped with maternally stored products, or if zygotic gene expression does not commence at the correct time, the embryo will die. One of the major abnormalities observed during early development is cellular fragmentation. We showed previously that cellular fragmentation in human embryos can be attributed to programmed cell death (PCD). Here, we demonstrate that the PCD that occurs during the 1-cell stage of mouse embryogenesis is likely to be regulated by many cell death genes either maternally inherited or transcribed from the embryonic genome. We have demonstrated for the first time the temporal expression patterns of nine cell death regulatory genes, and our preliminary experiments show that the expression of these genes is altered in embryos undergoing fragmentation. The expression of genes involved in cell death (MA-3, p53, Bad, and Bcl-xS) seems to be elevated, whereas the expression of genes involved in cell survival (Bcl-2) is reduced. We propose that PCD may occur by default in embryos that fail to execute essential developmental events during the first cell cycle.

Expression of Hex mRNA in early murine postimplantation embryo development

The onset of Hex expression and its role in early murine development was analyzed using in situ hybridization. Hex mRNA was first detected in the chorion of the ectoplacental cavity and weakly at the visceral endoderm of the future yolk sac at embryonic age (E) 7.5. Expression in embryonic tissues was detected exclusively in the hepatic anlage and thyroid primordium at E 9.5. At E 12.5 and E 15.5, Hex expression persisted in the fetal liver and thyroid, and was also detected in the fetal lung. These results suggest that Hex has its role in differentiation and/or organogenesis of several embryonic tissues.

Effect of maternal age and conditions of fertilization on programmed cell death during murine preimplantation embryo development.

One of the major morphological anomalies observed in many human pre-embryos is extensive cellular fragmentation. Previously we confirmed that embryo fragmentation seemed to be associated with the activation of programmed cell death (PCD). The purpose of our experiments was to establish a rate for murine embryo fragmentation in vivo after hormonal stimulation in young versus older females and to compare it with the rate of embryo fragmentation during in-vitro fertilization (IVF). While murine maternal age beyond 40 weeks increased the rate of embryo fragmentation following in-vivo fertilization (P = 0.001), oocytes from females of all ages had a uniformly high rate of fragmentation when fertilized in vitro (33%). None of the fragmented murine embryos proceeded further in development. In the mouse, fragmentation occurs exclusively during the first cell cycle. Furthermore, IVF significantly reduced the rate of blastocyst formation (P = 0.0001) and decreased the mean cell number at the blastocyst stage in comparison with embryos produced in vivo (P < 0.0001). The cell death index was significantly affected by both maternal age (P = 0.005) and IVF (P = 0.0001). Identification of specific factors which trigger PCD, especially those associated with IVF, may enable us to lower the rates of fragmentation in preimplantation embryos and thereby increase pregnancy rates after human IVF.

Effect of human hydrosalpinx fluid on murine embryo development and implantation

Objective: To determine the effect of hydrosalpinx fluid-containing medium on murine embryo development and implantation. Design: The development of one-, two-, and four-cell mouse embryos in medium containing 5%, 10%, and 20% of human hydrosalpinx fluid was observed. Implantation rates of mouse embryos transferred into the uterine horn with hydrosalpinx fluid-containing media were determined. Setting: Private hospital-based fertility center and IVF program. Main Outcome Measure(s): Percentage of embryos continuing cell division and implantation rates after ET. Result(s): Hydrosalpinx fluid in culture medium affected embryo development in a dosedependent fashion. The injection of hydrosalpinx fluid-containing medium into the uterine horn did not affect embryo implantation. Conclusion(s): Hydrosalpinx fluid negatively affects murine embryo development, but its presence in the uterine horn at ET did not affect implantation.

Anti-activin A antibody (IgY) specifically neutralizes various activin A activities.

Activin A (beta A beta A), originally isolated from ovarian follicular fluids as a follicule-stimulating hormone (FSH) secretion stimulator, has also been identified as an erythroid differentiation factor (EDF), a neuron survival factor and a mesoderm-inducing factor. Thus, activin A is a multifunctional factor, and further studies on its physiological function are important. However, it is very difficult to produce a specific antibody to neutralize the activity of activin A because of its highly conserved amino acid sequence across mammalian species. In this study, we succeeded in generating an antibody against activin A, which can neutralize several activities of activin A, such as the stimulation of FSH secretion from pituitary cells and the induction of the differentiation of erythrocytes in vitro. This antibody did not affect the activity of activin B (beta B beta B), which induces the differentiation of erythrocytes in vitro, and the activity of inhibin A (alpha beta A), which inhibits FSH secretion from pituitary in vitro, but slightly neutralized that of activin AB (beta A beta B). Western blotting analysis showed that this antibody recognized both dimeric and monomeric forms of the beta A subunit of activin and inhibin. These results suggest that this antibody recognizes the beta A subunit of activin and specifically neutralizes the activity of a dimer of the beta A subunit, activin A. Furthermore, by the addition of this antibody to the culture medium, the development of murine embryos was suppressed, suggesting that endogenous activin A plays an important role in murine development. These results indicate the usefulness of this antibody for studies of endogenous activin actions.

Thymosin alpha-1 and FA-1 monoclonal antibody affect murine preimplantation embryo development by modulating protein phosphorylation

The effects of thymosin α1 (Tα1) and FA-1 monoclonal antibody (anti-FA-1 mAb) on murine preimplantation embryonic development were investigated by performing 2-cell embryo bioassay and by studying ova/embryos protein phosphorylation pattern (by 32P metabolic labeling and by in vitro kinase assay) and protein synthesis (by in vitro [ 35S]methionine labeling). Tα1 treatment (0.1, 0.5 and 5 ng/100 μl) significantly increased blastulation rates ( P < 0.01), blastocyst hatching rate ( P < 0.0001), blastocyst diameter ( P < 0.001) and number of cells per blastocyst ( P < 0.0001) of the in vitro cultured 2-cell stage embryos. Anti-FA-1 mAb reduced blastulation rates ( P < 0.001) primarily due to an arrest of development at morula stage. In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into 4 major protein bands of murine ova ( M r 125, 90, 68 and 31 kDa, respectively), 7 protein bands of 2-cell ( M r 90, 68 and 31; and 145, 52, 38 and 32 kDa, respectively), 10 protein bands of morula ( M r 150, 110, 92, 82, 70, 54, 39, 34, 30 and 29 kDa, respectively), and 15 protein bands of blastocyst (150, 110, 92, 70, 68, 54, 39, 34 and 30; and 131, 105, 52, 44, 43 and 33 kDa, respectively) stage embryos. Tα1 treatment (0.1–0.5 ng/100 μl) resulted in a general increase in 32P labeling in all proteins of 2-cell, morula and blastocyst stage embryos. Anti-FA-1 mAb completely blocked 32P labeling of various proteins of murine ova, 2-cell, morula and blastocyst stage embryos, whereas control mouse myeloma IgG did not affect phosphorylation of these proteins. In vitro kinase assay performed directly on various ova/embryos extracts revealed 6 phosphoproteins ( M r 105, 82, 55, 38, 34 and 33 kDa, respectively) that were common to ova and 2-cell embryos, besides a 43 kDa protein detected only in the ova extract. Of these phosphoproteins, Tα1 treatment specifically enhanced whereas anti-FA-1 mAb inhibited autophosphorylation of a 55 kDa protein of 2-cell embryos. Similarly, Tα1 treatment enhanced 6 phosphoproteins of morula ( M r 102, 60, 58, 49, 35 and 32 kDa, respectively) and 4 phosphoproteins of blastocyst ( M r 102, 60, 49 and 35 kDa, respectively) stage embryo extracts in the kinase assay. In [ 35S]methionine labeling experiments, Tα1 treatment did not affect labeling of any of the 5 proteins ( M r 170, 95, 86, 62 and 42 kDa, respectively) of 2-cell embryos, however, it enhanced labeling of 1 protein band of ova (46 kDa), 7 proteins of morula ( M r 95, 86, 70, 43, 42, 39 and 35 kDa, respectively) and 9 proteins of blastocyst ( M r 95, 86, 70, 43, 42, 39 and 35; and 51 and 33 kDa, respectively) stage embryos. Anti-FA-1 mAb did not affect 35S labeling of murine ova and 2-cell, morula or blastocyst proteins compared to control myeloma IgG. These results suggest that Tα1 enhance murine preimplantation embryonic development by modulating protein phosphorylation pattern as well as protein synthesis. On the other hand, anti-FA-1 mAb inhibit embryonic development by modulating protein phosphorylation pattern without affecting protein synthesis.

Dorso-ventral and rostro-caudal sequential expression of M-twist in the postimplantation murine embryo

M-twist is the murine homolog of the Drosophila twist gene which is a zygotic target for maternal genes that establish embryonic dorso-ventral polarity and is necessary for mesoderm formation. We recently showed that before gastrulation, M-twist transcripts are detected in morulae and blastocysts, then in extra-embryonic tissues of early implanted mouse embryos before the onset of gastrulation, and we suggested that M-twist might be involved in embryonic polarity (Stoetzel et al., submitted). Here, using in situ hybridization on whole mount embryos, we present the expression pattern of M-twist from primitive streak stage up to 10.5 days p.c. In implanted embryos, M-twist is first expressed in extra-embryonic tissues, then in embryo proper around egg cylinder stage within some embryonic ectodermal cells of the primitive streak. Slightly later, scattered cells within the amniotic cavity apparently detached from the primitive streak also express the gene. Then, M-twist transcripts accumulate in head mesenchyme, the first aortic arches, somites and lateral mesoderm and, as development procedes, successively the second, third and fourth branchial arches, the anterior limb buds and, finally, the posterior limb buds. Thus M-twist expression in implanted embryos occurs first along a dorso-ventral gradient pattern until the headfold stage, then it is gradually observed along the rostro-caudal axis of the embryos as development procedes in the mesodermal cell layer and in neural crest cell derivatives. In addition, we show the existence of some previously undescribed subsets of scattered cells that express M-twist and thus might participate in murine embryo development.

Modification of in vitro mouse embryogenesis by X-rays and fluorochromes.

In view of planned studies using single particle irradiation at the Institute for Medical Radiobiology (IMR), confocal microscopy will become an important tool to visualise subtle changes in embryo morphology. Therefore, the influence of X-rays and that of three different fluorochromes on the in vitro development of murine 2-cell stage embryos was investigated. Embryos of B6C3F1 mice were cultured at 32 h post-conception (pc) and treated with X-rays, acridine orange (AO), ethidium bromide (EB) or propidium iodide (PI), respectively, in various doses (0.0-3.0 Gy) or concentrations (AO: 0.05-2.00 micrograms/ml; EB and PI: 0.50-10.00 micrograms/ml). Additional experiments using combinations of AO and irradiation were performed. The embryos were cultivated for a total of 7 days and checked for their vital status by morphological endpoints such as the percentage of embryos that reached the blastocyst stage and the hatching rate. After treatment of the 2-cell embryos with AO, EB and PI, the dose-effect curves with the endpoint 'hatching rate' showed a 23-fold reduction of the ED50 for AO (0.23 microgram/ml) compared with EB (5.30 micrograms/ml). Despite the higher toxicity, AO had much better staining qualities in subtoxic concentrations than EB. PI showed no toxicity in these experiments and was used as an inverse control for embryo vitality. No synergistic modification of the radiogenic effects could be seen in combination experiments (AO+X-rays).

Protein phosphorylation pattern and role of products of c-erbB-1 and c-abl proto-oncogenes in murine preimplantation embryonic development.

To investigate the protein phosphorylation pattern and role of products of c-erbB-1 and c-abl proto-oncogenes with known tyrosine kinase activity in preimplantation embryonic development in mice. The protein phosphorylation pattern was studied by in vitro 32P metabolic labeling of murine ova/embryos as well as by in vitro kinase assay performed directly on various ova/embryos extracts. The role of products of c-erbB-1 (170 kDa, receptor for epidermal growth factor [EGF]) and c-abl proto-oncogenes (150 kDa) was examined by in vitro culturing murine embryos in the presence of monoclonal antibodies to respective protein products and by co-culturing with EGF, the ligand for EGF receptor (EGF-R). In vitro metabolic labeling of murine ova/embryos showed 32P incorporation into at least two protein bands of murine ova (M(r) 81 and 36 kDa), six protein bands of two-cell (M(r) 81, 36; and 97, 52, 22 and 19 kDa, respectively), six protein bands of morula (M(r) 81, 36; 97, 22, and 19; and 33 kDa, respectively), and eight protein bands of blastocyst (81, 36; 97, 22, 19; and 115, 58, and 15 kDa, respectively), stage embryos; there were some specific bands in each stage. Prolonged labeling from 2 to 4 h not only resulted in a relative increase in 32P incorporation into these proteins but also revealed additional bands in morula (M(r) 133 and 115 kD) and blastocyst (M(r) 49, 33, and 31 kD) stage embryos. In vitro kinase assays performed directly on various ova/embryos extracts revealed at least three phosphoproteins (M(r) 58, 36 and 33, respectively) that were common to ova, two-cell, morula, and early/late blastocyst stage embryos. Additionally, three protein bands each in murine ova and two-cell embryos (M(r) 108, 81, 73 kDa, respectively), and four protein bands of late blastocyst (M(r) 108, 73; 133 and 18 kDa, respectively) stage embryos were also revealed. Culture of two-cell embryos in the presence of EGF, the ligand for EGF-receptor, resulted in a concentration dependent increase (P < .001) in the number of cells per blastocyst. Monoclonal antibody to c-erbB-1 170 kDa protein (receptor for EGF) did not affect development of in vitro cultured murine embryos from two-cell to morula, but significantly (P < .001) inhibited the in vitro development of morula to late blastocyst stage. Monoclonal antibody to c-abl protein inhibited the development of murine embryos from two-cell to morula (P < .017), as well as, from morula to late blastocyst stage (P < .002 to .01). These results suggest that the stage-specific protein phosphorylation pattern and specific products of c-erB-1 and c-abl proto-oncogenes may have a role in preimplantation embryonic development in mice.

Induced thermotolerance during early development of murine and bovine embryos

During early development, elevated temperatures have deleterious effects on embryonic viability and development. The primary objective of the current study was to determine the ontogeny of induced thermotolerance during early murine embryonic development. Embryos were either retrieved from superovulated ICR female mice at the 2 cell and 4 cell stages and cultured thereafter or were retrieved from oviducts or uterine horns at the desired stage of development. Induction of thermotolerance was detected by evaluating viability and further development after embryos were exposed to homeothermic temperature (37 degrees C), mild heat shock (40 degrees C for 1 h), severe heat shock (42 degrees C for 1 h or 43 degrees C for 2 h), or mild heat shock followed by severe heat shock (to induce thermotolerance). Induction of thermotolerance was observed beginning at the 8 cell stage when embryos were developed in culture from the 2 cell to 4 cell stage. When embryos were developed in vivo (i.e., were retrieved from the reproductive tract at the desired stage of development), thermotolerance was not induced until the blastocyst stage of development. The induction of thermotolerance was dependent on serum supplementation since induction of thermotolerance was not observed when embryos were placed in medium without serum. Induced thermotolerance could also be demonstrated in bovine blastocysts. In conclusion, embryos acquire the ability to undergo thermotolerance as they progress through development. The timing of processes leading to acquisition of thermotolerance can, however, be hastened by exposure of embryos to in vitro conditions.

Enhancement of in vitro murine embryo development by recombinant leukemia inhibitory factor.

Human recombinant leukemia inhibitory factor (rLIF) has been shown to stimulate hatching of murine and ovine embryos in vitro. The temporal and dose-dependent effects of murine rLIF (mrLIF) and human rLIF (hrLIF) on embryo development in two different mouse strains were investigated in this work. Two-cell embryos were recovered from the fallopian tubes of superovulated/mated females and cultured in Krebs medium plus bovine serum albumin in microdroplets under oil. In the B6CBF1 strain, mrLIF significantly stimulated blastocyst formation and decreased embryo fragmentation/degeneration when added simultaneously at the initiation of culture or 24 hours thereafter. Human rLIF also had a positive effect on development. In the CD1 strain (lower fecundity), mrLIF dose-dependent effects were observed, with enhanced developmental stimulation achieved with higher doses. These findings confirm that hrLIF stimulates mouse embryo development in vitro and that different mouse strains show distinct responses to the cytokine. In addition, mrLIF enhances blastocyst formation and decreases embryo fragmentation when added to the embryo culture as early as the two-cell stage.

Embryo survival after pronuclear microinjection and trophectoderm biopsy

OBJECTIVE: Our purpose was to compare murine embryo development after pronuclear microinjection of a gene construct, followed by trophectoderm biopsy at the blastocyst state, with development after a single micromanipulation, and with cultured controls. STUDY DESIGN: α-Myosin heavy-chain gene sequence was microinjected into the murine embryo pronucleus and cultured to blastocyst. After trophectoderm biopsy the embryos were allowed to reexpand. Reexpanded embryos were transferred to pseudopregnant females; implantation and live birth rates were recorded. In this study group the rates were compared with three control groups of embryos simultaneously cultured after (1) pronuclear microinjection only, (2) trophectoderm biopsy only, and (3) nonmicromanipulated, culture only. RESULTS: A total of 1222 embryos were divided among the four groups. In the study group 472 embryos underwent pronuclear microinjection and trophectoderm biopsy. Of these, 203 (43%) reached the blastocyst stage and underwent biopsy; 183 (38.8%) reexpanded after biopsy. Of 275 pronuclear microinjected only (control 1) embryos, 113 (41.1%) reached the blastocyst stage. Of 336 embryos 148 (44.0%) reached the blastocyst stage and underwent trophectoderm biopsy only (control 2); 129 (39.2%) survived biopsy. The cultured only group (control 3) consisted of 139 pronuclear embryos; 67 (48.2%) developed to the blastocyst stage. CONCLUSIONS: Murine embryos can survive two micromanipulations, pronuclear microinjection followed by trophectoderm microbiopsy. Although there is minimal effect of these procedures on embryonic development in vitro, the live birth rate is tenuous. (AM J OBSTET GYNECOL 1994;170:1199-203.)

Preimplantation embryo development enhanced by epidermal growth factor

Enhanced embryo maturity and advanced stages of cleavage at the time of embryo transfer are associated with superior pregnancy rates in in vitro fertilization procedures. This study was performed to assess the potential usefulness of epidermal growth factor (EGF) to enhance the development of murine preimplantation embryos cultured in vitro. Two-cell stage mouse embryos were cultured for 72 hr with EGF at concentrations of 0.1 to 100 ng/ml. The percentage of embryos which developed to the expanded and hatching blastocyst stage at 72 hr was determined. The percentage of fully expanded and hatching murine blastocysts at 72 hr was significantly higher following incubation with EGF at concentrations of 2 ng/ml (44 +/- 4.1%; P < 0.02), 20 ng/ml (41 +/- 3.2%; P = 0.04), 50 ng/ml (43 +/- 2.5%; P < 0.04), and 100 ng/ml (46 +/- 3.6%; P = 0.001) compared to controls. This effect of enhanced embryonic development by EGF was neutralized by coincubation with 1.0 micrograms/ml of anti-EGF antibody. EGF at concentrations of 2 to 100 ng/ml significantly enhanced the percentage of expanded and hatching murine blastocysts at 72 hr.