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PubMed1996

Distribution of blood flow between embryo and vitelline bed in the stage 18, 21 and 24 chick embryo.

OBJECTIVE: We defined the distribution of blood flow between the embryo and the extraembryonic vascular bed as an initial step in understanding the control of flow distribution in the early developing heart. METHODS: Dorsal aortic blood flow of stage 18, 21, and 24 chick embryo (n > or = 7 at each stage) was measured with a 20 MHz pulsed-Doppler velocity meter. Analog waveforms were digitally sampled at 500 Hz. 1-5 x 10(3) yellow microspheres in saline suspension were injected into the vitelline vein. The embryo and the extraembryonic vascular bed were harvested and separated from each other. The dye on the microspheres from each portion was extracted and extrapolated from the standard curve of the absorbance of dye concentrations per number of microspheres quantified by spectrophotometry. Blood flow was calculated from the integral of blood velocity and aortic cross-sectional area multiplied by the fraction distribution of microspheres in the embryo and extraembryonic vascular bed. Data were presented as mean +/- standard error of the mean. RESULTS: The proportion distribution of microspheres between embryo and extraembryonic vascular bed shifted from 18.7 +/- 2.5 vs. 81.3 +/- 2.5% at stage 18, 25.1 +/- 3.0 vs. 74.9 +/- 3.0% at stage 21, and 34.2 +/- 2.4 vs. 65.8 +/- 2.4% at stage 24. Indices of blood flow normalized to wet weight (mean +/- 95% confidence interval) were similar between the embryo and the extraembryonic vascular bed, but increased throughout the stages. CONCLUSION: During embryogenesis, blood flow per unit mass is evenly distributed between the metabolically active embryo and the extraembryonic vascular bed.

Animals↗
PubMed2002

[Embryos and embryo-like entities: problem of definition in the draft of the Swiss embryonic research law].

At the end of May 2002, the draft of the Swiss "Federal Act on Research on Surplus Embryos and Embryonic Stem Cells" (EFG, Embryonic Research Act) reached the pre-legislative consultation stage. Under certain conditions, it would allow research on "surplus" embryos from in-vitro fertilization, and the derivation of embryonic stem cells from surplus embryos for research purposes. The EFG draft defines an embryo as "the developing organism from the point of nuclear fusion until the completion of organ development". New technological developments show that embryo-like entities can also be created without nuclear fusion having taken place. It remains unclear how to treat embryonic entities that don't fall under the draft's narrow definition of an embryo. Expanding this definition would be a welcome improvement.

Animals↗
PubMed1993

Computer analysis of the human embryo growth curve: differences between published ultrasound findings on living embryos in utero and data on fixed specimens.

Accurate information on the normal growth rate of the human embryo is fundamental to a better understanding of the embryonic period of pregnancy. Crown-rump length measured previously in utero (N = 227) with vaginal ultrasound in 107 in vitro fertilization (IVF) or gamete intrafallopian transfer (GIFT) singleton pregnancies was compared to the greatest length of fixed human embryos from the Carnegie collection, of known developmental stage whose postovulatory ages were estimated from menstrual histories. Average crown-rump length in utero was 60% of the greatest length of the fixed specimens prior to postovulation day 33, but were equal after postovulation day 40. The growth rate of in utero embryos and fixed specimens, analyzed by computer using exponential equations, was compared to linear and polynomial equations used in previously published embryo growth tables. The exponential equation, length = exp(a + B/age), fit in utero measurements best, while the equation length = exp[a + b/exp(age)] fit the fixed specimens best. Differences between length in utero and in fixed specimens may be related to distortion of the fixed embryos resulting from the formalin fixation, to ultrasound distortion, to curling of the embryo, or to incorrectly estimated ages of the fixed specimens. Study of human embryos in utero is now practical with vaginal ultrasound.

Embryonic and Fetal Development↗
PubMed1990

Potential of hypertonic medium treatment for embryo micromanipulation: I. Survival of rabbit embryos in vitro and in vivo following sucrose treatment.

Rabbit zygotes and embryos were exposed to hypertonic sucrose in phosphate-buffered saline (SPBS). In experiment one, 144 zygotes shrank to 32-36% of their initial volume in 1.0 M SPBS within 30 min. Neither hypertonic treatment with 0.5 M or 1.0 M SPBS nor micropuncture of the zona pellucida after shrinkage affected embryo development into blastocysts in vitro (88%, 83%, and 82%, respectively), compared to that of the controls (93%, P greater than .05). In experiment two, 252 two- to four-cell- and 177 morula-stage embryos were exposed to isotonic PBS control or 0.5 M, 1.0 M, or 1.5 M SPBS for 30, 60, 90, 120, and 150 min before transfer to PBS (290 mOsm). Embryo development was significantly reduced (P less than .05) when embryos were exposed in 0.5 M and 1.0 M SPBS for more than 60 min or in 1.5 M SPBS for more than 30 min. In experiment 3, morulae exposed for 60 min to 0.5 M or 1.0 M SPBS shrank to 37-39% or 32-35% of their initial volume and then expanded to 87-94% or 81-90% of their initial volume, respectively, after being returned to isotonic PBS for 60 min, but embryos in 1.5 M SPBS had erratic osmotic behavior. In experiment four, 192 two- to four-cell embryos exposed to 0.5 M SPBS for 0, 30, and 60 min before transfer to oviducts of recipients resulted in the production of 39%, 42% and 31% young, respectively (P greater than .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Embryo transfer as a means of controlling the transmission of viral infections. IX. The in vitro exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus.

When zona pellucida-intact porcine embryos were exposed to 10(7) plaque-forming units (pfu)/ml of swine vesicular disease virus (SVDV) and then washed, infectious virus could be isolated from all of the embryos. Culturing the embryos for 24 or 48 h or treating the embryos with pronase, trypsin, or antiserum after virus exposure and washing reduced the number of embryos carrying virus and lessened the amount of virus on each of the embryos. None of the treatments, however, was capable of disinfecting every embryo.

Journal Article↗
PubMed1994

In vitro culture of bovine IVM-IVF embryos: Cooperative interaction among embryos and the role of growth factors.

The objective of this study was to determine whether there is a cooperative interaction among bovine embryos during in vitro culture. Furthermore, culture medium was supplemented with the growth factors, epidermal growth factor (EGF) and transforming growth factor-beta1 (TGF-beta1), to determine if these factors had a stimulatory effect on bovine embryo development similar to that seen in mouse development. In vitro matured - in vitro fertilized bovine embryos (2- to 8-cell) were cultured singly and in groups of five in 25 mul of medium (CR1 + amino acids + fatty acid-free bovine serum albumin) with or without EGF and TGF-beta1. Bovine embryos cultured in groups had a significantly higher rate of development to the blastocyst stage than embryos cultured singly. Neither EGF (10 ng/ml) nor TGF-beta1 (2 ng/ml) affected blastocyst development, hatching or the cell number of the embryos cultured in groups. Epidermal growth factor stimulated hatching of embryos cultured singly from the 8-cell stage, but did not significantly affect blastocyst development.

Journal Article↗
PubMed2003

Dynamic dissolved oxygen concentration control for enhancing the formation rate of torpedo-stage embryos in carrot somatic embryo culture.

In utilizing somatic embryogenesis for transplant production, torpedo-stage embryos are harvested. In order to enhance the formation rate of torpedo-stage embryos to total embryos of all developmental stages in the culture at the time of harvest, a dynamic dissolved oxygen concentration (DO) control algorithm is proposed. The algorithm is based on the difference in developmental response of somatic embryogenesis to DO level depending on developmental stages, and the culture period was divided into three phases of different DO levels. The timing of the phase change was determined based on the formation rate of the embryos in each developmental stage in the suspension assessed by noninvasive monitoring of the culture. The induction of carrot (Daucus carroa L.) somatic embryogenesis resulted in doubled formation rate of torpedo-stage embryos by dynamic DO control compared to the 20% oxygen gas aeration, and 1.4 times higher formation rate compared to 6% oxygen gas aeration, while the total number of embryos did not differ among DO treatments. Plant conversion rate of torpedo-stage embryos cultured by the dynamic DO control was 70%, and was approximately the same as that in the control cultures. The relations between variations of medium pH and somatic embryogenesis were also analyzed.

Journal Article↗
PubMed1987

Analysis of Qa-2 antigen expression by preimplantation mouse embryos: possible relationship to the preimplantation-embryo-development (Ped) gene product.

The preimplantation-embryo-development (Ped) gene, a gene that controls the cleavage rate of preimplantation mouse embryos, maps to the Qa-2 subregion of the mouse major histocompatibility complex (MHC). A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure was used to detect Qa-2 antigens on mouse embryos. The use of a monoclonal antibody specific for Qa-2 antigens showed that Qa-2 antigens were present on oocytes, 2-cell, 8-cell, and blastocyst-stage embryos, with the greatest expression found on blastocysts. Expression of Qa-2 antigens by the embryos correlated completely with Ped gene phenotype. Those embryos expressing the fast Ped allele showed the presence of Qa-2 antigens (Qa-2a mice), whereas those embryos expressing the slow Ped allele showed the absence of Qa-2 antigens (Qa-2b mice). It is hypothesized that the Qa-2 antigen may be the Ped gene product.

Alleles↗
PubMed1995

Expression of genes encoding antioxidant enzymes in preimplantation mouse and cow embryos and primary bovine oviduct cultures employed for embryo coculture.

Preimplantation embryos from a variety of mammalian species contrast markedly in their response to culture in vitro. Murine preimplantation embryos display a wider tolerance than other mammalian species to culture environments, and this has contributed to the development of several effective defined culture media. Embryo coculture on somatic cells remains the most effective method of supporting reasonable rates of bovine preimplantation development in vitro. The patterns of gene expression for several antioxidant enzymes during preimplantation murine and bovine development were examined by use of the reverse transcription-polymerase chain reaction technique to determine whether the differential developmental capacity of mammalian preimplantation embryos in culture may reflect variations in the patterns of expression for a series of antioxidant enzymes. Transcripts for catalase, CuZn-containing superoxide dismutase (CuZn-SOD), Mn-SOD, glutathione peroxidase (GPX), and glutamylcysteine synthetase (GCS) were detected in mouse embryos at all stages of development regardless of in vivo or in vitro development. Preimplantation cow embryos produced by in vitro procedures expressed mRNAs for catalase, CuZn-SOD and GPX, whereas transcripts for Mn-SOD were not detected at any stage. GCS transcripts, although present in stages up to the morula, were not detected in cow blastocysts. Analysis of antioxidant gene expression in both bovine primary oviductal cell monolayer cultures and nonattached, ciliated oviductal cell vesicle cultures revealed a constitutive pattern of expression of all five enzymes for the 8-day culture interval. These experiments suggest that differences in gene expression may contribute to the variation in the ability of embryos to develop in vitro with respect to levels of oxygen and dependence on coculture.

Animals↗
PubMed1992

Germin isoforms are discrete temporal markers of wheat development. Pseudogermin is a uniquely thermostable water-soluble oligomeric protein in ungerminated embryos and like germin in germinated embryos, it is incorporated into cell walls.

Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water-soluble, pepsin-resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has been named pseudogermin. Pseudogermin accumulates, maximally, between 20-25-days postanthesis, then declines appreciably in amount by 30-days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze-cleaved embryos by scanning electron microscopy. Wall-associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination-related change in the distribution of cell-wall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potentials during embryogenesis. The N-terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDS/PAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water-soluble, pepsin-resistant protein, but pseudogermin has unprecedented disulphide-independent thermostability properties that have never been previously reported for a water-soluble oligomeric protein. Polysaccharides that co-purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino-acid sequence that suggest a possible relation between germin and a proton-specific ion pump: gastric ATPase.

Amino Acid Sequence↗
PubMed1986

[Toxicological evaluation of an in vitro culture of mouse embryos; effect of non-carcinogenic mutagens for the development of embryos].

Short term drug toxicities were investigated using cultures of mouse embryos in the early stage of development. These embryos were collected at the two- or eight-cell stage. They were exposed to bleomycin (B1) or 6-mercaptopurine (MP) for 24 hr, thereafter, they were grown in BMOC-3 medium without these agents until the blastocyst stage. Total culturing period was 72 hr for the two-cell embryos and 48 hr for the eight-cell embryos. At the end of the culture periods, the number of cells, mitotic index and frequencies of sister chromatid exchange in these embryos after these exposures were unaltered. However, the death rate of embryos was elevated by the exposure to either B1 or MP. These agents are regarded as non-carcinogenic mutagens; therefore, it is suggested that these compounds are lethal to the embryos through an induction of mutation.

Animals↗
PubMed2003

Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin.

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.

Animals↗
PubMedDate not supplied

[Cytogenetic analysis of sister chromosome sets in the second polar body and in pronuclei of unicellular mouse embryos. I. Frequency and origin of aneuploidy in embryos heterozygous for the reciprocal chromosome translocation T[14;15]6Ca].

We carried out a cytogenetic study of ovulating oocytes and unicellular embryos, heterozygous by reciprocal chromosomal translocation T[14;15]6Ca. Okadaic acid was used to induce premature condensation of the interphase chromosomes in the embryos, and the number of G1 chromosomes was counted in the second polar body and pronuclei. It was shown that cytogenetic analysis of the sister chromosomal sets adequately determines the frequency of chromosomal segregation errors during oocyte meioses I and II. Trisomy and monosomy were observed in 36.2% embryos, while 2.2% featured tetrasomy or double monosomy. Errors of the first meiotic division caused aneuploidy in 28.5% embryos; trisomy and monosomy resulted from the homologs non-disjunction and chromatid presegregation in 17.6 and 10.9%, respectively. Numeral chromosomal aberrations in 4.1% of the embryos resulted from abnormal chromosomal segregation during oocyte meiosis II, while paternal chromosomal aberrations were found in 5.8% embryos. The main advantage of the proposed method is not only the higher accuracy in estimating the meiotic error frequency, but also the possibility to trace the origin of aneuploidy in mammalian embryos.

Aneuploidy↗
PubMed2006

Preferable correlation to blastocyst development and pregnancy rates with a new embryo grading system specific for day 3 embryos.

PURPOSE: To investigate the cleavage stage embryo quality by the correlation between the morphological features and blastocyst development rate to develop a new embryo grading system. METHODS: A retrospective analysis, including 216 cycles of cleavage stage embryo transfer and 251 cycles of blastocyst transfer. The correlation with blastocyst development of the embryo cleavage stage, fragmentation and uniformity of blastomeres was evaluated. RESULTS: There were significant differences in the blastocyst development rate between > or =7 cells and < or=6 cells (68.8% vs. 30.7%), <50% fragmentation and > or =50% fragmentation (51.9% vs. 25.7%), and evenly sized blastomeres and unevenly sized blastomeres (48.7% vs. 30.1%) on day 3. The new grading system defined by these 3 parameters showed a preferable correlation to the pregnancy rate. CONCLUSIONS: The new grading system specific for day 3 embryos is useful for the selection of good quality embryos and may improve the pregnancy rate.

Adult↗
PubMed2005

Extended embryo culture reduces the implantation rate on day 4 and day 5 when only a maximum of three embryos are cultured beyond the pronuclear stage.

OBJECTIVES: To study the potential of embryo transfer after 3, 4 or 5 days of embryo culture under the German embryo protection law according to which only a maximum of three zygotes are allowed to be cultured for embryo transfer. STUDY DESIGN: In a prospective study, 273 patients with assisted reproductive treatment were randomly allocated for transfer on days 3, 4 or 5. Pregnancy and implantation rates were evaluated in regard to day of transfer and results were compared by Chi-square or ANOVA test. RESULTS: Out of 234 transfer cycles, 79 were performed on day 3, 76 on day 4 and 79 on day 5. Pregnancy and implantation rates were 41.8%/27.1% for transfer on day 3, 27.6%/14.1% for day 4 transfer and 16.5%/8.8% for transfer on day 5. These results were significantly different for pregnancy rates on day 3 versus day 5 (P < 0.001) and for implantation rates on day 3 versus day 4 (P < 0.005) and day 3 versus day 5 (P < 0.001). CONCLUSIONS: These findings suggest that extended embryo culture is not beneficial when the option for embryo selection at later stages of development is not available.

Adult↗
PubMed1986

The successful use of human amniotic fluid for mouse embryo culture and human in vitro fertilization, embryo culture, and transfer.

The development of mouse and human embryos was assessed in human amniotic fluid to determine its suitability as a culture medium for human in vitro fertilization (IVF). Two-cell mouse embryos developed to blastocysts after 72 hours at rates similar to that in Whittingham's T6 + 10% fetal calf serum. Significantly more mouse embryos hatched in amniotic fluid. No difference was found between individual patient's amniotic fluids obtained at 16 to 21 weeks' gestation. A preliminary trial comparing amniotic fluid with T6 + maternal serum in human IVF showed no significant difference in fertilization rate and embryo development during 42 to 48 hours in vitro. Expanded blastocysts were obtained in amniotic fluid after 5 days in vitro. Four pregnancies were obtained in 9 patients' transferred embryos grown in amniotic fluid and with 2 or 12 patients' transferred embryos grown in T6 + maternal serum.

Amniotic Fluid↗
PubMed2001

Ovarian response, embryo recovery and results of embryo transfer in a Hungarian native pig breed.

The objective of the study was to use embryo transfer (ET) for propagation of the Swallow Belly Mangalica population. Mangalica is a native Hungarian pig breed adapted to extreme climate and housing conditions and distinguished for excellent meat and fat quality. However, due to their weak reproductive characteristics and relatively high fat proportion, Mangalica pigs have been replaced by modern breeds. Now, there is an increased interest again to safeguard the properties of this breed. We conducted two experiments. First, we used a total of 18 puberal Mangalica gilts to determine an optimal superovulatory treatment. Following estrus synchronization with Regumate, we injected gilts with either 750, 1000 or 1250 IU PMSG, followed by 750 IU hCG 80 h later. We scanned ovaries endoscopically 3 days after hCG administration. The application of 1000 and 1250 IU PMSG resulted in a higher rate of ovulation compared to 750 IU (24.2 +/- 3.6 and 21.0 +/- 2.3 vs. 13.7 +/- 2.7 P<0.05). The number of follicular cysts increased after administration of 1250 IU PMSG compared to 750 and 1000 IU (2.0 +/- 1.3 vs. 0.3 +/- 0.7 and 0.2 +/- 0.3, P<0.05). Thus, we chose 1000 IU PMSG for further stimulation of Mangalica gilts. In the second experiment, we induced superovulation in 10 Mangalica donor gilts by 1000 IU PMSG and 750 IU hCG. Gilts were fixed-time inseminated, and then five days later embryo collection was carried out surgically (n=6) or endoscopically (n=4). Out of the 187 ova recovered, 92.5% were at the morula/blastocyst stage. The embryo recovery rate was higher following surgical flushing than following endoscopy (91.5 +/- 4.4% vs. 71.4 +/- 12.7%, P<0.05). Altogether 143 embryos were transferred surgically or endoscopically into 8 Landrace recipients. Surgical and endoscopic transfer of Mangalica embryos into Landrace gilts resulted in pregnancies in 3 and 2 gilts, respectively; thus the overall farrowing rate was 62.5%. The birth of 59 Mangalica piglets from 5 embryo recipients equals an average litter size of 11.8 +/- 1.3, which is two times larger than usual in this breed. Therefore, we concluded that an appropriate inter-breed ET program is a suitable tool to propagate the endangered Mangalica breed.

Animals↗
PubMed1997

The effect of a propofol-based sedation technique on cumulative embryo scores, clinical pregnancy rates, and implantation rates in patients undergoing embryo transfers with donor oocytes.

STUDY OBJECTIVE: To determine the effect, if any, of a propofol-based sedation technique on the reproductive outcomes of patients undergoing embryo transfers with donor oocytes. These ova recipients form a unique subgroup, whose clinical outcomes are unrelated to direct anesthetic effects on their reproductive tracts. DESIGN: Retrospective chart review. SETTING: A 1200-bed university medical center. PATIENTS: 117 patients who received fresh embryo transfer cycles between January 1991 and December 1995. MEASUREMENTS AND MAIN RESULTS: The anesthesia records of 106 women who donated ova were reviewed for propofol usage during the transvaginal needle aspiration of the ova. The medical records of the 117 patients who received these donated embryos were reviewed for cumulative embryo scores, clinical pregnancy rates, and implantation rates. Fourteen patients received ova from women who were sedated with fentanyl and midazolam during ovum retrievals, while 103 patients received ova from women who had been given fentanyl, midazolam, and propofol in doses of 1.87 mg/kg to 8 mg/kg. The pregnancy rate among all patients who received ova from women who received propofol (44 of 103 = 42.7%) was 14.1% greater than those whose ovum donors did not receive propofol (4 of 14 = 28.6). 78.6% of both propofol and non propofol-exposed groups had cumulative embryo scores of greater than 50. Among patients who became pregnant, 52.3% of propofol-exposed and 50% of nonpropofol-exposed cases had greater than 20% implantation rates. CONCLUSION: There is no evidence from our data that the administration of propofol during the aspiration of ovarian follicles for oocyte donation had a negative impact on the oocytes as measured by cumulative embryo scores, probability of a clinical pregnancy, or implantation rate.

Anesthetics, Intravenous↗