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Pre-implantation embryos of Chinese hamster. I. Incidence of karyotype anomalies in 226 control embryos.

Karyotyes were determined in 226 pre-implantation embryos (4--8-cell stages) of Chinese hamster. The study was carried out under controlled natural breeding conditions, without superovulation and with the embryos developing in their mothers. A total of 5.3% karyotypically abnormal embryos were found. Over half, 3.1%, were due to ploidy mutations, 5 cases of triploidy and 2 cases of haploidy. Only 0.9% genome mutations were present, consisting of one autosomal trisomy and one autosomal monosomy. Structural aberrations were found in 1.8%, half of these probably due to a balanced maternal aberration and the rest appearing the mosaic condition only. These results are compared with the scarce body of mammalian data from the literature. Compared with the situation in man, the spontaneous aberration rates in the Chinese hamster and other experimental mammals are extremely low. This may be due, in part, to optimal timing of copulation in respect to estrus and ovulation prevailing in these animals but not in man. The low spontaneous aberration rate in the reported system is a valuable asset for purposes of mutagen testing.

Animals↗

Ovarian differentiation and human embryo quality. 1. Molecular and morphogenetic homologies between oocytes and embryos in Drosophila, C. elegans, Xenopus and mammals.

Knowledge on the formation of oocytes and follicles in Drosophila, C. elegans and Xenopus, and the genetic regulation of polarities and embryo growth, has been related to comparable data in mammalian oocytes and embryos. Initially, details of the nature of the regulatory processes in the non-mammals are described, with considerable attention being paid to the role of individual genes and their specific functions. The molecular genetic aspects of these developmental processes are discussed in detail. Attention then turns to mammals, to identify, describe and evaluate their homologies with the lower animals and flies. Several of these homologies are described, including genes regulating primary ovarian failure and various aspects of early embryonic growth. The polarized distribution of genes in mammalian oocytes and embyros is discussed, together with the implications in the form of differentiation in the early embryo. Morphogenetic systems operative during follicle maturation, fertilization and cleavage are described and related to similar processes in lower forms. These events include ooplasmic and pronuclear rotations, the form of ooplasmic inheritance in early blastomeres and the establishment of embryonic axes. Models of early mammalian development are considered.

Journal Article↗

Wheat embryo ribonucleates. VII. Rapid, efficient and selective formation of 5S-18S and 5.8S-26S hybrids in an aqueous solution of the four ribosomal polynucleotides, and the results of a search for the corresponding hybrids in wheat embryo ribosomes.

(1) If wheat embryo 5S and 5.8S rRNA are differentially labelled, it can be shown that there is highly selective association of 5S [14C]RNA with 18S rRNA, and of 5.8S [3H]RNA with 26S rRNA when a solution (0.3 M NaCl) that contains approximately equimolar amounts of the four ribosomal polynucleotides is heated briefly (3 min) at 60 degrees C. (2) Comparison of Tm values and melting profiles for laboratory-prepared and natural 5.8S-26S rRNA hybrids suggests that restoration of the natural union between 5.8S and 26 rRNA can be achieved with facility and fidelity in the laboratory. (3) Union between 5.8S rRNA remains intact when wheat embryo ribosomes are disintegrated either by digestion with pronase or by treatment with sodium dodecyl sulphate, but the same treatments release 5S and 18S rRNA as freely migrating electrophoretic components. (4) Intact 18S and 26S rRNA can be prepared from small and large subunits, respectively, when wheat embryo ribosomes are dissociated by treatment with 0.5 M KCl. (5) Incidental to the principal investigation, it has been shown that, even after storage for more than 6 years at -70 degrees C, commercial supplies of roller-milled wheat germ yield S23 extracts that are very active in the cell-free translation of globin mRNA. (6) The physicochemical and possible biochemical significance of various types of intermolecular complexing between pairs of ribosomal polynucleotides is a subject of discussion.

Molecular Weight↗

Wheat embryo ribonucleates. VIII. The presence of 7-methylguanosine 'cap structures' in the RNA of imbibing wheat embryos.

1. By imbibing wheat embryos in media that contain methyl-labelled methionine, it is possible to label both terminal and nonterminal 7-methylguanosine constituents in NaCl-insoluble (2.5 M, 0 degrees C) RNA (iRNA). 2. Most of the 7-[Me-14C]methylguanosine in wheat embryo i[Me-14C]RNA is present in nonterminal positions of polynucleotide chains, probably in ribosomal RNA. 3. By passage through a column of oligo-dT-cellulose, it is possible, to show that most of the 7-[Me-3H]methylguanosine in a 'bound' fraction of i[Me-3]RNA from imbibing wheat embryos is present in terminal 'cap' structures, probably in messenger RNA. 4. Although most of the 7-[Me-3H]methylguanosine in the 'unbound' (to oligo-dT-cellulose) fraction of i[Me-3H]RNA was present in nonterminal positions, there was also a highly significant fraction of 7-[Me-3H]methylguanosine in terminal 'cap' structures. Although it will be a subject of continued investigation, possible reasons why a large fraction of the total 7-[Me-3H]-methylguanosine was present in the 'unbound' fraction, in this present study, are a subject of discussion. 5. Careful analysis failed to reveal the presence of any N6,O2'-di[Me-3H]methyladenosine in the 'unbound' fraction of i[Me-3H]RNA. 6. Factors that might influence the binding of 'cap' oligonucleotides to DEAE-cellulose are the subject of a brief discussion.

Base Sequence↗

Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos.

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS -- polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.

Animals↗

Cryopreservation of zygotic embryo axes and somatic embryos of European chestnut.

This work describes experiments demonstrating the feasibility of long-term conservation of Castanea sativa germplasm through cryopreservation of embryonic axes or somatic embryo clumps. Between 93 % and 100 % of excised embryonic axes of recalcitrant chestnut seeds survived storage in liquid nitrogen (LN) following desiccation in a laminar flow cabinet to moisture contents of 20-24 % (on a fresh weight basis), and some 63 % subsequently developed as whole plants. Desiccation to moisture contents less than 19 % produced damage resulting in loss of organized plant development after cryostorage, allowing only root growth. When 6-8 mg clumps of globular or heart-shaped somatic embryos were precultured for 7 days on high-sucrose medium and then desiccated to a moisture content of 25 % before storage in LN, the embryogenesis resumption level after thawing was 33 %. When the embryo clumps were precultured for 3 days on high-sucrose medium followed by 60 min application of PVS2 vitrification solution before cryostorage, the post-storage embryogenesis resumption level was 68 %.

Cryopreservation↗

Transformation of rodent immortalized and embryo cells by oncogenes. II. Rat embryo fibroblasts (REF).

Rat embryo fibroblasts were transformed by co-transfer of two plasmids carrying the oncogenes ras and myc, respectively. In contrast to immortalized cells gene transfer of ras alone was not sufficient for transformation of rat embryo cells. Embryo cells transformed by both oncogenes showed an altered morphology and produced colonies in soft agar. Cell lines were established from transformation foci. These cells are characterized by a high proliferation capacity, they expressed the oncogenes ras and myc and produced regressing tumors in syngeneic newborn rats. Oncogene expression was demonstrated by in situ and blot hybridization. A cell line was established from a rat tumor induced by oncogene transformed cells. This cell line, designated FTD5, showed in addition to an increased expression of the transferred oncogenes ras and myc an elevated expression of the cellular oncogene fos.

Animals↗

The impact of embryo quality on pregnancy outcome in elderly women undergoing in vitro fertilization-embryo transfer (IVF-ET).

Most IVF-ET units limit the procedure to women below age 38. Nevertheless, demands for infertility treatment, including IVF therapy, are more frequent nowadays. We compared 46 cycles for ovulation induction for IVF in 46 women aged 40 or more (Group I) to 51 induced cycles for this procedure in younger women of mean age 30.2 years (Group II). Cancellation rates due to early luteinization or ovulation were significantly higher in group I than in group II (28.2 and 17.6%, respectively) (P less than 0.001). Also, significantly higher abortion rates were observed in older women (62.5%), in comparison to the younger control group, (25%), (P less than 0.001). It is suggested that the high abortion rate considered to be due to genetic factors in older women, may possibly also be due to the aging uterine environment. Furthermore, while embryos with fragmentations may often produce clinical pregnancies in the young, the aging uterus in the elderly woman does not encourage the development of clinical pregnancies in such embryos. Consequently the higher abortion rate in the elderly woman.

Abortion, Spontaneous↗

Treatment with piroxicam before embryo transfer increases the pregnancy rate after in vitro fertilization and embryo transfer.

OBJECTIVE: To examine the effect of beta-cyclodextrin piroxicam treatment for priming of the uterus on the pregnancy outcome of IVF-embryo transfer (ET) programs. DESIGN: Prospective, randomized, double-blinded placebo-controlled clinical study. SETTING: Large urban medical center. PATIENT(S): One hundred eighty-eight consecutive cycles of fresh IVF-ET and 78 cycles of frozen-thawed ET. The patients underwent IVF because of tubal, male infertility, unexplained, or endometriosis factors. They were randomly divided into treatment and control groups. INTERVENTION(S): In the treatment group, 94 cycles in fresh ET and 39 cycles in frozen-thawed ET the patients received an oral dose of 10 mg of piroxicam. In the control group, the same number cycles corresponding to the treatment group were treated with placebo. Both groups started piroxicam or placebo treatment 1-2 hours before ET. Patients and staff were blinded to the treatment. MAIN OUTCOME MEASURE(S): Implantation rate (IR) and pregnancy rate (PR). RESULT(S): Piroxicam increased significantly IR (18.7%) and PR (46.8%) compared to the control group (8.6% and 27.6%, respectively) in fresh cycles. With the exception of an unexplained factor, patients with the tubal, male infertility, or endometriosis factor had significantly higher PR in the treatment group compared to the control group. The beneficial effect of piroxicam was found in patients less than 40 years old, but was not found in patients more than 40 years. In frozen-thawed cycles, there were statistically significant differences between the treatment group and the control group in IR (9.4% vs. 2.3%) and PR (25.6% vs. 7.7%), respectively. CONCLUSION(S): Our study showed that piroxicam increases IR and PR after IVF-ET in both fresh and frozen-thawed ET cycles. The beneficial effect seems to be more remarkable in patients less than 40 years old with tubal, male infertility, or endometriosis factors. These results suggest that piroxicam treatment before ET is very effective in the priming of a uterus suitable for embryo implantation. This is the first study to investigate the possible consequence of piroxicam for improving the PR after IVF-ET.

Adult↗

A randomized study of transcervical intrafallopian transfer of pronucleate embryos controlled by ultrasound versus intrauterine transfer of four- to eight-cell embryos.

OBJECTIVE: To establish the value of transcervical intrafallopian transfer of zygotes and the accuracy of fallopian cannulation. DESIGN: A prospective randomized study, comparing ultrasonically controlled transcervical intrafallopian transfer of zygotes with intrauterine transfer of cleaved embryos. SETTING: Department of IVF of the Rotterdam Academic Hospital. PATIENTS: One hundred forty-five patients with patent tubes entered the IVF program. MAIN OUTCOME MEASURE: Pregnancy rates in both groups and ultrasound (US) assessment during fallopian cannulation and ET. RESULTS: Transvaginal cannulation of the tube appears not to be sufficiently accurate when performed without US guidance. Catheter damage occurred in many cases. The previously reported superior implantation rate after intrafallopian transfer in comparison with intrauterine transfer could not be confirmed. CONCLUSIONS: Intrauterine transfer of cleaved embryos remains the method of choice in IVF.

Adult↗

Hormonal profiles and embryo quality in women with severe endometriosis treated by in vitro fertilization and embryo transfer.

A study was undertaken comparing the outcomes of 30 women with infertility due to untreated severe (grade IV) pelvic endometriosis with a comparable series of 28 women whose infertility was caused solely by irreversible tubal disease. There were no significant differences in either the follicular phase or luteal phase hormonal profiles of estradiol and progesterone, but there was a significantly reduced pregnancy rate in those women with severe endometriosis. In part, this was due to the recovery of fewer oocytes from the endometriosis patients (P less than 0.001) despite the fact that the peak estradiol levels and ovarian accessibility were similar in the two groups. However, there were no significant differences in the proportion of oocytes that fertilized or the number that demonstrated normal embryo growth and high-grade embryo quality. There also appears to be an implantation inhibitory factor in patients with severe endometriosis as the pregnancy rate/embryo transferred and number of gestational sacs identified/embryo transferred were significantly reduced (P less than 0.05).

Embryo Transfer↗

Ultrasound-guided embryo transfer and the accuracy of trial embryo transfer.

BACKGROUND: Studies have suggested that ultrasound-guided embryo transfer (UG-ET) may improve the outcome in IVF; however, several factors may account for the improvement in pregnancy rate. This study examines the use of ultrasound to determine the accuracy of trial transfer (TT) in preparation for ET. METHODS: Sixty-seven consecutive patients prospectively underwent UG-ET over a 2 month period. Total cavity length by US was compared with the length noted by TT. A difference of > or = 1 cm was considered significant. All embryos were placed within 1-2 cm of the fundus by US. RESULTS: Twenty patients (29.9%) had a difference of > or = 1 cm and 13 patients (19.4%) had a difference of < or = 1.5 cm. Patients with a difference of > or = 1 or > or = 1.5 cm had a significantly greater depth at transfer (P < 0.001) and uterine cavity length (P < 0.001) when compared with patients without a difference. Clinical pregnancy, implantation, delivery and overall miscarriage rates did not differ between patients with a difference of > or = 1 or > or = 1.5 cm versus no difference. There were no ectopic pregnancies. CONCLUSIONS: Nineteen percent of patients had a discrepancy of > or = 1.5 cm and approximately 30% had a difference of > or = 1 cm from TT at UG-ET, suggesting a benefit to UG-ET. A large prospective randomized trial comparing UG-ET with blind transfer is required to assess further if UG-ET should be used in all cases of ET.

Abortion, Spontaneous↗

A homologue of snail is expressed transiently in subsets of mesenchyme cells in the sea urchin embryo and is down-regulated in axis-deficient embryos.

Vertebrate members of the zinc finger transcription factor family related to Drosophila snail are expressed in neural crest and paraxial mesoderm along the left-right axis of the embryo. As simple deuterostomes, echinoderms are an important sister phylum for the chordates. We have identified populations of patterned, nonskeletogenic mesenchyme in the sea urchin Lytechinus variegatus by their expression of a sea urchin member of the snail family (Lv-snail). Lv-snail mRNA and protein are detectable at the midgastrula stage within the archenteron. At the late gastrula stage, a contiguous cluster of cells on the left side of the tip of the archenteron is Lv-snail-positive. At the early prism stage, two small clusters of mesenchyme cells near the presumptive arm buds are also Lv-snail-positive. At the pluteus stage, staining is detectable in isolated mesenchyme cells and the ciliated band. Based on fate mapping of secondary mesenchyme cells (SMCs) and double-label immunostaining, these patterns are consistent with expression of SNAIL by novel subsets of SMCs that are largely distinct from skeletogenic mesenchyme. In radialized embryos lacking normal bilateral symmetry, mesenchymal expression of Lv-SNAIL is abolished. These results suggest that transient expression of Lv-snail may be important for the differentiation of a subset of axially patterned nonskeletogenic mesenchyme cells and suggest conserved functions for snail family members in deuterostome development.

Animals↗

Effects of dimethylsulphoxide on mice arsenite-induced dysmorphogenesis in embryo culture and cytotoxicity in embryo cells.

Dimethylsulphoxide (DMSO) is a widely used vehicle for water insoluble compounds in experimental studies. Nevertheless, little is known about its potential impact on dysmorphogenesis caused by reactive oxygen species (ROS). In order to evaluate if DMSO at concentrations used as vehicle can alter in vitro sodium arsenite (Asi) teratogenicity and cytotoxicity, mouse embryos with 4-5 somites were grown for 48 h in Asi 0.4 and 4 microM, with and without 0.1% DMSO (v/v). Also embryonic mesenchymal cell were cultured, using mesenchymal mouse embryo cells obtained from gestation day 11 and treated with DMSO 0.1%, 0.2% and 0.5% (v/v) 15 min before Asi was added at final concentrations of 0.4 and 4 microM. Cytotoxicity and intracellular ROS production, were evaluated with MTT and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), respectively. Results indicated that Asi produced growth retardation and abnormal development. Main malformations involved neural tube closure defects, abnormal rotation and optic vesicle defects among others. Co-treatment with DMSO partially reduced neural tube defects as well as facial dysmophology. Asi reduced cell viability inversely to the level of ROS production, and DMSO returned cellular viability to control values by reducing ROS intracellular production. In summary, the protective effect observed for DMSO appeared to reflect free radical scavenger properties, though other mechanisms independent to ROS production may have also been involved.

Abnormalities, Drug-Induced↗

Monitoring of postimplantation embryo viability following successful in vitro fertilization and embryo transfer by measurement of placental proteins.

Serum levels of pregnancy-associated plasma protein A (PAPP-A), human chorionic gonadotropin (hCG), and pregnancy-specific beta 1-glycoprotein (SP1) were measured in 21 women after successful in vitro fertilization and embryo transfer. Of the 21 pregnancies, 14, including 1 twin gestation, progressed successfully to term. The remaining seven, composed of tubal (n = 3), anembryonic (n = 1), and spontaneously aborted (n = 3) pregnancies, failed during the first half of pregnancy. Placental protein measurement was of no diagnostic value in the detection of anembryonic pregnancy. Similarly, measurement of hCG and SP1 could not readily distinguish tubal ectopic from normal intrauterine pregnancies. By contrast, the predictive value (38.9%) of a depressed PAPP-A level in conjunction with superior diagnostic sensitivity (70%) and relative risk factor (23.6) proved to be of greater diagnostic value in this potentially lethal condition. In the absence of ultrasonography, the biochemical diagnostic indices were comparable in the prediction of spontaneous abortion. However, in the presence of a live fetus, PAPP-A levels were consistently depressed (sensitivity, 91.7%) many weeks before pregnancy demise. The relative risk factor of depressed PAPP-A levels was 29 times greater than the risk associated with a depressed hCG level. These findings further demonstrate the potential diagnostic value of PAPP-A measurement for monitoring postimplantation embryo viability.

Abortion, Spontaneous↗

Dummy embryo transfer: a technique that minimizes the problems of embryo transfer and improves the pregnancy rate in human in vitro fertilization.

Three hundred thirty-five patients selected for in vitro fertilization (IVF) were randomly divided into two groups. Group A (n = 167) was subjected to dummy embryo transfer (ET) before the start of IVF treatment to choose the most suitable catheter for each patient. Group B (n = 168) started their IVF treatment without dummy ET. Embryo transfer technique was difficult in 50 cases (29.8%) in group B, whereas no difficulty was met in group A. Pregnancy rate and implantation rate (22.8%, 7.2%) in group A were significantly higher than in group B (13.1%, 4.3%). The lower pregnancy rate in group B is due to the very low pregnancy rate (4%) in difficult ET cases. Dummy ET is a simple procedure that determines the most suitable ET catheter for each patient and avoids unexpected difficult and failed ET.

Adult↗

Setting standards for the levels of endotoxin in the embryo culture media of human in vitro fertilization and embryo transfer.

OBJECTIVE: To clarify the optimum set standards for the levels of endotoxin in a culture medium for human IVF-ET programs. DESIGN: Retrospective and randomized study. SETTING: One hundred patients underwent 163 cycles of IVF-ET at Fukuoka University Hospital. INTERVENTIONS: Measurements for endotoxin were performed using the Limulus Amoebocyte Lysate test. The negative group was administered media with < 1 pg/mL of endotoxin and the positive group was given media with > or = 1 pg/mL of endotoxin. MAIN OUTCOME MEASURES: Human embryo development, clinical outcomes, and pregnancy outcomes were evaluated in each group and also the levels of endotoxin. RESULTS: The rates of clinical pregnancy (26.1%) and the live birth rates (20.7%) in the negative media were significantly higher than those in the positive media (9.9% and 5.6%, respectively). Rates of embryo development were decreased and a gestational sac and fetal heart beat were not detected in media with > 2 pg/mL of endotoxin. CONCLUSION: The set standard for the level of endotoxin in a medium should be < 1 pg/mL to obtain the best outcome. The allowable levels of endotoxin in a medium should be < or = 2 pg/mL in human IVF.

Adult↗

Handling and culture of bovine embryos: survey of media used by 26 embryo transfer companies in the USA.

Embryo transfer has evolved from a few highly centralized clinics to a widespread number of clinics offering both clinic and on-the-farm services. Practitioners in the field have made very few changes in the handling and culture of embryos. A short review of general handling techniques, plus a survey of media used by 26 companies in the USA, is presented here. Currently, phosphate-bufferend saline (PBS) plus two other commercially available media (EMCARE and ViGro) are used by these companies. Fourteen different combinations of these products were reported and only 11 of the 26 consistently used the same medium for flushing, culture and freezing.

Animals↗