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PubMed2004

Subzonal older adult fibroblast insertion in both in vivo-fertilized and nuclear transfer rabbit zygotes and embryos: effects on further in vitro embryo development.

In the present work, we evaluated the effect on further in vitro embryo development of inserting rabbit adult fibroblasts into in vivo-fertilized rabbit embryos. To this end, we inserted either 4 or 15-20 rabbit adult fibroblasts in two different early embryo stages of development, 1-cell stage and 4-8-cell stage embryos. We observed that fibroblast insertion not only did not negatively affect further embryo development, but also may have exerted a positive effect on development on it. Therefore, in forthcoming works were where we intend to study a possible cell helper role on early embryo development. The early embryo microenvironment may reprogram somatic cell gene expression of fibroblasts inserted within the embryo, making them suitable as nuclear donors.

Animals↗
PubMed2004

Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5.

BACKGROUND: Chromosomal mosaicism in human embryos may give rise to false positive or false negative results in preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Therefore, we have investigated whether the results obtained from a 2-cell biopsy of frozen-thawed embryos and fluorescence in situ hybridization (FISH) analysis are representative for the chromosome constitution of the remaining embryo on day 5. METHODS: Cryopreserved day 3 embryos were thawed and from surviving embryos two blastomeres were biopsied. FISH analysis was performed for chromosomes 1, 7, 13, 15, 16, 18, 21, 22, X and Y. After biopsy, the embryos were cultured until day 5 and further analysed using the same probe panels. RESULTS: In all, 17 embryos were available with a diagnosis based on two blastomeres on day 3 and confirmatory studies on day 5. In 10 of these 17 cases the initial diagnosis could be confirmed. However, in only six cases cytogenetic results were concordant. Besides the 10 cases with a 'correct' diagnosis, there were six false positive results and one false negative, all involving mosaicism. CONCLUSIONS: Investigating the chromosomal constitution of two blastomere nuclei offers a good opportunity to study the incidence of chromosomal mosaicism in early embryo development. The confirmation rate of the results obtained on day 3 depends on the interpretation and is higher when considered from a clinical than from a cytogenetic point of view.

Aneuploidy↗
PubMed2006

The CryoLoop facilitates re-vitrification of embryos at four successive stages of development without impairing embryo growth.

BACKGROUND: Vitrification has been shown to be an effective method of cryopreservation, but little is known about re-vitrification of embryos. This study investigated the effect of re-vitrification on mouse embryo preimplantation development and viability post-transfer. METHODS: Mouse embryos at the 1-cell stage were vitrified using the CryoLoop technique. Embryos were warmed and then re-vitrified successively at the 2-, 8-cell and blastocyst stages. The effects of multiple rounds of vitrification on development, differentiation and viability were assessed and compared with non-vitrified embryos. RESULTS: Development to the 8-cell stage on day 3 and blastocyst on day 5 were not affected by re-vitrification. However, better hatching rates were observed in the non-vitrified control group. Total cell number and the number of cells allocated to the inner cell mass (ICM) were not different between treatments. The percentage of ICM development was also not different between treatments. Implantation rate and fetal weights were the same between treatments. However, overall there were fewer fetuses per embryo transferred in the re-vitrified group. CONCLUSION: Re-vitrification of mouse embryos has minimal effect on preimplantation embryo development or implantation potential.

Animals↗
PubMed1985

Multiple embryo-transplant offspring produced from quartering a bovine embryo at the morula stage.

A non-surgical embryo collection was completed on a day 7 superovulated Chianina donor cow. Because all but two of the ova from the collection were unfertilised and a surplus of potential recipients was available, one embryo (an excellent quality late morula) was dissected into four equal portioned 'quarter' embryos using a simplified micromanipulation procedure. Each quarter embryo was then placed in a 0.25 ml French straw and non-surgically transplanted to four different crossbred beef recipient females. The remaining embryo was similarly transplanted to a herd mate recipient as an intact embryo. One recipient returned to oestrus, one recipient had an extended post transfer cycle and the two remaining recipients produced a live quarter embryo transplant calf each within 24 hours of the other. The intact embryo placed in a herd mate recipient did not produce a transplant calf. To the authors' knowledge, these transplant offspring are the first live births reported from a non-surgically collected later-stage bovine morula (day 7), which had been dissected into quarters and then individually transplanted non-surgically to recipient females. The procedure was relatively simple to perform and was completed in less than one hour.

Animals↗
PubMed2004

Effect of six-and ten-day-old chick embryo amniotic fluid on development of two-cell mouse embryos.

The purpose of this study was to investigate the effect of six-and ten-day-old chick embryo amniotic fluid (CEAF) on the development of two-cell mouse embryos. Six- and ten-day-old CEAF (6-AF, 10-AF) were aspirated separately from the amniotic cavity and two experiments were performed. In the first experiment, two-cell mouse embryos were cultured in different supplements of heat-inactivated 6-AF, pure heat-inactivated 6-AF and pure active 6-AF. The second experiment was also carried out in the same manner using the 10-AF. The rate of the development of embryos in all groups were daily determined and statistically compared with that of control (Ham's F-10 supplemented with 5 mg/ml human serum albumin). During the cultivation period, more embryos reached to the blastocyst stage in all groups of CEAF compared to the control. The result hatched blastocysts embryos also increased from days 3 till 5 of cultivation in all groups of CEAF. The developmental potential of embryos appears to be almost identical in six- and ten-day-old CEAF. In conclusion, CEAF as a supplement or a natural medium could support the development of two-cell mouse embryos.

Amniotic Fluid↗
PubMed1992

Aggrecan core protein is expressed in membranous bone of the chick embryo. Molecular and biomechanical studies of normal and nanomelia embryos.

The recessive mutation nanomelia blocks the synthesis of a large aggregating proteoglycan (aggrecan) by avian embryo chondrocytes. Lack of aggrecan is associated with short stature, multiple morphological defects in cartilage, and embryo lethality. Bony defects have also been described, but were assumed to be a secondary consequence of the cartilage defect. However, two lines of evidence presented in this paper indicate that the aggrecan deficiency directly affects intramembranous bone. First, the morphology (i.e. projected area and shape) of certain membranous bones of nanomelia embryos was abnormal. Second, membranous bone from nanomelia embryos proved to be significantly stiffer in biomechanical tests that measured functional properties of the extracellular matrix. These findings were unexpected because intramembranous bones normally develop from mesenchyme and not from a cartilage intermediate, and they prompted a search for evidence of aggrecan expression in the bone of normal chick embryos. We report that: 1) aggrecan mRNA was identified by PCR analysis of total RNA isolated from day-13 chick embryo calvarium, 2) the PCR method successfully amplified aggrecan mRNA from primary chick embryo osteoblasts in culture, 3) in situ hybridization of membranous bone tissue sections demonstrated aggrecan expression by chick embryo osteoblasts in vivo, and 4) the aggrecan message was identified in Northern blots of calvarial mRNA probed at high stringency. The results of the molecular and biomechanical studies provide evidence that aggrecan is indeed expressed in membranous bone as well as cartilage. Altogether, these results suggest that aggrecan may contribute to the functional properties and the normal growth and development of avian membranous bone.

Aggrecans↗
PubMed1986

Size regulation in the mouse embryo. II. The development of half embryos.

The study describes an analysis of the development of mouse embryos halved at the 2-cell stage by the destruction of one blastomere, in comparison with control embryos of parallel derivation, at 2.5-13.5 days post coitum. The results showed that: (1) half embryos achieve size regulation some time between 7.5 and 10.5 days; (2) there is an indication that by 13.5 days half embryos may have again dropped back significantly in size relative to controls; (3) preregulation half embryos are slightly retarded developmentally, but this does not wholly account for their smaller size: morphogenesis is not size-dependent; (4) early postimplantation half embryos contain a significantly decreased proportion of inner cell mass derivatives and increased proportion of trophectoderm derivatives when compared with controls. A comparison is also made between the up-regulation of half embryos and the down-regulation of aggregate embryos, and it is suggested that size regulation may occur by delaying a change in the normal growth rate.

Animals↗
PubMed1995

Phenytoin-initiated DNA oxidation in murine embryo culture, and embryo protection by the antioxidative enzymes superoxide dismutase and catalase: evidence for reactive oxygen species-mediated DNA oxidation in the molecular mechanism of phenytoin teratogenicity.

A murine embryo culture model was used to investigate phenytoin-initiated embryonic DNA oxidation and dysmorphogenesis and to determine the embryoprotective potential of superoxide dismutase and catalase, which detoxify reactive oxygen species. Gestational day 9.5 CD-1 embryos were cultured for up to 24 hr at 37 degrees in medium containing phenytoin (20 micrograms/ml, 80 microM) or its vehicle (0.002 N NaOH). Embryos cultured for 24 hr were examined for embryotoxicity. After varying durations of incubation, embryonic DNA was isolated and purified, and DNA oxidation was determined from the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-2'-dG). Control embryos showed an early increase in 8-OH-2'-dG levels that was maximal between 2 and 4 hr, followed by a small but significant decrease over 24 hr, with no evidence of embryopathy. Phenytoin-treated embryos within 4 hr also demonstrated maximal 8-OH-2'-dG formation, which was substantially greater than that of controls, with a maximal 3-fold increase over controls at 24 hr (p < 0.05). In wash-out studies, embryos removed from the phenytoin-containing medium after 4 hr and then cultured in phenytoin-free medium for an additional 20-hr period showed no decrease in either 8-OH-2'-dG levels or embryotoxicity, compared with embryos incubated in the presence of phenytoin for 24 hr. Embryos exposed to phenytoin demonstrated substantial dysmorphogenesis, as evidenced by decreases in anterior neuropore closure, turning, yolk sac diameter, crown-rump length, and somite development (p < 0.05). Superoxide dismutase and catalase virtually eliminated phenytoin-initiated 8-OH-2'-dG formation and reduced or completely eliminated all phenytoin-initiated dysmorphological anomalies (p < 0.05). These results suggest that embryonic DNA oxidation constitutes teratologically important molecular target damage, and they provide the first direct evidence that free radical-mediated oxidative stress plays a critical role in phenytoin teratogenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2007

The pre-cycle blind mock embryo transfer is an inaccurate predictor of anticipated embryo transfer depth.

PURPOSE: To assess if the uterine cavity depth measured by a blind pre-cycle mock transfer changes after gonadotropin stimulation. METHODS: This is a retrospective cohort study at an academic IVF program involving 128 infertility patients. The main outcome measures were uterine cavity depth measured at the blind pre-stimulation mock transfer and the ultrasound-guided embryo transfer. RESULTS: A >or= 1 cm increase in uterine cavity depth was found in 57.9% of the patients. The mean pre-cycle blind mock transfer uterine depth significantly differed from the mean uterine depth measured at embryo transfer. Based on the mock transfer, the anticipated embryo transfer depth was significantly less than the actual ultrasound-guided embryo transfer depth. CONCLUSION: Uterine depth significantly differed between the blind pre-cycle mock transfer measurement and the ultrasound-guided embryo transfer measurement. The mock transfer may predict a difficult embryo transfer but it is an inaccurate predictor of the final embryo transfer depth.

Cohort Studies↗
PubMed1995

Psychological study of in vitro fertilization-embryo transfer participants' attitudes toward the destiny of their supernumerary embryos.

OBJECTIVE: To study the motivations underlying IVF-ET participants' choice to donate or destroy their supernumerary embryos. DESIGN: Couples' opinions are studied through a questionnaire and a psychological interview. PARTICIPANTS: Two hundred couples about to undergo IVF-ET. SETTING: The fertility unit of an academic hospital. MAIN OUTCOME MEASURES: Couples' choice for supernumerary embryos' destiny; opinions on embryo status, on importance of genetic lineage in the filial bonding, on gamete donation, and on multiple pregnancy risk. RESULTS: Donation is the most frequent choice but destruction is tolerated by almost all the couples (92%). Couples considering the embryo as a child choose destruction as frequently as donation but refuse experimentation on the embryo. Donation is highest among couples who stress education more than genetic lineage in parental bonding. This is confirmed by the choice of the couples requiring donor gametes. Couples express differing attitudes toward risks of twins and risks of triplets: twins are much more desired than triplets, which are frequently refused. CONCLUSIONS: Couples' opinions on the respective importance of genetic lineage and education in defining parental bonding are more determinant in their decision to destroy or to donate their supernumerary embryos than their opinions on the in vitro embryo status, which only determines their attitude toward experimentation.

Attitude↗
PubMed2004

Improved embryo development with decreased apoptosis in blastomeres after the treatment of cloned bovine embryos with beta-mercaptoethanol and hemoglobin.

In preliminary experiments, the treatment of donor somatic cells with beta-mercaptoethanol (ME) or hemoglobin (Hb) improved in vitro-development of bovine cloned embryos. This study was subsequently evaluated whether the exposure to Hb and/or ME during in vitro-maturation or embryo culture could further promote the development of embryos cloned with ME-treated donor cells. A prospective, randomized study was conducted and, embryo development, cell number, and apoptosis in blastocysts were monitored. A significant (P < 0.05) effect was found after the combined treatment of cloned embryos with Hb (1 microg/ml) and ME (10 microM); the development of morulae (53 vs. 35%) was greatly improved, which resulted in enhanced blastocyst formation (38%). However, cell number and apoptosis in blastocysts were predominantly affected by ME rather than Hb; a significant increase in total cell number of blastomeres (142-154 vs. 123 cells/embryo), inner cell mass (ICM) (39-41 vs. 27), and trophectoderm (TE) (103-114 vs. 98), and the ratio of ICM to TE cell number (0.26-0.27 vs. 0.22) was found. Also, the apoptosis index indicating the ratio of apoptotic cell to normal blastomere number was greatly reduced after ME treatments (0.85 vs. 0.056-0.069). When embryos cloned with ME-treated cells were cultured in Hb + ME-containing medium, any of the treatments to recipient oocytes before enucleation did not further promote the development. In conclusion, combined treatment of cloned embryos with Hb + ME not only improved in vitro-development but also decreased blastomere apoptosis. The use of ME-treated donor cells and the culture of cloned embryos in Hb + ME-containing medium yielded the optimal results for promoting the production of blastocysts with improved quality.

Animals↗
PubMed1993

Embryo sex selection by a rat male-specific antibody and the cytogenetic and developmental confirmation in cattle embryos.

Embryos of mouse, rabbit, goat, sheep, and cattle were separated into 2 groups on the basis of their morphology when incubated with a male-specific antibody (qualified here as the H-Y antibody) prepared from newborn rat testis. When morula-stage embryos were cultured in the presence of this H-Y antibody, the development of roughly one half of the embryos was arrested at that stage, whereas the other half continued to develop to the blastocyst stage. The developmentally arrested group of embryos resumed their development into blastocysts when cultured in antibody-free medium. Eighty to 90% of cattle embryos whose development was unaffected by the antibody were shown to possess a female karyotype (XX), and close to 80% of those embryos whose development was arrested possessed a male karyotype (XY). Cattle embryos whose sex had been presumptively identified by development in the presence of the H-Y antibody were cryopreserved and transferred, and the sex of the calves was examined. The overt sex of the young born from sexed embryos was found to be the same as that determined by chromosomal analysis.

Animals↗
PubMed1987

Differential effects of activators of cAMP-dependent protein kinase and protein kinase C on cleavage of one-cell mouse embryos and protein synthesis and phosphorylation in one- and two-cell embryos.

Membrane-permeable cAMP analogs or elevation of intracellular cAMP by cyclic nucleotide phosphodiesterase (PDE) inhibitors activates cAMP-dependent protein kinase. Biologically active phorbol esters or diacylglycerol activate the calcium-, phospholipid-dependent protein kinase, protein kinase C (PK-C). We report that membrane-permeable cAMP analogs, PDE inhibitors, biologically active phorbol esters, or a synthetic diacylglycerol inhibited cleavage of 1-cell mouse embryos to the 2-cell stage. The cAMP analogs and PDE inhibitors were effective only when added prior to S of the first cell cycle, whereas PK-C activators inhibited cleavage when added up until late G2/M. The PDE inhibitor Ro 20 1724/1 inhibited both DNA and protein synthesis in 1-cell embryos, whereas the phorbol ester, 12-O-tetradecanoyl-phorbol-13 acetate, or alpha-amanitin did not. In addition, 1-cell embryos prevented from cleaving by PDE inhibitors did not show specific changes in the pattern of protein phosphorylation associated with the 2-cell embryo, whereas such changes occurred in 1-cell embryos inhibited from cleaving with PK-C activators. Transcription in the 2-cell embryo results in the synthesis of a specific set of proteins, which is inhibited by alpha-amanitin. Although treatment of 1-cell embryos with aphidicolin or PK-C activators during G1 did not inhibit the synthesis of these proteins, treatment with cAMP analogs or PDE inhibitors during G1 inhibited the appearance of these proteins. These results are discussed in terms of how the synthesis of transcription-dependent proteins in the 2-cell embryo may be regulated by protein phosphorylation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Embryo transfer as a means of controlling the transmission of viral infections. VII. The in vitro exposure of bovine and porcine embryos to foot-and-mouth disease virus.

When 169 zona pellucida-intact bovine embryos were exposed to 10(6) pfu/ml of foot-and-mouth disease virus and then washed, no infectious virus was detected on any of the embryos. FMD viral infectivity was found, however, in association with 14 of 42 hatched (zona pellucida-free) bovine embryos and in a small number of zona pellucida-intact porcine embryos. The porcine embryos were assayed individually and in groups of 8 embryos. Four of the 124 individual embryos and 2 of the 9 groups of embryos carried the infectious virus.

Journal Article↗
PubMed1987

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

Two experiments involving the transfer of embryos from donors infected with swine vesicular disease virus (SVDV) to "clean" recipients were carried out. In Experiment 1, 47 embryos were collected from 4 SVDV-infected donors and transferred to 2 recipients that subsequently produced 10 piglets. All of the recipients and piglets remained seronegative for SVDV. In addition to the transfers, 10 embryos and 58 unfertilized eggs from the infected donors were assayed in vitro and found to be negative for SVDV infectivity. A fifth donor was also inoculated with SVDV in this experiment, but it could not be demonstrated that infection had occurred. This SVDV-exposed donor provided two embryos for transfer and one embryo and two unfertilized eggs for in vitro assay. In Experiment 2, 158 embryos from 9 infected donors were transferred to 7 recipients, resulting in 12 piglets. A total of 7 embryos and 37 unfertilized eggs were assayed in vitro. The recipients, piglets, and embryos/eggs were all negative for SVDV infectivity. Although a final conclusion on the safety of using embryo transfer for the control of swine vesicular disease (SVD) is not possible, the results obtained justify additional studies.

Journal Article↗
PubMed1987

Embryo transfer as a means of controlling the transmission of viral infections. XI. The in vitro exposure of bovine and porcine embryos to vesicular stomatitis virus.

Infectious virus was isolated from both porcine and bovine zona pellucida-intact embryos that had been exposed to the Indiana strain of vesicular stomatitis virus (VSV) and then washed. The amount of virus isolated from embryos depended on their initial exposure level. Porcine embryos always retained more virus than bovine embryos. When embryos were cultured for 24 h after viral exposure and washing, the number of embryos carrying VSV and the amount of virus on each of the embryos was reduced. Trypsin (0.25%) was also found to be effective in inactivating/removing the VSV from embryos, suggesting that most, if not all, of the virus was bound to the zona pellucida.

Journal Article↗
PubMed1991

Embryo survival in pseudopregnant and in pregnant but genetically semi-sterile recipients after nonsurgical embryo transfer in the mouse.

A new nonsurgical embryo transfer technique was used in the mouse that yielded survival rates of between 40 and 70% depending on embryo stage and, possibly, on the degree of synchrony between the embryo and recipient. Three variables were tested using this embryo transfer technique: a) pseudopregnant recipients vs pregnant but genetically semi-sterile recipients, b) embryos resulting from superovulation vs embryos from natural ovulation, and c) 12-hour vs 24-hour asynchrony between donors and recipients. None of these variables significantly affected the pregnancy rate or the percentage of transferred embryos developing to term. The pregnancy rates were between 77 and 90% in 6 experimental groups of 8 to 13 females. Survival rates were between 41 and 63% when all recipients were considered and between 53 and 68% when only the pregnant recipients were included. The embryo transfer procedure influenced litter size composition of the endogenous conceptuses of the semi-sterile recipients. Too many females were devoid of these. Recipients of expanded blastocysts had significantly better transfer results than recipients that also received morulae and early blastocysts. It was concluded that the transfer success rates were influenced by the recipients and possibly by their preparation for transfer.

Journal Article↗
PubMed1991

Fluorescein diacetate assessment of embryo viability after ultrarapid freezing of human multipronucleate embryos.

OBJECTIVE: To assess embryo viability after ultrarapid freezing-thawing. DESIGN: We studied the fluorescence pattern of 35 ultrarapidly frozen-thawed multipronucleate human embryos exposed to fluorescein diacetate. SETTING: All the embryos were obtained from the Medical Center for Fertility Diagnostics and In Vitro Fertilization and Embryo Transfer at Leuven (Belgium), a private care center. PATIENTS, PARTICIPANTS: None. INTERVENTIONS: None. MAIN OUTCOME MEASURE: The fluorescence pattern was evaluated at room temperature after a 1-minute incubation in fluorescein diacetate solution, 4 hours and 24 hours after thawing. RESULTS: Healthy human multipronucleate embryos, when exposed to fluorescein diacetate, accumulated intracellular fluorescein and fluoresced brightly under ultraviolet light. On the other hand, embryos presenting submicroscopic cell membranes damage caused by different processes (e.g., high or low temperatures) lost the ability to accumulate intracellular fluorescein. All the ultrarapidly frozen embryos with normal looking blastomeres fluoresced brightly after a short exposure to fluorescein diacetate. CONCLUSIONS: Our experiments indicate an intact cell membrane permeability and an integrity of the intracytoplasmatic esterase enzyme activity of human embryos ultrarapidly frozen.

Blastocyst↗