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PubMed2000

Micromanipulation of cryopreserved embryos and cryopreservation of micromanipulated embryos in PGD.

The possibility to employ cryopreservation in Preimplantation Genetic Diagnosis (PGD) should enlarge the opportunities for research and clinical activity. For these purposes, we tried three kinds of approaches on human abnormal embryos: (1) cryopreservation of biopsied embryos; (2) biopsy of thawed embryos; and (3) biopsy of embryos derived from thawed oocytes. Our preliminary results show that: (1) biopsy of thawed embryos is feasible and FISH analysis is possible on both survived and lysed cells; (2) Optimization of freezing/thawing procedures are necessary to obtain better survival rate after thawing of biopsied embryos; (3) Biopsy and FISH are feasible on embryos derived from thawed oocytes and they could be a good way to study the chromosomal arrangement of these poorly investigated embryos.

Biopsy↗
PubMed2000

Repeated surgical embryo recovery and embryo production in rabbits.

The purpose of this study was to assess the embryo production after repeated surgical recovery of embryos in Gigante de España does. A total of 195 ovulatory treatments and embryo recoveries were performed from 1995 to 1999. Ovulation was induced by an intramuscular injection of 20 microg GnRH immediately after mating. Each doe was induced to ovulate up to four consecutive times at intervals of at least 50 days. Embryos were surgically collected from oviducts 68-69 h post-coitus. An average of 8.6 corpora lutea and 6.4 recovered embryos (90% of them classified as viable) were recorded from the 195 treatments. The process seemed to be less efficient in the fourth treatment, with a drop of more than two recovered and viable morulae with reference to the third (P<0.05 for both parameters) or the second recovery (P<0.1 and P<0.05, respectively). More than 20 recovered embryos and 18 viable embryos per donor doe were recorded considering the three first ovulatory treatments performed in 33% of the does (30/90). Results indicate that the methodology used in the present study could be an efficient way to maximize in vivo embryo production from rabbits.

Animals↗
PubMed2005

Predicting embryo quality: mRNA expression and the preimplantation embryo.

To overcome the low implantation rate (10-20%) following IVF in humans, more than two embryos are commonly replaced, potentially leading to high order multiple pregnancies with associated significantly elevated risks. Selecting the most viable embryos and transferring fewer of them could reduce this risk. Prolonged culture of embryos in vitro to the blastocyst stage may expose the embryo to hazards not normally encountered in the female reproductive tract. Recent studies comparing bovine oocyte maturation, fertilization and embryo culture in vivo and in vitro have demonstrated that the origin of the oocyte is the main factor affecting blastocyst yield, while the post-fertilization culture environment is crucial in determining blastocyst quality, measured in terms of cryotolerance and relative transcript abundance, irrespective of the origin of the oocyte. Production of embryos in vitro, particularly when using an extended period of in-vitro culture may predispose the embryo to phenomena such as 'large offspring syndrome', which is probably linked to altered gene expression, particularly of imprinted genes. Post-fertilization culture environment clearly has a profound effect on the relative abundance of gene transcripts within the embryo. Culture under sub-optimal conditions for even one day can lead to perturbations in the pattern of expression.

Amino Acids↗
PubMed1996

Alterations of intraembryonic metabolites in preimplantation mouse embryos exposed to elevated concentrations of glucose: a metabolic explanation for the developmental retardation seen in preimplantation embryos from diabetic animals.

Preimplantation mouse embryos exposed to hyperglycemia, whether in vivo or in vitro, experience delayed development from the 2-cell to blastocyst stage. By comparing metabolites from embryos exposed to high vs. normal glucose conditions, a metabolic explanation for the delayed growth pattern was sought. Fertilized 1-cell embryos obtained from superovulated B5 x CBA F1 mice were cultured for 96 h in medium containing 2.8 mM glucose (C) or in medium with added glucose to give 10 mM, 30 mM, or 52 mM glucose (HG). After incubation, each embryo was quick-frozen and freeze-dried. Metabolites were assayed by the ultramicrofluorometric technique and enzymatic cycling to obtain measurable levels in single embryos. Embryos cultured in HG exhibited 7-fold higher intracellular glucose levels than those cultured in C (C: 2.25 +/- 0.6 vs. HG: 16.61 +/- 2.4 mmol/kg wet weight; p < 0.001; C, n = 9; HG, n = 16). This accumulation of glucose was dose-related and stage-dependent. Citrate (C: 1.07 +/- 0.14 vs. HG: 1.98 +/- 0.12; p < 0.001), sorbitol (C: 0.41 +/- 0.06 vs. HG: 0.57 +/- 0.03; p < 0.01), malate (C: 0.81 +/- 0.13 vs. HG: 1.72 +/- 0.17; p < 0.001), and fructose (C: 2.1 +/- 0.3 vs. HG: 5.3 +/- 0.6; p < 0.001) were all significantly higher in HG. Also, these metabolites were highest in the most delayed embryos. Glycogen and 6-phosphogluconate levels were not significantly different. In conclusion, intraembryonic levels of glucose, and polyol pathway and Krebs cycle metabolites are elevated and correspond to the degree of developmental delay. These findings suggest that a metabolic abnormality may be responsible for retarded development experienced by embryos exposed to high glucose.

Animals↗
PubMed1998

PEI1, an embryo-specific zinc finger protein gene required for heart-stage embryo formation in Arabidopsis.

We used virtual subtraction, a new gene isolation strategy, to isolate several genes of interest that are expressed in Arabidopsis embryos. These genes have demonstrated biological properties or have the potential to be involved in important biological processes. One gene isolated by virtual subtraction is PEI. It encodes a protein containing a Cys3His zinc finger domain associated with a number of animal and fungal transcription factors. In situ hybridization results showed that PEI1 is expressed throughout the embryo from globular to late cotyledon stage. Transgenic Arabidopsis plants expressing a PEI1 antisense gene produced white seeds in which embryo development did not progress through heart stage. Aberrant embryos failed to form cotyledons, but the embryonic root appeared to be normal. Aberrant embryos did not turn green, and the expression of genes involved in photomorphogenesis was drastically attenuated. In culture, aberrant embryos did not form true leaves, but root formation was apparently normal. These results suggest that PEI1 is an embryo-specific transcription factor that plays an important role during Arabidopsis embryogenesis, functioning primarily in the apical domain of the embryo.

Amino Acid Sequence↗
PubMed2000

Inoculation of somatic embryos of sweet potato with an arbuscular mycorrhizal fungus improves embryo survival and plantlet formation.

Responses of somatic embryos of sweet potato (Ipomoea batata (L.) Poir., cv. White Star) at different developmental stages to in vitro inoculation with Glomus etunicatum (Becker and Gerdemann) (isolate INVAM FL329) were evaluated. Somatic embryos were grown in glass tubes containing sterilized vermiculite and sand. A layer of natrosol plus White's medium was used as a carrier for arbuscular mycorrhizal (AM) fungal spores. Survival of embryos inoculated with AM fungi was significantly (P < 0.05) greater than that of noninoculated embryos at the rooted-cotyledonary-torpedo and rooted-elongated-torpedo developmental stages. Mycorrhizae significantly (P < 0.05) increased plantlet formation only when inoculation occurred at the rooted-elongated-torpedo developmental stage. The growth stage at which the embryos were inserted into the glass tubes exerted a significant influence upon plantlet formation, and plantlet formation was further enhanced by inoculation with G. etunicatum. Plantlet formation was greatest at the rooted-elongated-torpedo stage. These results demonstrate that inoculation of somatic embryos with AM fungi improves embryo survival and plantlet formation, and could enhance use of somatic embryos as synthetic seeds.

Fungi↗
PubMed1996

Influence of oocyte aging on developmental ability of reconstituted embryos produced from oocyte cytoplast and single blastomeres of two-cell stage embryos.

The present study was conducted to investigate the influence of aging of recipient oocyte on the developmental ability of reconstituted mouse embryos produced from the cytoplast of oocytes and single blastomeres of early or late 2-cell stage embryos by electrofusion. Oocytes were obtained at 14 (newly ovulated oocytes), 18, 22 (oocyte that time passed after ovulation; aged oocyte) hr after hCG injection and oocyte cytoplast was produced by manual enucleation using a fine glass needle under the dissecting microscope. The aging of the oocytes significantly influenced on the fusion rate of the reconstituted embryos (14 hr: 42.7-47.2% vs. 22 hr: 75.3-77.6%). Similarly, the cleavage rate of reconstituted embryos increased with aging of the oocytes (14 hr: 50.8-56.3% vs. 22 hr: 82.2-90.7%). The percentage of reconstituted embryos produced from cytoplast of aged oocytes (22 hr post hCG) and single blastomeres of late 2-cell stage embryos developing to the blastocyst (20.8%) was significantly higher than that of reconstituted embryos produced by other combinations (2.0-8.2%: P < 0.01). Although cell cycle stage of donor nuclei influenced to developmental ability of reconstituted embryos, these results are probably related to the aging of the oocytes since aged oocytes can be activated more easily by electrical stimulation than newly ovulated oocytes.

Animals↗
PubMed2003

Inositol transport in preimplantation rabbit embryos: effects of embryo stage, sodium, osmolality and metabolic inhibitors.

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used [3H]inositol to investigate the transport of inositol, an essential component of the phosphatidylinositol signal transduction system, over the 6 days of preimplantation development by rabbit embryos. In the presence of 15 micromol inositol-1 in the incubation medium, there was a small linear increase in inositol uptake from 0.07 pmol per embryo per h at the one-cell stage (day 1) to 0.135 pmol at the late morula (day 3) stage. Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavage-stage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V(max) and 0.045 and 0.038 mmol-1 [corrected] for Km were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.

Animals↗
PubMed2004

Influence of embryo transfer depth on in vitro fertilization and embryo transfer outcomes.

OBJECTIVE: To investigate the influence of transfer distance from the fundus (TDF) on clinical pregnancy rate (PR) and ectopic pregnancy rate. DESIGN: Retrospective cohort. Between January 2000 and December 2001, 699 ultrasound (US)-guided embryo transfers were conducted. Mock transfer was performed to measure uterine cavity depth 1 month before treatment. Cavity depth was measured by abdominal US before the transfer, from the vaginal stripe to the fundus. Transfers were performed with a Wallace embryo transfer catheter (Cooper Surgical, Shelton, CT) using US and physician's judgment of cavity depth. Transfer distance from the fundus was calculated by subtracting the depth of catheter insertion from the cavity depth, as determined by US or by mock transfer. Statistical analyses were performed by building a multivariable logistic regression model to calculate odds ratios and 95% confidence intervals (CI). SETTING: Women aged 23 to 43 years who are in a university-affiliated, community-based IVF program in Springfield, Massachusetts. PATIENT(S): All patients enrolled in IVF program undergoing embryo transfer. INTERVENTION(S): No patient received any additional procedure or intervention. All of the measurements obtained with the embryo catheter and the transvaginal ultrasound were part of the program's protocol for the embryo transfer. MAIN OUTCOME MEASURE(S): Odds ratio examining relationship between embryo transfer depth and PR.Clinical, implantation, and ectopic PR were 37%, 20%, and 2.1%. Cavity depth by US differed from cavity depth by mock by at least 10 mm in >30% of cases. The TDF by US was highly predictive of PR; TDF by mock was not predictive of PR. Increasing the TDF by US resulted in significantly increased PR as well as lower ectopic rates. Using regression analysis, the odds ratio for TDF by US was 1.11 (95% CI: 1.07-1.14). This suggests that for every additional millimeter embryos are deposited away from the fundus, the odds of clinical pregnancy increased by 11%. CONCLUSION(S): After controlling for potential confounders, the clinical PR is significantly influenced by the transfer distance from the fundus. Cavity depth by US is clinically useful to determine the depth beyond which catheter insertion should not occur.

Adult↗
PubMed2004

Middle to lower uterine segment embryo transfer improves implantation and pregnancy rates compared with fundal embryo transfer.

OBJECTIVE: To assess differences in pregnancy and implantation rates as a function of the embryo placement. DESIGN: Prospective cohort study. SETTING: A tertiary care center. SUBJECT(S): All fresh, nondonor IVF cycles performed in 2001. INTERVENTION(S): Alteration in embryo transfer (ET) target location from the fundal region to the middle to lower uterine segment. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (sonographic sac evidence/number of transfer cycles), implantation rate (number of sacs/number of embryos transferred), patient age, peak E(2), and fertilization rate. RESULT(S): A total of 393 fundal and 273 lower to middle uterine segment ETs were performed. The pregnancy (PR), implantation, and birth rates were significantly higher after a middle to lower uterine segment ET compared with fundal ET (39.6% vs. 31.2%; 21% vs. 14%; and 34.1% vs. 26.2%, respectively). Groups did not differ regarding patient age, basal FSH, peak E(2), number of intracytoplasmic sperm injection (ICSI) cycles, fertilization rate, embryo quality, or number of embryos transferred. CONCLUSION(S): Both PR and implantation rates are favorably affected by directing embryo placement to the lower to middle uterine segment. By some unknown mechanism, it appears that this endometrial location provides a more favorable region for embryo deposition.

Cohort Studies↗
PubMed2005

Removal of lysed blastomeres from frozen-thawed embryos improves implantation and pregnancy rates in frozen embryo transfer cycles.

OBJECTIVE: To evaluate the effect of degenerated (lysed) blastomere removal on implantation and pregnancy rates in cleavage-stage cryo-embryo transfer (ET) cycles. DESIGN: Randomized clinical trial. SETTING: Private reproductive medical center. PATIENT(S): A total of 88 patients who received frozen-thawed ET, divided into two groups. INTERVENTION(S): Embryo freezing and thawing; opening of the zona pellucida and removal of cryodamaged blastomeres (in the study group), followed by same-day ET. MAIN OUTCOME MEASURE(S): Extent of survival of cleavage-stage embryos after the freeze-thaw procedure; embryo implantation and clinical pregnancies. RESULT(S): Oocyte number per patient, fertilization rate, embryo development rate (and quality), and freezing rates were similar in the two groups in the fresh cycle. In the control group, a total of 55 embryos (25%) of the 217 thawed remained fully intact, and 53 (26%) of the 207 in the study group remained intact. The average number of embryos transferred per group was similar (control, 3.4 +/- 0.9; study, 3.3 +/- 0.9). Implantation rates were 12% and 26% in the control and study groups, respectively. The clinical pregnancy rate was 23% in the control group and 64% in the study group when lysed cell removal was performed. CONCLUSION(S): The results show that pregnancy and implantation rates are higher in the study group; therefore, the removal of degenerated blastomeres may be beneficial to all patients who undergo cleavage-stage, frozen-thawed ET.

Adult↗
PubMedDate not supplied

Is embryo-cryopreservation really neutral? A new long-term effect of embryo freezing in mice: protection of adults from induced cancer according to strain and sex.

BACKGROUND: Beneficial or harmful effects of embryo freezing have been described in man and animals, raising the question of the neutrality of this technique. OBJECTIVE: We examined, in mice, the possibility that embryo freezing influences the probability of the emergence, in adults, of an induced urinary bladder cancer. METHODS: The experiment was conducted in mice derived from embryos of two different genotypes. Females receiving embryos were parsed into two groups according to whether these embryos were cryopreserved or not. The derived adults received the chemical carcinogen N-butyl-N-4hydroxybutylnitrosamine (BBN), in the drinking water. Time to death since the onset of treatment was measured for each animal until the 300th day. RESULTS: In females from one of the two strains tested, embryo freezing led to a favorable long-term effect on the probability of resistance to induced cancer. CONCLUSION: This beneficial effect, taken together with other effects reported in the literature be they beneficial or harmful, suggests that embryo freezing in mice may not be neutral.

Animals↗
PubMed2003

Assessment of embryo viability: the ability to select a single embryo for transfer--a review.

By being able to select the most viable embryo(s) within a given cohort it will be possible to reduce the number of embryos transferred in a given IVF procedure. Several morphological scoring systems have been proposed for the successive stages of human embryo development. Other indicators of embryo viability include rate of cleavage. Finally, non-invasive methods of assessing nutrient uptake and utilization have been developed that can be used to measure the health of individual embryos. A sequential scoring system has therefore been proposed that uses the above parameters in order to create a history for each embryo during the preimplantation period. Such systems will help lead to single embryo transfers for the majority of IVF patients.

Adult↗
PubMed1995

The probability of pregnancy after embryo transfer is affected by the age of the patient, cause of infertility, number of embryos transferred and the average morphology score, as revealed by multiple logistic regression analysis.

Because the process of conception is affected by many variables, a multiple logistic regression analysis was performed to assess (i) the impact and relative weight of both patient and embryo variables and (ii) their possible effects on the probability of a vital pregnancy after embryo transfer. A statistical model was constructed predicting the probability of pregnancy after embryo transfer. The variables that contributed significantly to the predictive value of the model were the age of the patient, the cause of infertility, the number of embryos transferred and the average morphology score of the transferred embryos. Embryo variables appeared to have a significant but modest value in predicting the probability of pregnancy after embryo transfer. Other variables, such as the thickness of the endometrium, were found to have no prognostic value. Moreover, we found that their effect could be explained by the variables already included in the model.

Adult↗
PubMed2002

A study to determine if embryo cryopreservation influences the potential of rapidly growing embryos to successfully implant in uterine environments not influenced by controlled ovarian hyperstimulation.

PURPOSE: To determine if transferring at least one embryo with eight blastomeres at 72 hours improves prognosis of donor oocyte recipients. The study aim was to verify if cryopreservation increases or decreases the advantage of rapidly growing embryos. The study could exclude the influence of controlled ovarian hyperstimulation (COH) on the uterine environment. METHODS: All transfers, fresh or frozen, using exclusively embryos that resulted from fertilization of donor oocytes over a three-year period were evaluated. RESULTS: Significantly higher pregnancy rates (PRs) and delivery rates were found in donor oocyte recipients receiving at least one eight-cell embryo compared to transfers without any eight-cell embryos. These differences were not found when comparing frozen embryo transfers (ETs). The data could not be explained by confounding variables. CONCLUSIONS: The inclusion of at least one 8-cell embryo on day 3 fresh ET resulted in higher PRs even without the influence of COH. However, higher blastomere number did not influence frozen ET outcome.

Blastomeres↗
PubMed1985

Mitochondrial alterations in embryos exposed to B-hydroxybutyrate in whole embryo culture.

The ketone body B-hydroxybutyrate (B-OHB) produces malformations and ultrastructural alterations in mitochondria of mouse embryos exposed for 24 hours to the compound in whole embryo culture. The present study was conducted to establish the time-course of the mitochondrial changes to determine whether the changes are reversible, and to relate these changes to the malformations produced by the compound. Since mitochondria also play a key role in the metabolism of ketone bodies, the capacity of the early somite embryo to metabolize B-OHB was investigated in an effort to link the morphological alterations in the mitochondria to a biochemical process. Early somite embryos were cultured 4, 8, or 24 hours in the presence of 32 mM DL-B-OHB and then cultured for an additional 24 hours in control serum. Finally, embryonic tissue during the teratogenic period was assessed for its capability to oxidize B-OHB using D-(3-14C)-B-OHB. The treated embryos showed progressive alterations in the mitochondria, beginning at 4 hours with a loss of matrix density and culminating at 24 hours with high-amplitude swelling, complete loss of matrix density, and disappearance of cristae. These alterations were reversible following removal of the embryos after 24 hours of exposure to B-OHB and culturing for an additional 24 hours in control serum. Metabolism studies demonstrated that the early somite embryo possesses a limited capacity to oxidatively metabolize B-OHB. The biochemical implications of these findings are discussed with respect to the possible role of ketone bodies in the mechanism of diabetes-induced congenital malformations.

3-Hydroxybutyric Acid↗
PubMed1995

Developmental toxicity of formate and formic acid in whole embryo culture: a comparative study with mouse and rat embryos.

Acute methanol (MeOH) toxicity in primates is attributed to the conversion of MeOH to formate and the resulting acidosis. MeOH has been shown to be developmentally toxic in mice and rats both in vivo and in vitro, but rodents neither accumulate formate nor develop acidosis after MeOH exposure. To further assess the potential human developmental toxicity of MeOH exposure, we evaluated the developmental effects of sodium (Na) formate and formic acid in rodent whole embryo culture (WEC). Day 9 rat embryos were cultured for 24 or 48 hours and day 8 mouse embryos were cultured for 24 hours in the presence of Na-formate or formic acid. Rat and mouse embryos exposed to either agent for 24 hours exhibited a trend toward reduced growth and development and the number of abnormalities increased at the higher concentrations. Rat embryos exposed for 48 hours to either Na-formate or formic acid showed a trend toward reduced growth and development with increasing concentration. Embryo lethality and incidence of abnormal embryos were also increased at the higher concentrations. The anomalies observed in both species after exposure to either compound were primarily open anterior and posterior neuropore with less frequent incidence of rotational defects, tail anomalies, enlarged pericardium and delayed heart development. Exposure to Na-formate or formic acid for comparable periods of time results in comparable degrees of embryotoxicity at concentrations (mMolar) at least 4-fold lower than those previously reported for methanol exposure.

Abnormalities, Drug-Induced↗
PubMed1996

Chick embryos as an alternative experimental animal for cardiovascular investigations: stable recording of electrocardiogram of chick embryos in ovo on the 16th day of incubation.

Recording of electrocardiogram (ECG) tracings in developing chick embryos often fails because of spontaneous motion of the embryos in the egg shell. We attempted to record ECG of chick embryos in ovo. When we injected a mixture of 450 mg/ml urethane and 45 mg/ml alpha-chloralose into the air sac of fertile eggs at volumes of 0.1 to 0.3 ml, the spontaneous motor activity of chick embryos was decreased and stable ECG tracings could be obtained from at least 10 min after the injection. The P, QRS, and T waves were noted in the electrograms, and the QT interval was positively correlated to the RR interval. The heart rate (HR) could be analyzed for the RR interval in fertile eggs after the 8th day of incubation. The HR of the 16-day fertile embryos was linearly increased with incubation temperature in the range from 31 to 41 degrees C. Using this system, cardiac effects of some drugs were examined. Isoprenaline and acetylcholine increased and decreased the HR in a dose-dependent manner, respectively, and these effects were inhibited by respective antagonists, propranolol and atropine. These ECG responses of chick embryos were similar to those of mammals or humans. In conclusion, stable ECG tracings could be obtained from chick embryos anesthetized by urethane and alpha-chloralose in ovo and this method may be applicable for the investigation of the developing heart and the evaluation of cardiovascular drugs.

Age Factors↗