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PubMed2005

Production of rabbit chimeric embryos by aggregation of zona-free nuclear transfer blastomeres.

The objective of this study was to compare in vitro developmental capacity of zona-free aggregated rabbit chimeric embryos and the allocation of EGFP (enhanced green fluorescence protein) gene expression to the inner cell mass (ICM). We produced chimeric embryos by synchronous aggregation of zona-free blastomeres from embryonic cell nuclear transfer (EMB-NT) or somatic cell nuclear transfer (SC-NT) and blastomeres from normal zona-free embryos (N) at the 16-cell stage. In the control group, transgenic (TR) and normal zona-free embryos were used to produce chimeric embryos (TR<>N). EMB-NT embryos were produced by fusion of enucleated oocytes with embryonic cells, which were derived from 32-cell stage transgenic embryos bearing the EGFP gene. The SC-NT embryos were produced by fusing enucleated oocytes with cumulus cells, which were derived from homozygotes transgenic for the EGFP gene female oocytes at 16h post-coitum. Nuclei of transgenic blastomeres emitted a green signal under fluorescence microscopy. Zona-free EMB-NT or zona-free SC-NT rabbit embryos, both with EGFP fluorescence, as well as TR and zona-free rabbit embryos with no fluorescence (EMB-NT<>N, SC-NT<>N, TR<>N) were aggregated on day 2.5 and evaluated on day 5. The proportion of EMB-NT<>N embryos that developed to the blastocyst stage was significantly higher compared with SC-NT derived cells (p < 0.05), but significantly lower than in TR<>N chimeric blastocysts (p < 0.001). Similarly, a higher proportion (p < 0.001) of EGFP-positive cells allocated to ICM of chimeric blastocysts was revealed in TR<>N chimeras (55%), compared with EMB-NT<>N (35%) and SC-NT<>N (21%). Our results indicate that synchronous chimeric embryos reconstructed from TR embryos were better able to develop and colonize the ICM area than EMB-NT and SC-NT embryos. In this study we have demonstrated for the first time that rabbit NT-derived embryos are able to develop into chimeric blastocysts and participate in the ICM area.

Animals↗
PubMed2001

Alternative embryo transfer on day 3 or day 5 for reducing the risk of multiple gestations.

PURPOSE: This study was carried out to reduce the possibility of high-order multiple gestations and the failure of embryo transfer by determining their replacement date based on the number and quality of 2-day embryos. METHODS: All zygotes were cocultured with cumulus cells in 10 microliters of YS medium containing 10% human follicular fluid (hFF) for 48 or 96 hr. In period I, all embryos were transferred on day 3 (1032 cycles). In period II, the embryos were transferred on either day 3 or day 5 by determining their replacement date based on the number and quality of 2-day embryos: there were 2701 patients in whom embryos were replaced on day 3 (in the case that the number of zygotes was less than eight and the number of good-quality embryos was less than three) and 1952 patients less than 40 years old in whom embryos were replaced on day 5 (in the case that the number of zygotes was eight or more and/or the number of good-quality embryos was three or more). On the other hand, patients who were 40 years old or more were alloted to day 3 transfer cycles, regardless of the number and quality of the 2-day embryos, due to the possibility of their not producing blastocyst-stage embryos in vitro. RESULTS: The number of embryos transferred in period II was 2.9 +/- 0.6, while that in period I was 3.7 +/- 0.5. The multiple pregnancy rate was significantly decreased in period II (30.7%) compared to that (49.6%) in period I, while the pregnancy and implantation rates in period II (36.1 and 16.4%, respectively) were not lower than those (34.9 and 16.1%, respectively) in period I. The rate of triplet or more gestations was significantly minimized in period II (2.3%) compared to that in period I (26.5%). CONCLUSIONS: We propose that determination of the date on which embryos should be transferred based on the number and quality of embryos on day 2 may help to maintain an acceptable pregnancy rate, while minimizing embryo transfer failure and high-order multiple gestations.

Adult↗
PubMed1990

Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos.

The allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) was investigated at 6-h intervals from 78 h to 102 h after hCG injection in 3/4 mouse embryos to determine the effect of removal of a single blastomere at the 4-cell stage on early differentiation. The procedures used to produce 3/4 embryos had little effect on embryo development. Embryos that had a single blastomere removed and then re-aggregated (RA embryos) had the same total number of cells as untreated (UT) embryos except at 78 h (P less than 0.05) and 102 h (P less than 0.01) post hCG where there were slightly less cells in RA embryos. Three-quarter embryos always had significantly fewer cells than RA embryos (P less than 0.001), with an average of 74% of the total cell number of RA embryos. As expected, 3/4 embryos always had significantly fewer cells in the ICM and TE compared with RA embryos (P less than 0.001). However, the ICM:TE ratio was also significantly lower in 3/4 embryos compared with RA embryos at 84, 96, and 102 h post hCG, indicating that the allocation of cells to the ICM and TE was disturbed. The ICM:TE ratio of 3/4 embryos could not be manipulated if either an early- or late-dividing blastomere was selectively biopsied at the 4-cell stage; this suggests that the known preferential contribution of an early-dividing blastomere to the ICM is not cell autonomous.

Animals↗
PubMed1997

Early luteal phase administration of mifepristone inhibits preimplantation embryo development and viability in the rhesus monkey.

It is generally believed that progesterone is essential for inducing the changes in oviduct and uterus necessary for embryo viability and implantation in a number of mammalian species. The aim of this study was, in the rhesus monkey, to examine in conception cycles with and without early luteal phase antiprogestin (mifepristone; RU 486) treatment: (i) the growth status of preimplantation embryos and (ii) the implantation ability of the preimplantation embryo after transfer to a synchronous-cycle surrogate recipient. A total of 43 proven fertile rhesus monkeys were randomly placed in the control (group 1, n = 18) and mifepristone (group 2, n = 25) groups. All monkeys cohabited with proven fertile male monkeys on cycle days 8-16 and were injected with vehicle alone [benzyl benzoate:olive oil, 1:4 (v/v), s.c.] for group 1 and with mifepristone (2 mg/kg body weight s.c.) for group 2, on day 2 after the presumed day of ovulation. A total of 12 preimplantation embryos [premorula (n = 1), morula (n = 2), zona-encased (n = 7) and zona-free (n = 1) blastocysts and degenerate embryos (n = 1)] were recovered from 17 ovulatory, mated cycles in group 1 on day 6 after ovulation. In group 2, of the 23 ovulated cycles, 12 preimplantation embryos [premorula (n = 2), morula (n = 7), zona-encased blastocyst (n = 1), and degenerate embryos (n = 2)] were retrieved. Despite no significant difference in the recovery rate between the two groups, early luteal phase RU 486 exposure induced delay (P < 0.01) in preimplantation embryo growth, primarily at the morula-blastocyst transition stage. Nine of the embryos from group 1 and seven of the embryos from group 2 recovered on day 6 were transferred to naturally synchronized, non-mated and untreated surrogate recipients. In group 1, five embryos implanted (55%) and, of these, three (60%) gave rise to live infants through natural delivery; implantation was assessed from extension of the cycle (i.e. no menstrual bleeding) and rise in concentrations of oestradiol and progesterone from day 10 of conception; rectal palpation was performed on cycle day 50 to confirm clinical pregnancy. In group 2, however, there was not a single case of establishment of pregnancy following transfer of embryos retrieved from mifepristone-exposed monkeys. Thus, preimplantation embryos recovered from RU 486-exposed monkeys failed to establish evolutive implantation and pregnancy, while significant (P < 0.02) success was observed in transfers of embryos from the control group. We postulate that progesterone-mediated actions are involved in mediating the growth and viability of preimplantation-stage embryos in the rhesus monkey.

Animals↗
PubMed1998

Fluorescent in-situ hybridization on human embryos showing cleavage arrest after freezing and thawing.

Our current freezing-thawing policy is to transfer only embryos that cleave further in the 24 h following thawing. The purpose of our study was to investigate the incidence of numerical abnormalities for chromosomes X, Y and 1 in blastomeres of human preimplantation embryos that survived cryopreservation but did not cleave further after thawing. A total of 63 embryos surviving a freezing-thawing cycle but not cleaving further within 24 h after thawing were screened. Of the 63 screened embryos that showed cleavage arrest 24 h after thawing, 56 embryos (88.9%) remained arrested 48 h after thawing and slightly more than half of these (29/56; 51.8%) showed further deterioration in morphological quality. Seven embryos (11.1%) showed signs of further cleavage; five embryos showed additional cleavage of one blastomere and two developed a blastocoelic cavity. Fluorescent in-situ hybridization (FISH) with three specific probes for simultaneous detection of chromosomes X, Y and 1 was performed and was successful in 60 out of 63 embryos. Of these successfully labelled embryos, 26 (43.3%) were in the diploid range: 12 (20%) were uniformly diploid for the chromosomes X, Y and 1; three embryos showed aneuploidy in all their blastomeres (two were XXY-karyotype and one was monosomy 1) and in 11 embryos nondisjunction was detected. Thirteen embryos were categorized as being either haploid, triploid, tetraploid or hexaploid. Nine embryos were classified as mosaic and 12 as being highly abnormal or chaotic. These preliminary results suggest that a large proportion of embryos that do not cleave further after freezing and thawing carry chromosomal aberrations. This finding supports our policy of not transferring cryopreserved embryos which do not cleave further 24 h following thawing.

Aneuploidy↗
PubMed2006

Stimulation of embryo hatching and implantation by prostacyclin and peroxisome proliferator-activated receptor delta activation: implication in IVF.

BACKGROUND: Successful IVF depends in part on quality embryos. Recent work suggests that prostaglandin I(2) (PGI(2) or prostacyclin) promotes the development of embryos in vitro and enhances their implantation potential. The mechanism underlying the effects of PGI(2) is unclear. It has been reported that peroxisome proliferator-activated receptor delta (PPARdelta) mediates the effects of PGI(2) at the implantation sites. METHODS: The expression of PPARdelta in the preimplantation embryos was examined by RT-PCR, western blot analysis and immunohistochemistry. Synthetic PPARdelta ligand (L-165041) and PPARdelta targeted (PPARdelta(-/-)) embryos were used to reveal the roles of PPARdelta in PGI(2)-stimulated and spontaneous embryo development. RESULTS: Preimplantation embryos express PPARdelta, which is essential for the enhancing effect of PGI(2) and the spontaneous progression of preimplantation embryos. Enhanced blastocyst hatching by PGI(2) (P < 0.05) was abrogated by PPARdelta deletion. Blastocyst formation and embryo hatching were impaired in PPARdelta(-/-) embryos. PPARdelta deletion significantly reduced embryo cell proliferation (P < 0.01); PPARdelta activation increased embryo cell proliferation (P < 0.05). PPARdelta activation enhanced the implantation of wild-type (WT) embryos (P < 0.05); PPARdelta deletion reduced embryo implantation (P < 0.05). CONCLUSIONS: PPARdelta is essential for spontaneous and PGI(2)-stimulated embryo development and blastocyst hatching. The implantation of cultured embryos is enhanced by PPARdelta activation. PPARdelta represents a novel therapeutic target to improve IVF outcome.

Acetates↗
PubMed1996

Insulin-like growth factor-binding proteins produced by Vero cells, human oviductal cells and human endometrial cells, and the role of insulin-like growth factor-binding protein-3 in mouse embryo co-culture systems.

Co-culturing embryos on helper cells can mimic the in-vivo environment, thereby enhancing embryo development in vitro. Insulin-like growth factors (IGF) and their binding proteins (IGFBP) also enhance embryo development. To investigate the kinds of IGFBP produced by various cell monolayers and the effects of IGFBP-3 on mouse embryo co-culture systems, 2-cell ICR mouse embryos were cultured in either human tubal fluid medium alone or in the presence of Vero cells, human oviductal cells or endometrial cells. The helper cells were analysed immunohistochemically to investigate the types of IGFBP produced by various cell monolayers. The concentrations of IGF-I and IGFBP-3 in media obtained from the culture of embryos alone, cells alone or cells plus embryos were determined by radioimmunoassays. On day 7, more blastocysts hatched in the co-culture groups (73% in the Vero cell group, 76% in the endometrial cell group and 74% in the oviductal cell group) than in the control group (43%) (P < 0.0001). The results of immunohistochemistry revealed that (i) all three cell groups produced a lot of IGFBP-1, -2 and -3, but only a little of IGFBP-4 and -5; and (ii) IGFBP-1, -2, and -3 were present in blastocysts in either the presence or absence of helper cells. The IGF-I secreted by cell monolayers or embryos was undetectable (detection limit 0.83 microg/l). The IGFBP-3 concentrations in media obtained from co-cultured embryos and cells were significantly higher than in media without embryos (median values in oviductal cell culture medium, 165 versus 127 microg/l, P = 0.04; median values in endometrial cell culture medium, 277.5 versus 183.5 microg/1, P = 0. 0002; median values in Vero cell culture medium, 219 versus 120 microg/l, P = 0.011). Although IGFBP-3 concentration in the medium that contained embryos alone was undetectable by radioimmunoassay (detection limit 1.1 microg/l), immunohistochemistry demonstrated the presence of IGFBP-3 in the embryos. Co-culture in systems in which there was an increased production of IGFBP-3 led to an improved development of mouse embryos. IGFBP can improve the binding of IGF to cell surface receptors of target tissue, and thus enhance the effect of limited IGF concentrations in promoting embryo development in a co-culture system. We conclude that Vero cells, human endometrial cells and oviductal cells produce IGFBP-1, -2, -3, -4 and -5. IGFBP-3 may play a role in embryotrophic potential by either regulating the action of IGF or directly enhancing embryo development.

Animals↗
PubMed1995

A retrospective analysis of the in-vitro development of 'spare' human in-vitro fertilization preimplantation embryos using 'in-house' prepared medium and 'Medi-Cult' commercial medium.

In-house prepared medium was used routinely in our in-vitro fertilization (IVF) facility prior to the introduction of the commercial 'Medi-Cult' products. A comparative study of the in-vitro development of embryos cultured in two [T6 and Earle's balanced salt solution (EBSS)] human-inactivated serum (HIS)-supplemented media from days 0 to 5 showed that 44.7% (46/103) of the embryos developed to the blastocyst stage in the T6 medium compared with 22.3% (23/103) in EBSS. Following the introduction of the commercial Medi-Cult IVF M2 medium, which is used routinely to culture fertilized eggs from days 0 to 2, new baseline data were required for the in-vitro development of 'spare' embryos from days 2 to 5. When Medi-Cult M3 medium was used, 35.6% (37/104) of the 'spare' day 2 embryos achieved the blastocyst stage. However, if morphologically similar (four normal nucleated blastomeres with no fragmentation) day 2 embryos were selected, an increase in the blastocyst rate to 50.0% (33/66) was achieved. This compared favourably with the 45.0% blastocyst rate (published in the Medi-Cult literature) for M2/M3 medium cultured human embryos. A small series of experiments with T6 + HIS medium and human serum albumin (HSA)-supplemented Ham's F-10, MCDB 302 and M3 media was undertaken to identify a suitable medium which could be used for the culture of M2 medium day 2 embryos. Results show that M2 medium cultured embryos placed in Ham's F-10 medium supplemented with 10 mg/ml HSA gave an acceptable 37.8% (14/45) blastocyst rate. Therefore, this medium could be substituted for M3 medium in an emergency. A total of 483 IVF embryos donated by patients, which were surplus to the therapeutic IVF programme, were used for these studies over a period of 30 months. Late day 2 IVF spare embryos were assigned an embryo score based on a high-power phase-contrast microscopic examination prior to being placed in culture. The embryo score provides an effective in-vitro parameter with which embryos from different patients can be compared. The cleavage and development of individual embryos were monitored on days 2 to 5. In some cases, the continuing normal development and viability of the day 5 cultured embryo were assessed by monitoring the hatching, attachment and outgrowth of the cavitated blastocyst.

Blastocyst↗
PubMed1991

Human preimplantation embryo cryopreservation: selected aspects.

This report describes the results of cryopreserving human preimplantation zygotes and cleaved embryos (2-4 cells) in our in-vitro fertilization programme. Cryopreserved zygotes and cleaved embryos resulted in similar post-thaw survival rates (74.8 versus 70.9%). Pregnancy rates per retrieval cycle (RC) and embryos transferred per pregnancy for frozen-thawed zygotes versus frozen-thawed cleaved embryos were 21.8 versus 11.5% (P less than 0.2) and 12.6 versus 17.5 (P less than 0.2), respectively. Pregnancy rates increased significantly for both fresh (P less than 0.0005) and frozen-thawed (P less than 0.05) embryos as the number of embryos replaced per transfer increased from one to three or more. Frozen-thawed embryos resulted in multiple implantation rates per transfer of 25 compared to 6.4% (P less than 0.1) for fresh embryos when two embryos were replaced. Pregnancy rates were reduced for fresh (P less than 0.05) and frozen-thawed (P less than 0.1) embryos obtained from patient retrieval cycle numbers greater than 3. The method of follicular stimulation during the retrieval cycle did not affect frozen-thawed embryo survival rates. There was no difference in pregnancy rates from frozen-thawed embryos replaced during natural or clomiphene citrate transfer cycles. Patients with cryopreserved embryos had cumulative pregnancy rates of 37.1% (66/178) compared to 23.5% (110/468) (P less than 0.01) for patients with no embryos cryopreserved; cryopreservation of preimplantation embryos is a reliable therapeutic procedure that enhances achievement of pregnancy through in-vitro fertilization.

Blastocyst↗
PubMed1997

Taurine acts as an osmolyte in human and mouse oocytes and embryos.

Taurine transport in mouse embryos has been shown to be osmotically regulated. We studied release of taurine from mouse and human oocytes and embryos when exposed to conditions that created osmotic imbalances, either by incubation in anisosmotic media or by inhibition of Na(+)-K(+)-ATPase with ouabain. Furthermore, we studied the effect of cleavage in mouse embryos on release of taurine. When human oocytes that remained unfertilized after in vitro fertilization, human embryos (2- to 8-cell), and mouse 2-cell embryos were loaded with [3H]taurine and subsequently incubated for 4 h in hyposmotic media (200 and 240 mOsm/kg), they showed significantly lower radioactivity as compared to those incubated in media of 280, 320, and 360 mOsm/kg and higher radioactivity of the incubation media. Incubation with 1.5 mM ouabain resulted in decreased radioactivity of mouse embryos and increased radioactivity of incubation medium. When mouse 2-cell embryos were cultured for 24 h after loading with [3H]taurine, radioactivity of embryos that cleaved to the 4-cell stage was significantly lower than that of uncleaved embryos. This finding is in accordance with the theory that cell division induces cell volume-regulatory mechanisms. In contrast, when 1-cell embryos were cultured for 24 h, radioactivity of embryos developing to the 2-cell stage was significantly higher than that of uncleaved embryos. These data support the theory that taurine is released by embryos when they have to adjust their cell volume because of either extracellularly induced or intracellularly occurring osmotic imbalances. When culture is performed without taurine, the resultant taurine depletion of embryos may be disadvantageous, either because the embryo has to rely more on its inorganic osmolytes for volume regulation or because taurine can no longer provide its other protective functions.

Animals↗
PubMed1993

Neural crest ablation versus sham surgical effects in a chick embryo model of defective cardiovascular development.

In chick embryos, ablation of premigratory neural crest destined for the third, fourth, and sixth pharyngeal arches results in persistent truncus arteriosus and interrupted aortic arch. Studies of cardiogenesis in these embryos have shown decreased ejection fraction and ventricular dilation with normal cardiac output. The experimental embryos that survive to d 11 of incubation have a higher cardiac output than nonsurvivors at the same earlier stage of development. We hypothesize that this survival is due to a surgically induced decrease in vascular resistance of the embryonic and vitelline vessels. Embryos from 15 opened eggs, eight sham-operated embryos, and 13 neural crest-ablated embryos were examined at stage 18. The sham-operated embryos were treated identically with the experimental embryos except that neural crest was not ablated. Ejection fraction and cardiac output were determined by cinephotography. Mean dorsal aortic and diastolic ventricular (approximates mean atrial) pressures were determined by a servo-null pressure technique and used to calculate vascular resistance. The experimental embryos had a significantly decreased ejection fraction in comparison with either sham-operated embryos or embryos from eggs that remained unopened until just before cinephotography. The experimental and sham-operated embryos had a higher cardiac output than embryos from unopened eggs. The calculated vascular resistance was also significantly lower in both the experimental and sham-operated embryos than in the embryos from unopened eggs. Neural crest ablation appears to cause a decreased ejection fraction. Sham surgery is associated with a higher cardiac output and lower vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2004

Effects of delipidation and oxygen concentration on in vitro development of porcine embryos.

The effects of delipidation and the oxygen (O(2)) concentration in the atmosphere during culture on in vitro development and H(2)O(2) content were investigated in porcine in vivo fertilized embryos and embryos after in vitro maturation and in vitro fertilization (IVM/IVF embryos). There was no significant difference in the developmental rates to the blastocyst stage between the intact and delipidated IVM/IVF embryos. However, the mean number of cells in blastocysts derived from delipidated IVM/IVF embryos (19.8 +/- 0.8 cells) was significantly smaller than that from intact embryos (24.2 +/- 1.2 cells). Although there were no significant differences in the developmental rates to the blastocyst stage of intact and delipidated IVM/IVF embryos between the cultures under 5% O(2) and 20% O(2), the developmental rate of intact IVM/IVF embryos cultured under 5% O(2) (27.1%) was significantly higher than that of the delipidated embryos cultured under 20% O(2) (19.3%). On the other hand, there was no difference in the developmental rate to the blastocyst stage between in vivo fertilized embryos cultured under 5% O(2) and 20% O(2). Hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), is thought to cause damage to embryos. The H(2)O(2) content per embryo derived from oocytes cultured under 5% O(2) (in vivo fertilized, 58.0 +/- 2.5 pixels; IVM/IVF, 79.6 +/- 3.2 pixels) was significantly lower than that (in vivo fertilized, 100.2 +/- 3.8 pixels; IVM/IVF, 103.9 +/- 3.2 pixels) under 20% O(2). Furthermore, the level of H(2)O(2) in delipidated IVM/IVF embryos (94.7 +/- 3.9 pixels) was significantly lower than that in intact embryos (103.9 +/- 3.2 pixels) cultured under 20% O(2). The present results indicate that the delipidation of porcine IVM/IVF embryos and reduction of the O(2) concentration decreased the H(2)O(2) level rather than the in vitro developmental rate to the blastocyst stage.

Animals↗
PubMed2001

The transfer of fresh and frozen embryos in an elite swamp buffalo herd.

To investigate the development of fresh and frozen swamp buffalo embryos after transfer to synchronized recipients, 14 fresh embryos and 28 frozen embryos, collected from Thai swamp buffalo cows of an elite herd at the Surin Breeding Center, were transferred nonsurgically to 31 synchronized recipients buffalo cows. One fresh embryo was transferred to each of 14 recipients. Twenty eight frozen embryos were transferred to 17 recipients of which 7 cows received I embryo, 9 cows received 2 embryos and 1 cow received 3 embryos. Pregnancy was diagnosed by real time B-mode ultrasonography one month after transfer and confirmed by rectal palpation one and two months later. The pregnant cows were kept under observation until calving. The results of fresh embryo transfer showed that 5/14 (35.7%) were pregnant after 30 days, 4/14 (28.6%) remained pregnant until the 3rd month and 2/14 (14.3%) calved. With the frozen embryos, only one cow which received three embryos became pregnant and remained so for 3 months although the embryo did not survive to full term. The overall pregnancy rate using frozen embryos was 5.9% (1/17). The study demonstrated the possibility of performing embryo transfer in elite buffalo herds for genetic improvement, however the use of frozen embryos needed further investigation.

Animals↗
PubMed1993

Cytogenetic analysis of day-7 bovine embryos by cytophotometric DNA measurements.

A Feulgen staining procedure that stains the DNA of individual fixed nuclei stoichiometrically was used to analyse cytophotometrically the incidence of total ploidy and mixoploidy in 28 day-7 bovine embryos that had been fixed after collection ('non-cultured' embryos). The influence of culture on the incidence of chromosome abnormalities was further studied in another group of 24 embryos ('cultured' embryos) by culturing them for 24 h in Whittingham's medium. Of the total 52 embryos studied, two appeared to be entirely abnormal: one embryo was completely haploid, whereas the other embryo was completely triploid. Individual hyperdiploid nuclei and hypodiploid nuclei were frequently observed in the otherwise diploid embryos. As haploid polar bodies can still be present in morulae and blastocysts (to a maximum of three), only embryos with more than three hypodiploid nuclei were considered as abnormal. Of the 'non-cultured' embryos, 33.3% had one or more hyperdiploid nuclei, whereas 51.9% had more than three hypodiploid nuclei. In this latter group, 35.7% of the embryos also had hyperdiploid nuclei. The results also showed that day-7 bovine embryos that are completely haploid, completely triploid or mixoploid cannot be detected only by examining their morphology. It is concluded that the incidence of, especially, mixoploidy in embryos can be better studied by measuring the DNA content of the individual nuclei of an embryo rather than by analysing chromosomes, as in the latter method only dividing cells can be analysed. The presence of hyperdiploid and hypodiploid nuclei may indicate the frequent occurrence of mitotic segregation failures during mitosis in bovine embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system.

Oxygen consumption is a useful parameter for evaluating embryo quality, since it provides a valuable indication of overall metabolic activity. Over the years, several approaches have been used to measure the respiration rates of individual embryos, but a convincing method has not yet been reported. In this study, we introduce and have validated a novel high resolution microsensor technology to determine the respiration rates of individual embryos at different developmental stages. We have employed this technology to investigate the correlation between respiration rate and embryo morphology, diameter and sex. Following morphological evaluation, individual respiration rates of day 3 (n = 18) and day 7 (n = 60) bovine in vitro-produced embryos were determined. Of the measured embryos, 64 were lysed for sex diagnosis by PCR. Average respiration rates of day 7 embryos (1.30 +/- 0.064 nl/h) were 3.4-fold higher than day 3 embryos (0.38 +/- 0.011 nl/h). On day 7, the average respiration rate of quality 1 blastocysts was significantly higher than the respiration rates of the lower qualities. For both day 3 and day 7 embryos, respiration rates were directly influenced by embryo diameter but did not differ between sexes. These results have demonstrated that the novel microsensor technology can be used to accurately and rapidly (8 min) measure the respiration rates of individual embryos at different developmental stages. Respiration rates were only in partial agreement with embryo morphology, suggesting a slight discrepancy between these two methods in assessing embryo quality. It is likely that a combined assessment of embryo respiration and morphology would improve embryo classification and subsequent selection.

Animals↗
PubMed1989

A diabetic-like condition of turkey embryos maintained in shell-less culture.

Serum insulin concentration and pancreatic insulin content were determined for turkey embryos incubated in ovo and in long-term shell-less culture (ex ovo). Insulin was undetectable (less than 10 pg) in serum from 87% of the ex ovo embryos compared with their in ovo counterparts. This was evident at all incubation ages, although insulin was detectable in more of the ex ovo embryos on Day 24. Insulin increased in the embryos incubated in ovo from 122 (Day 15) to levels exceeding 2000 pg/ml at hatching. Total pancreatic insulin content was greater in the cultured embryos on Days 15, 17, and 22 compared with their in ovo counterparts. Serum glucose was significantly greater (P less than 0.05) in the ex ovo embryos at all ages. In response to an infusion of L-arginine, serum insulin increased from 566 to 1256 pg/ml in the in ovo embryos, whereas no change was evident in the ex ovo embryos (233 vs 257 pg/ml). When embryos incubated in ovo were injected with insulin, a significant (P less than 0.05) reduction of serum glucose was observed at 60 min after injection. Serum glucose concentrations remained elevated in the embryos incubated ex ovo despite the insulin injection. Liver glucose 6-phosphatase activity, assessed on Days 15 and 22 of incubation, was found to be significantly (P less than 0.05) lower in the ex ovo embryos. Turkey embryos incubated in shell-less culture exhibited chronic hyperglycemia in concert with extremely low circulating levels of insulin. The pancreatic beta cells of these embryos were not responsive to arginine or elevated glucose. Taken together these findings suggest the occurrence of a diabetic-like condition in the ex ovo embryos. This defect in insulin secretion may, in part, be responsible for some of the developmental abnormalities characteristic of the turkey embryo cultured ex ovo.

Animals↗
PubMed1992

Postcranial morphological features of homozygous tetraploid mouse embryos.

The postcranial morphological features of 22 homozygous tetraploid mouse embryos were studied. The latter were produced by electrofusion of blastomeres at the 2-cell stage in vitro, and were transferred to appropriate recipients that were autopsied on d 12-16 of gestation. Seven embryos were isolated on d 15 or d 16 of gestation and were developmentally equivalent to control diploid embryos of 13-14.5 d p.c. In each of these embryos, their sex could be determined from the histological analysis of their gonads. A further 15 embryos were isolated on d 12-14 of gestation and most were developmentally equivalent to controls of between 11-11.5 and 12-12.5 d p.c. The sex of these embryos could not be determined from the analysis of their gonads, as these were all at the 'indifferent' stage. Twelve of the 22 embryos had a normal postcranial axial morphology, while 10 had an abnormal postcranial axial morphology associated with an enormous omphalocele which contained most of the abdominal viscera and often, particularly in the more advanced specimens, the heart as well. In each of the 7 developmentally most advanced embryos studied, a major congenital abnormality involving at least one organ system was present, but none of these was believed to be life-threatening. The most interesting finding was that 3 of these embryos had abnormalities of the aortic arch arterial system; in 2 embryos the arch of the aorta was retro-oesophageal and retrotracheal, while a 3rd embryo had transposition of the great vessels associated with a right-sided descending aorta. In a 4th embryo, an aneurysmal dilatation of the posterior cardinal vein was present, and this was associated with the anomalous persistence of the left subcardinal/supracardinal venous system. These abnormalities represent a degree of mirror imaging of the normal vascular arrangement. Analysis of the gonads revealed that all of the embryos with vascular abnormalities in this group were male (XXYY), and no vascular abnormalities were present in the females (XXXX). The histological features of the viscera were normal. In 5 of the 15 developmentally less advanced embryos, the distal part of the bulbus cordis region of the heart was displaced medially, and consequently the outflow tract was located on the left part of the pericardial cavity rather than on the right where it is normally found. This may represent an early stage in the differentiation of the aortic arch arterial vascular anomalies indicated above. In 2 additional embryos, abnormally located (i.e. retro-oesophageal) innominate vessels were present.(ABSTRACT TRUNCATED AT 400 WORDS)

Abdomen↗
PubMed2005

Developmental potential of fully intact and partially damaged cryopreserved embryos after laser-assisted removal of necrotic blastomeres and post-thaw culture selection.

OBJECTIVE: Partially damaged frozen and thawed embryos are currently considered to have a lower viability than intact ones. This study was undertaken to compare the performance of intact frozen and thawed embryos with that of partially damaged embryos after removal of the necrotic blastomeres. DESIGN: Observational clinical series. SETTING: Private hospital. PATIENT(S): Three hundred twenty-six infertile couples undergoing frozen embryo transfer. INTERVENTION(S): Removal of necrotic blastomeres from frozen-thawed human embryos. MAIN OUTCOME MEASURE(S): Pregnancy and implantations rates. RESULT(S): Outcomes of frozen embryo transfer cycles in which all embryos were fully intact (group 1) were compared with those in which all embryos have lost 1-2 blastomeres (group 2) or 3-4 blastomeres (group 3). Laser-assisted hatching was performed in all embryos, and necrotic blastomeres were removed from partially damaged embryos on this occasion. Only embryos that resumed mitotic activity after thawing were transferred. Comparable clinical pregnancy rates (PR) (38.7%, 39.6%, and 29.4%), delivery rates (34.4%, 34.0%, and 29.4%), and implantation rates (21.6%, 21.4%, and 17.2%) were obtained in groups 1, 2, and 3, respectively. CONCLUSION(S): The developmental potential of partially damaged frozen and thawed embryos can be equivalent to fully survived embryos if the necrotic blastomeres are removed from the partially damaged embryos and only those of them that show post-thaw cleavage are selected for transfer.

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