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PubMed1997

Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates.

We investigated whether further in-vitro culture of human multicellular embryos that survive cryopreservation can select the viable embryos for transfer. Embryos for cryopreservation were supernumerary multicellular embryos obtained after in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) treatments, with <20% of their volume filled with anucleate fragments. These had been cryopreserved using a slow-freezing and slow-thawing protocol with 1.5 M dimethylsulphoxide as the cryoprotectant. From the start of our cryopreservation programme until September 12, 1994, the thawing strategy was to thaw frozen embryos up to the exact number needed for transfer. Embryos for transfer were selected on the basis of their morphological appearance and embryo transfer to the patient was done on the day of thawing. From September 12, 1994 onwards we used a more selective thawing strategy where a cohort of up to a maximum of 12 frozen embryos per patient is thawed from which embryos of the best morphological quality, and which are furthest advanced in terms of cleavage after a 24 h in-vitro culture period in Menezo B2 medium, are selected. We took delivery rates, embryo implantation rates and birth rates into account to see if there is any difference between the following three types of transfers used: 187 transfers exclusively of embryos having continued to cleave after thawing, 107 mixed transfers of embryos with and without further cleavage and 53 transfers exclusively of embryos with no further cleavage. The overall outcome in terms of delivery rate and embryo implantation and birth rates were not different between the new and the earlier thawing policies (6.6, 5.2 and 3.6% versus 6.0, 4.1 and 2.7% respectively). Only when a distinction was made between transfers on the basis of the presence of embryos with further cleavage, did the advantage of selection on the basis of cleavage capacity become evident. Significantly higher delivery and embryo implantation and birth rates (11.2, 7.7 and 6.5% respectively) were recorded with transfers exclusively of embryos with further cleavage versus mixed transfers of embryos with and without further cleavage (1.9, 2.9 and 0.6% respectively). Fifty-three transfers exclusively of embryos with no further cleavage did not lead to any delivery. Our results demonstrate that selection of human multicellular embryos which survive cryopreservation and continue to cleave in vitro can significantly improve the delivery rate per transfer and the implantation rate per transferred embryo.

Abortion, Spontaneous↗
PubMed2000

Application of vitrification to human embryo freezing.

To solve the problem of multiple pregnancies during the in vitro fertilization (IVF) and embryo transfer procedure, excess embryos must be cryopreserved for embryo transfer in future. We applied the vitrification method to cryopreservation of human embryos. A total of 31 frozen-thawed embryo transfer cycles were analyzed at the Yamagata University Hospital, Yamagata, Japan. The patients were introduced to IVF treatment and had an excess of valuable embryos to be frozen after the transfer of three fresh embryos that did not result in establishing a pregnancy. Excess human 8- to 16-cell stage embryos were exposed to vitrification solution and then frozen in liquid nitrogen. The cryoprotectant was removed by washing the embryos in media containing different concentrations of cryoprotectant. Three days after LH surge and/or 2 days after ultrasonographic ovulation the embryos were transferred. The rate of poor quality embryos significantly increased and the rate of good quality embryos decreased after thawing the embryos frozen by the vitrification method. In menstrual cycles with good quality embryo transfer, a higher rate of pregnancies was established than in the cycles in which fair or poor quality embryos were the highest grade of embryos transferred into the uterus. In total, 5 pregnancies were established from 31 embryo tansfers; 4 pregnancies were in cycles associated with the transfer of good quality embryos, and 1 pregnancy was in a cycle in which the highest grade of embryo was fair. When compared with slow embryo freezing methods, vitrification has marked advantages for clinical application in terms of cost and time. Vitrification will be an alternative method for embryo freezing.

Cryopreservation↗
PubMed2005

[The protective effect of stem cell factor (SCF) on in vitro development of preimplantation mouse embryos].

INTRODUCTION: The protective potential of stem cell factor (SCF) on preimplantation embryo development in vitro has not been well investigated. MATERIAL AND METHODS: The aim of the study was to determine the effect of SCF on preimplantation development in vitro of (1) frozen-thawed mouse embryos; (2) mouse embryos exposed to Fas-L. Female mice were stimulated intraperitoneally with 5 IU of pregnant mare serum gonadotropin. An injection of 5 IU of equine chorionic gonadotropin (eCG) was given 48 hours later and the animals were mated. 40 hours after eCG injection, embryos at the stage of 2 blastomeres were flushed out ofthe fallopian tubes. Experiment 1: The two-cell embryos were randomly divided into 3 groups: (A) cultured in standard IVF medium (control); (B) frozen-thawed and cultured without SCF; (C) frozen-thawed and cultured in medium with SCF (100 ng/mL). Experiment 2: two-blastomere embryos were divided into 3 groups: (A) cultured in standard IVF medium (control); (B) cultured in standard IVF medium with Fas-L (500 ng/mL); (C) cultured in standard IVF medium with Fas ligand (500 ng/mL) and SCF (100 ng/mL). Culture was performed for 96 h in groups of 10 embryos in 50 microl droplets under mineral oil at 37 degrees C and 5% CO2. In both experiments the dynamics of embryo development, outgrown blastocyst rates, total blastocyst cell numbers (TBC) and blastocyst cell viability were analyzed. In experiment 2, TUNEL reaction was additionally performed to detect apoptosis of the blastocyst cells. Statistics was based on ANOVA and log-linear analysis with significance taken at p < 0.05. RESULTS: In experiment 1 significantly more hatched and outgrown blastocysts were noted in the control group when compared to embryos exposed or not exposed to SCF after thawing. Control embryos had better cell viability and higher TBC than in the remaining two groups. Blastocyst rates, TBC and cell viability in the frozen-thawed embryos cultured with and without SCF did not differ significantly. Log-linear analysis revealed that the dynamics of embryo growth was highest in control group, followed by thawed embryos cultured with SCF and finally by thawed embryos cultured without SCF. In experiment 2, Fas-L decreased embryo growth, but the effect was not apoptosis-dependent as the TUNEL reaction in embryos exposed to Fas-L was only sporadically positive. The growth of embryos exposed to Fas-L was comparable to that observed in media enriched with both Fas-L and SCF. However, blastocysts grown in Fas-L enriched media had significantly smaller cell numbers (TBC) than when cultured in both Fas-L and SCF. CONCLUSIONS: SCF may improve the dynamics of post-thaw embryo development in vitro but not embryo quality. Fas-L deteriorates preimplantation embryo development in vitro. SCF seems to improve the quality of embryos exposed to Fas-L.

Animals↗
PubMed2002

Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos.

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.

Actin Cytoskeleton↗
PubMed2006

Improved felid embryo development by group culture is maintained with heterospecific companions.

Domestic cat embryos of excellent quality appear to improve development of conspecific embryos when cultured together, providing an avenue for improving development of embryos from valuable species or individuals. To have relevance to rare species, it would be useful to understand if this advantage could be conferred by heterospecific companions because there usually are severely limited numbers of conspecific embryos available from wildlife donors. In the first study, we incubated single test cat embryos alone (controls) or with 10 cat embryos or 10 or 20 mouse embryos under similar regimented conditions (each group shared 20 microl medium). In the second study, single test cat embryos were cultured alone, with 10 conspecific or 20 mouse embryos or 10 cattle embryos (each group shared 20 microl medium). Single test embryos in all treatment groups achieved similar (P>0.05) stages of compaction and blastocyst development. In the first study, only the test embryos incubated with 10 cat or 20 mouse companion embryos achieved blastocyst expansion. The average total cell number within test embryos incubated with 10 cat or 20 mouse companions was greater (P<0.05) than controls or those placed with 10 mouse embryos. In the second study, test embryos in all groups achieved blastocyst expansion and had more (P<0.05) total cells per embryo than the solitary controls. Inner cell mass to trophoblast cell ratio did not differ among treatments in either study. Thus, companion mouse and cattle embryos selected for excellent quality confer a benefit to singleton cat embryos, although the number of companions necessary to grant an advantage may be species dependent. If this phenomenon can be extrapolated across species, this may be an avenue for 'common animal embryos' to improve developmental potential of embryos from rare, unrelated taxa.

Animals↗
PubMed2005

Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses.

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19-20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94-96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p < 0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi- and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p < 0.001) and lower total cell number (p < 0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

Animals↗
PubMed2004

Cryopreservation and sexing of in vivo- and in vitro-produced bovine embryos for their practical use.

My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GL-Tip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GL-Tip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex pre-determination.

Animals↗
PubMed2003

Implantation rates after two, three, or five days of embryo culture.

Extended embryo culture together with amelioration of embryo selection methods and embryo culture conditions have allowed a substantial increase on both pregnancy and implantation rates. However, uterine embryo transfers are still performed after 2 to 6 days of egg retrieval. In this paper, we show the results of two studies, one prospective study comparing IVF outcome of day 2 and day 3 embryo transfers, and a retrospective study looking at blastocyst transfers versus day 3 embryo transfers in our egg donation program. Also, we test the predictive value of the presence of three or more seven cell-stage embryos on day 3 of development on blastocyst formation and pregnancy rates. No significant differences were found between day 2 and day 3 embryo transfers in terms of pregnancy, ongoing pregnancy, and implantation rates, as well as in multiple and in high order pregnancy. In general, day 6 embryo transfers resulted in significantly higher ongoing pregnancy and implantation rates compared with day 3 embryo transfers (41.1 per cent and 23.6 per cent versus 50.1 per cent and 38.1 per cent, respectively). No differences were found in terms of multiple gestations despite transferring significantly more embryos on day 3 compared with day 6 transfers. When less than three 7-cell embryos were present in the embryo cohort, day 6 embryo transfers did not improve the rates of ongoing pregnancy with regards to day 3 embryo transfer, although significant high implantation rates were obtained on the group of blastocyst transfer. The presence of three or more 7 cell-stage embryos improved significantly both ongoing pregnancy and rates on blastocyst transfers compared to day 3 embryo transfers (65.6 per cent versus 50.6 per cent and 37.4 per cent vs 24.7 per cent, respectively). In conclusion, at least in egg donation, day 3 embryo transfers do not improve either pregnancy or implantation rates when compared to day 2 transfers. Generally speaking blastocyst transfers give significantly higher chance of pregnancy and implantation rates per cycle and per transfer than early cleavage stage transfers. However, the absence of a good embryo cohort, that is having less than three 7 cell-stage embryos on day 3, blastocyst transfers will improve implantation rates but not ongoing pregnancy rates.

Adult↗
PubMed1992

Chromosome status of untransferred (spare) embryos and probability of pregnancy after in-vitro fertilisation.

Many spontaneous abortions are associated with chromosomal abnormality of the fetus. In in-vitro fertilisation (IVF) the chromosome status of untransferred ("spare") embryos and subsequent fate (pregnancy or not) of the transferred sibling embryos might be related. Since the spare and transferred embryos of a patient's cycle genetically are full siblings, the inherited chromosomal abnormalities in spare embryos have a 50% probability of also appearing in transferred embryos. We have tested whether chromosome analysis of spare embryos has predictive power for transferred embryos. 48 couples with a total of 437 embryos were selected because their spare embryos (1-4 per couple; 76 total) were successfully analysed for chromosome status. 16 patients became pregnant. These women produced a higher proportion of chromosomally normal spare embryos (9/24; 37.5%) than those who did not achieve pregnancy (1/52; 1.9%). The proportion of patients who had only normal embryos was significantly higher (p = 0.012) in the pregnant group than in the non-pregnant group, and the proportion of patients who had only abnormal embryos was significantly higher (p = 0.001) in the non-pregnant group. Patients with preclinical and clinical pregnancy losses had only chromosomally abnormal spare embryos; by contrast, 50% of spare embryos from patients with ectopic pregnancies were normal. The proportion of spare embryos that were normal (13%, 10/76), was similar to the livebirth rate of 11% per transferred embryo (19 infants from 171 transferred embryos). These results suggest that chromosome analysis of spare embryos may have predictive value for their transferred sibling embryos. We conclude that improving detection of chromosomally normal embryos for transfer should improve the success rate in IVF.

Chromosome Aberrations↗
PubMed2004

Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro.

The objective of this study was to evaluate the in vitro development of frozen-thawed bovine embryos held at room temperature or refrigerated for 2, 6 or 12 h prior to freezing. After recovery, embryos were randomly assigned to be placed in holding media for 2 h (n=131), 6 h (n=136) or 12h (n=133) prior to freezing. Approximately one-half of the embryos were refrigerated (5 degrees C; n=203) while the remaining half were held at room temperature (22 degrees C; n = 197) until freezing. Embryos were frozen in 10% ethylene glycol and stored in liquid nitrogen. After thawing, embryos were cultured for 72 h in Ham's F-10 media supplemented with 4% fetal bovine serum. Embryos were evaluated for quality and stage of development prior to freezing and after culture. At the end of culture, it was determined if each embryo had developed beyond the stage recorded prior to freezing and if the embryo had hatched from the zona pellucida. The percentage of embryos that developed during culture was greater (P < 0.001) for Grade 1 (81%) than for either Grade 2 (65%) or Grade 3 (48%) embryos. Likewise, a greater proportion (P < 0.001) of Grade 1 embryos developed to hatched blastocysts (60%) than either Grade 2 (40%) or Grade 3 (24%) embryos. The holding temperature from collection to freezing did not influence embryo development, regardless of the interval from embryo collection to freezing. The proportion of embryos that developed to expanded blastocysts and hatched was greater (P < 0.005) for embryos held 2 h prior to freezing (64%) than for embryos held for 12 h (33%). Hatching rate of embryos held 6 h prior to freezing (54%) tended (P < 0.08) to be lower than the hatching percentage for embryos held for 2 h. Thus, post-thaw embryonic development was impaired the longer embryos were held prior to freezing and temperature during the interval from collection to freezing did not affect post-thaw development.

Animals↗
PubMed2000

A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos.

The impact of cryopreservation on the implantation potential of early cleavage stage (day 2) embryos was assessed by analysing the outcome from > 5000 thawed embryos in relation to the outcome from a similar number of fresh embryos. Analysis of procedures in which all transferred embryos fulfilled equivalent defined criteria revealed no significant difference in the implantation rates (fetal hearts/100 embryos transferred) of fresh 4-cell embryos (16.6%) and fully intact thawed 4-cell embryos (16.9%). Although 2-cell embryos implanted at significantly lower rates, there was again no significant difference between fresh (6.5%) and fully intact thawed (7.2%) embryos. Similar analysis of all embryos (irrespective of cell number on day 2) demonstrated that the implantation potential of partially intact thawed embryos was related to the extent of blastomere loss with the implantation rate of embryos with 50% cell survival (5.4%) being approximately half the rate of fully intact embryos (11.3%). Combining the values obtained from 'pure' data for the implantation rates of embryos with defined levels of survival with their relative prevalence in the total population of thawed embryos gave a predicted number of implantations (441) which was similar to the observed outcome (463). This number was approximately 30% less than the number expected had the same embryos been transferred fresh (635). The results suggest that intact thawed embryos have the same implantation potential as equivalent fresh embryos and that the impact of cryopreservation is limited to blastomere loss which is directly related to loss of implantation potential. The observed frequency of blastomere loss results in a reduction of approximately 30% in the implantation potential of a population of embryos following cryopreservation.

Blastomeres↗
PubMed1995

Prospective randomized study on the cryopreservation of human embryos with dimethylsulfoxide or 1,2-propanediol protocols.

OBJECTIVE: To investigate the optimal protocol for cryopreservation of human embryos obtained from IVF. DESIGN: Prospective randomized study. SETTING: Consenting patients in an academic research environment. PATIENTS: Couples undergoing IVF. INTERVENTIONS: A cohort of 2,220 supernumerary multicellular embryos were obtained from 488 patients who were randomized over slow freezing protocols with dimethylsulfoxide (DMSO, 819 embryos), 1,2-propanediol (699 embryos) or a mixture of DMSO and 1,2-propanediol (702 embryos). A total of 725 embryos have been thawed (DMSO, 232 embryos; 1,2-propanediol, 250 embryos and DMSO and 1,2-propanediol, 243 embryos) for transfer in natural ovarian cycles. MAIN OUTCOME MEASURES: Embryo survival rate, embryo implantation rate, clinical pregnancy rate (PR), delivery rate, live-birth rate. RESULTS: The embryo survival rate was significantly higher with the DMSO protocol (52.6%) than with the 1,2-propanediol (32.0%) or the DMSO and 1,2-propanediol protocols (34.9%). The clinical PR per thawing cycle was significantly higher in the DMSO protocol (17.2%) than in the 1,2-propanediol protocol (3.9%). The clinical implantation rate per embryo thawed was significantly different between a DMSO-frozen embryo (4.7%) and a 1,2-propanediol-frozen embryo (1.2%). A DMSO and 1,2-propanediol-frozen embryo had a 3.7% chance of of implantation. The delivery rate per thawing cycle was significantly higher in the DMSO protocol (12.5%) than in the 1,2-propanediol protocol (2.6%). The live-birth rates per embryo thawed were 3.5%, 0.8%, and 2.9% in the DMSO, 1,2-propanediol, and DMSO and 1,2-propanediol groups, respectively. CONCLUSION: Supernumerary multicellular embryos as presented in daily clinical IVF practice have the highest chance of survival and of implantation after cryopreservation when DMSO has been used.

Birth Rate↗
PubMed1986

Cryobiology: preservation of mammalian embryos.

The preservation of mammalian embryos has become a routine procedure. Thousands of live offspring have been produced from frozen-thawed embryos transferred into recipient foster mothers. Species whose embryos have been successfully preserved include mouse, rat, rabbit, sheep, goat, cattle, horse, antelope, baboon, and human. During the past few years, novel procedures have been introduced that permit embryos to be frozen and thawed rapidly, and to be transferred into recipients under field conditions almost immediately upon thawing. Thus, the transfer of frozen-thawed embryos of domestic animals is becoming almost as efficient as is artificial insemination using frozen-thawed semen. Because of both the inherent fundamental interest and the practical applications of embryo freezing, a substantial understanding of the mechanisms responsible for freezing damage of embryos has been achieved. To survive freezing, embryos must be exposed to protective compounds; to function after thawing, embryos must be washed free of these compounds. Based on fundamental physiology, efficient methods to accomplish such washing have been developed. Furthermore, to survive freezing, embryos must be cooled under conditions in which intracellular ice does not form. This can be accomplished either by pretreating the embryo or by cooling it in such a way as to cause it to dehydrate during freezing. Maximum survival of embryos appears to be achieved when intracellular water does not crystallize during cooling or during warming. As a result of the growing efficiency of embryo preservation, this method is being applied to a variety of practical situations. For example, large banks of frozen embryos of laboratory animals are being established to preserve valuable research resources. The freezing of cattle embryos is being used with increasing frequency as an adjunct to commercial embryo transfer. Preservation of endangered species by embryo preservation is beginning. And finally, the preservation of human embryos is finding application in the field of in vitro fertilization.

Animals↗
PubMed2000

The influence of supernumerary embryos on the clinical outcome of IVF cycles.

PURPOSE: To assess the influence of the presence of quality supernumerary embryos on the clinical outcome and risk of multiple conception in patients having their first in vitro fertilization (IVF) cycle. METHODS: Retrospective cohort study of 1448 women having their first IVF treatment cycle who received 4004 embryos where at least six embryos were available for transfer treated in an Assisted Conception Unit based in a large teaching hospital. RESULTS: The replacement of three rather than two embryos to women under 35 years who had good-quality supernumerary embryos resulted in a higher twin (12.5 vs. 11.9%) and triplet birth rates (2.1 vs. 0%), without significantly improving the clinical pregnancy (50.5 vs. 45.2%) or total live birth rates (38.9 vs. 35.7%). In the absence of quality spare embryos, these women who had three rather than two embryos replaced had a significantly higher clinical pregnancy rate (39.3 vs. 28.8%; P = 0.04), total live birth (32.7 vs. 19.4%; P = 0.02) and singleton birth rate per cycle (20.8 vs. 14.4%; P = 0.04), without significantly influencing the multiple birth rate. In women over 35 years, the replacement of three instead of two embryos in the presence or absence of quality supernumerary embryos led to a significant improvement in clinical outcome, without being associated with a concurrent increase in the multiple birth rate. Women in both age groups who had either two or three embryos replaced in the presence of quality supernumerary embryos had a notably better clinical outcome compared with their counterparts who had the same number of embryos replaced, but with no quality embryos to spare. CONCLUSIONS: The presence of good-quality supernumerary embryos can be used as a reference to determine the optimal number of embryos to transfer and as an indicator of the probability of success of an individual couple in a given cycle. Optimal pregnancy rates and simultaneous reduction of multiple gestation can be achieved with a flexible embryo replacement policy that is based on embryo quality, maternal age, and the presence or absence of surplus quality embryos.

Adult↗
PubMed2004

Impact of retained embryos on the outcome of assisted reproductive technologies.

OBJECTIVE: To assess the impact of embryo retention in the embryo transfer catheter followed by "immediate" retransfer on pregnancy outcome in women undergoing assisted reproduction. DESIGN: Retrospective analysis of embryo transfer following in vitro fertilization. SETTING: Assisted reproductive technology practice in a university in vitro fertilization program. PATIENT(S): In vitro fertilization charts for 1,812 embryo transfer cycles representing 1,139 patients between January 1997 and March 2002 were reviewed. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy rate, implantation rate, delivery rate. RESULT(S): Three embryo transfer cycles were excluded from analysis because of missing data, leaving 1,364 embryo transfers during oocyte recovery cycles and 445 embryo transfer cycles using thawed embryos. Seventy-one embryo transfers (3.9% of all transfers) were complicated by finding retained embryos after the initial embryo transfer-all retained embryos were immediately retransferred. There was no difference in the frequency of retained embryos during oocyte retrieval versus thawed embryo cycles. The pregnancy, implantation, and delivery rates per embryo transfer were not negatively affected by embryo(s) retained in the transfer catheter. Age, fresh versus frozen embryo, use of ultrasound during the procedure, or transferring physician did not influence pregnancy outcome. CONCLUSION(S): Immediate retransfer of embryos retained in the catheter following the initial transfer attempt did not have an adverse effect on pregnancy outcome.

Biomarkers↗
PubMed2005

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle.

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.

Animals↗
PubMed1998

Cellular composition and viability of demi- and quarter-embryos made from bisected bovine morulae and blastocysts produced in vitro.

The cellular composition and viability of intact, IVP embryos were compared with those of demi- and quarter-embryos produced by bisection of IVP morulae and blastocysts. Embryos were produced by established techniques from oocytes harvested from slaughterhouse ovaries. In Experiment 1, morulae at Day 6 or blastocysts at Day 7 were bisected on an inverted microscope using a microsurgical steel blade. Demi-embryos were then cultured without a zona pellucida until Day 8, when they were morphologically assessed for quality (viability). A higher proportion of demi-embryos made from blastocysts than from morulae were classified as viable (381/420, 91% vs 164/267, 61%; P < 0.001). In Experiment 2, only Day 7 blastocysts were bisected, and some of the resulting demi-embryos were bisected a second time 24 h later to produce quarter-embryos. The remaining demi-embryos, the quarter-embryos, and control intact embryos were cultured until Day 9, at which time they were assessed for quality and subjected to immunosurgery and differential staining to count inner cell mass (ICM) and trophectoderm cells. A higher proportion of demi-embryos than quarter-embryos was classified as viable (408/459, 89% vs 223/319, 70%, respectively; P < 0.001). Total cell numbers decreased with successive bisections, but the proportion of surviving cells found in the ICM was significantly (P < 0.05) higher in the best quality demi- and quarter-embryos (35 and 32%, respectively) than in the controls (22%). Transfer of all 12 quarter-embryos derived from 3 blastocysts, in pairs, into 6 recipient heifers resulted in 2 pregnancies, each with a single viable fetus at 90 d of gestation. The fetuses originated from 2 different blastocysts. The results suggest that bisection of intact IVP embryos into demi-embryos and bisection of those into quarter-embryos can increase the number of transferable embryos by as much as 178 and 235%, respectively.

Animals↗
PubMed1987

Transfer of bovine demi-embryos with and without the zona pellucida.

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.

Animals↗