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PubMed2000

Sexing of porcine embryo by in situ hybridization using chromosome Y- and 1-specific DNA probes.

This study was carried out to determine if a rapid, simultaneous detection system using chromosome Y- and 1-bearing boar spermatozoa was applicable for sexing embryos. Porcine embryos were recovered from gilts and sows 4 to 6 d after mating, and whole embryos or biopsy cells were mounted on a glass slide with a small amount of fixative (methanol: acetic acid: distilled water = 9:1:4). The samples were then stained by means of a fluorescence in situ hybridization (FISH) procedure developed specifically for the detection of Y-bearing spermatozoa. Hybridization was performed using digoxigenin (dig)-labeled chromosome Y- specific DNA, and biotin-labeled chromosome 1-specific DNA sequences were detected as a signal of FITC and Texas Red on nucleus visualized DAPI-stain. Proportions of whole embryos labeled with chromosome 1-probe were 17 and 97% at the 3 to 16 and > or = 32 cell stage, respectively. Of the 93 biopsied embryos analyzed by FISH, 85 embryos (91%) could be accurately classified as male or female. Of the 65 biopsied embryos, 60 embryos (92%) had a clear blastocoele and a inner cell mass after 48 h of culture in vitro, and these embryos were evaluated as available embryos. One out of 4 recipient gilts which received sexed embryos at transfer farrowed 12 piglets of the expected sex. The results of this study demonstrated that porcine embryos at the > or = 32 cell stage can be sexed within 2 h using the FISH method. Moreover further development of the FISH technique could make it an effective tool for the study of early porcine embryos and for the control of porcine sex.

Animals↗
PubMed2002

Early embryonic cells from in vivo-produced goat embryos transmit the caprine arthritis-encephalitis virus (CAEV).

The aim of this study was to investigate whether cells of early goat embryos isolated from in vivo-fertilized goats interact with the caprine arthritis-encephalitis virus (CAEV) in vitro and whether the embryonic zona pellucida (ZP) protects early embryo cells from CAEV infection. ZP-free and ZP-intact 8-16 cell embryos were inoculated for 2 h with CAEVat the 10(4) tissue culture infectious dose 50 (TCID50)/ml. Infected embryos were incubated for 72 h over feeder monolayer containing caprine oviduct epithelial cells (COECs) and CAEV indicator goat synovial membrane (GSM) cells. Noninoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. Six days postinoculation, infectious virus assay of the wash fluids of inoculated early goat embryos showed typical CAEV-induced cytopathic effects (CPE) on indicator GSM monolayers, with fluids of the first two washes only. The mixed cell monolayer (COEC + GSM) used as feeder cells for CAEV inoculated ZP-free embryos showed CPE. In contrast, none of the feeder monolayers, used for culture of CAEV inoculated ZP-intact embryos or the noninoculated controls, developed any CPE. CAEV exposure apparently did not interfere with development of ZP-free embryos in vitro during the 72 h study period when compared with untreated controls (34.6 and 36% blastocysts, respectively, P > 0.05). From these results one can conclude that the transmission of infectious molecularly cloned CAEV-pBSCA (plasmid binding site CAEV) by embryonic cells from in vivo-produced embryos at the 8-16 cell stages is possible with ZP-free embryos. The absence of interactions between ZP-intact embryos and CAEV in vitro suggests that the ZP is an efficient protective embryo barrier.

Animals↗
PubMed2002

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment.

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.

Animals↗
PubMed2002

Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure.

The study was designed to evaluate the efficiency of a modified (sealed) open pulled straw (mOPS) method for cryopreserving rabbit embryos by vitrification or rapid freezing. An additional objective was to determine whether the mOPS method could cause the vitrification of a cryoprotectant solution generally used in rapid freezing procedures. Two consecutive experiments of in vitro and in vivo viability were performed. In Experiment 1, the in vitro viability of rabbit embryos at the morula, compacted morula, early blastocyst and blastocyst stages was assessed after exposure to a mixture of 25% glycerol and 25% ethylene glycol (25GLY:25EG: vitrification solution) or 4.5 M (approximately 25% EG) ethylene glycol and 0.25 M sucrose (25EG:SUC: rapid freezing solution). Embryos were loaded into standard straws or mOPS and plunged directly into liquid nitrogen. The mOPS consisted of standard straws that were heat-pulled, leaving a wide opening for the cotton plug and a narrow one for loading embryos by capillarity. The embryos were aspirated into the mOPS in a column positioned between two columns of cryoprotectant solution separated by air bubbles. The mOPS were then sealed with polyvinyl-alcohol (PVA) sealing powder. The vitrification 25GLY:25EG solution became vitrified both in standard straws and mOPS, whereas the rapid freezing 25EG:SUC solution crystallized in standard straws, but vitrified in mOPS. The total number of embryos cryopreserved was 1695. Embryos cryopreserved after exposure to each solution in mOPS showed higher rates (88.2%) of survival immediately after thawing and removal of the cryoprotectant than those cryopreserved in 0.25 ml standard straws (78.8%; P < 0.0001). After culture, the developmental stage of the cryopreserved embryos significantly affected the rates of development to the expanded blastocyst stage. Regardless of the cryoprotectant used, lower rates of in vitro development were obtained when the embryos were cryopreserved at the morula stage, and higher rates achieved using embryos at blastocyst stages. Based on the results of Experiment 1, the second experiment was performed on blastocysts using the mOPS method. Experiment 2 was designed to evaluate the in vivo viability of cryopreserved rabbit blastocysts loaded into mOPS after exposure to 25GLY:25EG or 25EG:SUC. Embryos cryopreserved in mOPS and 25GLY:25EG solution gave rise to rates of live offspring (51.7%) not significantly different to those achieved using fresh embryos (58.5%). In conclusion, the modified (sealed) OPS method allows vitrification of the cryoprotectant solution at a lower concentration of cryoprotectants than that generally used in vitrification procedures. Rabbit blastocysts cryopreserved using a 25GLY:25EG solution in mOPS showed a similar rate of in vivo development after thawing to that shown by fresh embryos.

Animals↗
PubMed2003

Factors affecting the survival of sheep embryos after transfer within a MOET program.

Multiple ovulation and embryo transfer (MOET) has the potential to increase the rate of genetic improvement in sheep. However, better realization of this potential requires maximum survival rates of transferred embryos of high genetic merit after transfer into recipient ewes. These studies were therefore conducted to investigate the effect of both embryonic and recipient ewe factors on the survival rate of transferred embryos. Survival rate was similar after transfer of morula or blastocyst stage embryos, and these were higher (P<0.05) than for very early morulae and early morulae. Advanced embryos (Day 5 blastocyst) had an advantage (P<0.05) in survival rate over retarded embryos (Day 6 morula). Grades 1 and 2 embryos survived significantly (P<0.05) better than Grades 3 or 4 embryos. There was no difference in embryo survival rate following transfer to recipients with different numbers of corpora lutea. In general, age or parity of recipient ewes did not affect embryo survival rate, although a higher (P<0.05) embryo survival rate was observed for yearling recipients. Buserelin (GnRH agonist) treatment of recipient ewes 5 or 6 days after transfer of embryos (Day 12 of the cycle) did not improve embryo survival rate. These results confirm that both embryonic and recipient factors can play an important role in the success of a MOET program in sheep.

Age Factors↗
PubMed1998

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer.

Supplementation of synthetic oviduct fluid (SOF) medium plus amino acids and bovine serum albumin (BSA) with either fetal calf serum (FCS) or charcoal-treated FCS (CT-FCS) from Day 5 of development was investigated to determine if either in vitro or post-transfer development was altered. Development to the compact morula stage or beyond was similar for all 3 treatments. However, blastocyst development at Day 7 was accelerated when serum was added to the medium (21.6, 40.1 and 39.4% blastocysts from cleaved embryos for BSA, FCS and CT-FCS, respectively; P < 0.01), but cell number of the resulting embryos was unaffected. Furthermore, addition of CT-FCS decreased the between replicate variation in embryo development and produced more Grade 1 and 2 quality embryos (25.8%) than BSA supplementation (18.1%; P < 0.05). The transfer of Grade 1 and 2 embryos at Day 7 following culture resulted in similar pregnancy and embryo survival rates for the 3 treatments, with a tendency for lower embryo survival of embryos cultured in FCS (embryo survival at Day 50 = 37.7% vs 53.3% and 57.6% for FCS, BSA and CT-FCS, respectively; P = 0.1). Significant fetal loss from Day 50 to term occurred within all 3 treatments. There were no birth weight differences for calves amongst the 3 culture treatments; however, one of the sires produced calves that were significantly heavier than expected, suggesting a possible sire-by-embryo interaction. These results demonstrate that addition of FCS may promote blastocyst development; however, there was also a tendency for lower embryo survival. Thus charcoal treatment of FCS is recommended, because it decreases variability in embryo development between runs and results in embryo survival rates to term similar to that BSA-supplemented media.

Animals↗
PubMed1998

Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths.

In vitro and in vivo developmental competence of fresh and cryopreserved in vitro produced (IVP) bovine embryos was evaluated up to birth. Three experiments were done. The objective in the first experiment was to develop an optimal vitrification procedure for IVP bovine embryos by determining effects of exposure time (2, 5, 10, 20 min) and temperature (4, 22, 27 degrees C) in cryoprotective agents prior to vitrification on their post-thaw viability. The best combination was used in Experiments 2 and 3. In the second experiment, the importance of post-thaw morphologic selection on pregnancy rates was determined by transferring either selected or unselected single embryos. In the third experiment, pregnancy initiation, maintenance and calving results of vitrified embryos were compared with fresh and conventionally frozen embryos. Fetal losses, birth weights, gestation lengths and frequency of dystocia in the third experiment were monitored. The interaction of exposure time and temperature on both post-thaw re-expansion and hatching rates was significant (P < 0.01). Five minute exposure at 27 degrees C was optimal. In the second experiment, post-thaw selected vitrified embryos had higher pregnancy rates than unselected embryos (P < 0.05). In the third experiment, the pregnancy rate of vitrified embryos did not differ from that of fresh embryos (P > 0.05). However, pregnancy rate of conventionally frozen embryos was lower than that of fresh or vitrified embryos (P < 0.05). Of 92 calves born, 53 were male and 39 were female. Birth weights and dystocia scores of single-born calves did not differ between sexes (P > 0.05). Twin-born calves were lighter than single-born calves (P < 0.05). Overall, the data demonstrate that the transfer of vitrified IVP bovine embryos can result in healthy, apparently normal calves similar to those derived from transfer of fresh and conventionally frozen IVP bovine embryos.

Animals↗
PubMed1998

Developmental kinetics of the first cell cycles of bovine in vitro produced embryos in relation to their in vitro viability and sex.

The development of bovine IVP-embryos was observed in a time-lapse culture system to determine cell cycle lengths of 1) embryos that developed into compact morulae (CM) or blastocysts (BL) within 174 h after insemination (viable), 2) embryos that arrested during earlier stages (nonviable) and 3) male and female embryos. In 4 replicates, inseminated oocytes were cultured on a microscope stage in 3 to 4 groups on a granulosa cell monolayer in supplemented TCM 199. Images were sequentially recorded and stored at 30-min intervals. All embryos that could be identified throughout the culture period were included (n = 392), and the times of cleavage events noted. After culture, 100 CM or BL were randomly selected for sexing by PCR. BL developed equally well in the time-lapse and control culture systems (36 vs 38%). The respective lengths of the first 4 cell cycles of viable embryos were 32.0 +/- 3.9, 8.8 +/- 1.6, 10.8 +/- 4.7 and 47.7 +/- 11.8 h. The subsequent intervals between the 9- to 16-cell, early morula, CM and BL stages lasted 16.2 to 18.2 h. Blastomeres of 2-, 4- and 8-cell embryos cleaved asynchronously with < 1, 2.6 +/- 2.5 and 9.2 +/- 4.5 h intervals, respectively, between the first and last blastomere to cleave. The interval from insemination to tight compaction and formation of a blastocoel was 128.4 +/- 10.7 and 145.8 +/- 12.5 h, respectively. The first 3 cell cycles were approximately 3 h shorter (P < 0.1) while the fourth cycle was 5 h shorter (P = 0.06) for the viable vs nonviable embryos. On this basis it was possible to define time windows in which the proportion of viable 2-, 3- to 4-, 5- to 8- and 9- to 16- cell embryos were at their highest. No differences were found between the cleavage intervals of male and female embryos. We conclude 1) that the time-lapse culture system allows for detailed observation of the developmental kinetics of several embryo groups at the same time, and 2) that these embryos can be manipulated at the end of culture, thus allowing a linkage between early cleavage events and other developmental parameters such as embryo sex or viability after transfer.

Animals↗
PubMed1999

The ovine uterus as a host for in vitro-produced bovine embryos.

A series of experiments were conducted to determine whether bovine blastocysts would develop beyond the blastocyst stage in the ovine uterine environment. In Experiment 1, in vitro matured, fertilized and cultured (IVM/IVF/IVC) expanded bovine blastocysts were transferred into uteri of ewes on Day 7 or 9 of the estrous cycle and collected on Day 14 or 15 to determine if the bovine blastocysts would elongate and form an embryonic disk. Springtime trials with ewes that were synchronized with a medroxyprogesterone acetate (MAP) sponge resulted in a 78% blastocyst recovery rate, and 68% of the recovered spherical or elongated embryos had embryonic disks. In Experiment 2, transfer of 4-cell bovine embryos to the oviducts of ewes at Day 3 resulted in a lower recovery (47 vs 80%) than the transfer of blastocysts at Day 7 when embryos were recovered at Day 14. However, the percentage of embryos containing embryonic disks was higher for embryos transferred at the 4-cell stage (71%) than for embryos transferred as blastocysts (50%). In Experiment 3, IVF embryos from super-ovulated cows or Day 8 in vitro produced embryos transferred to cows were collected at Day 14 and were found to be similar in size to those produced by transfer to ewes in Experiment 2. In Experiment 4, the transfer of bovine blastocysts to ewes did not prolong the ovine estrous cycle. In Experiment 5, extension of the ovine estrous cycle by administration of a MAP releasing intravaginal device allowed bovine embryos to elongate extensively and to become filamentous. In Experiment 6, uterine flushings on Day 14 or Day 16 contained elevated levels of interferon-tau when bovine blastocyst were transferred on Day 7. Transfer of bovine embryos to the reproductive tract of a ewe allows some embryos to develop normally to advanced perimplantation stages and may be a useful tool for studying critical stages of embryo development and the developmental capacity of experimental embryos.

Animals↗
PubMed2000

Blastocyst viability and generation of transgenic cattle following freezing of in vitro produced, DNA-injected embryos.

This study examined whether the viability, determined in vitro, of DNA-injected bovine embryos produced in vitro was affected by freezing, and if the frozen embryos developed to term following transfer to recipients. In vitro fertilized zygotes were injected with the pBL1 gene and then co-cultured with mouse embryonic fibroblasts (MEF) in CR1aa medium. Embryos were prepared for cryopreservation by exposure to a 10% (v/v) glycerol solution, loaded into 0.25 ml straws and then frozen by conventional slow freezing. Thawing was by rapid warming in water (37 degrees C) and embryos were rehydrated in PBS diluents of 6%, 3% and 0% (v/v) glycerol supplemented with 0.25 M sucrose and 0.5% (w/v) BSA. In Experiment 1, blastocysts that developed from DNA-injected embryos were individually classified into three morphological groups and three stages of development prior to freezing. DNA-injected blastocysts of excellent quality at freezing showed a higher survival rate (78.8+/-10.6%) after thawing than those of good (60. 9+/-16.4%) or fair (12.5+/-5.9%) quality (P<0.05). Post-thaw survival rate, judged in vitro, increased with more advanced stage of blastocyst development at freezing (early 48.8+/-15.9%, mid 52. 1+/-12.6% and expanded 71.2+/-1.1; P<0.05). In Experiment 2, the frozen/thawed embryos were transferred to recipients to examine in vivo viability. Following transfer of one or two embryos per recipient, pregnancy rates at 60 days of gestation were 13.6% (13/96) for frozen embryos and 26.5% (43/162) for fresh embryos (P<0. 05). Of the 12 live calves born from the frozen/thawed embryos, two males (18.3%) were transgenic. None of the live-born calves derived from fresh embryos exhibited the transgene. One of transgenic bulls did not produce transgenic sperm. Three out of 23 calves (13.0%) produced from cows inseminated with semen of the other bull were transgenic, suggesting that this animal was a germ-line mosaic. These studies indicated that the viability of in vitro produced, DNA-injected bovine blastocysts was affected by freezing and by both the quality and stage of development of the embryo prior to freezing. The generation of transgenic cattle demonstrates that it is feasible to freeze DNA-injected, in vitro produced embryos.

Animals↗
PubMed2002

Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified, in vivo or in vitro produced ovine blastocysts.

The aim of this study was to assess the effect of production system and of cryopreservation of ovine embryos on their viability when transferred to recipients. The experimental design was an unbalanced 2 x 2 factorial design of two embryo production systems (in vivo versus in vitro) and two embryo preservation conditions prior to transfer (transferred fresh versus transferred after vitrification/warming). For the production of blastocysts in vivo, crossbred donor ewes (n=30) were synchronised using a 13-day intravaginal progestagen pessary. Ewes received 1500 IU equine chorionic gonadotropin (eCG) 2 days before pessary withdrawal, and were mated 2 days after pessary withdrawal and embryos were recovered surgically (6 days after mating). Blastocysts were produced in vitro (IVP) using standard techniques. Recipients (n=95) were synchronised using a progestagen pessary and received 500 IU eCG at pessary removal and were randomly assigned to receive (two per recipient) in vivo fresh (n=10), in vivo vitrified (n=10), in vitro fresh (n=35) or in vitro vitrified (n=40) blastocysts. Recipients were slaughtered at day 42 of gestation and foetuses recovered. Pregnancy and embryo survival rates were recorded and analysed using CATMOD procedures. Foetal weights and crown-rump lengths were recorded and analysed using generalised linear model (GLM) procedures. There were no statistically significant interactions between the effects of embryo production system and preservation status at transfer on pregnancy rate and embryo survival. The pregnancy rate following transfer of fresh IVP blastocysts was lower (P<0.07) than that of in vivo embryos (54.3% versus 90.0%, respectively). Vitrification resulted in a decrease in pregnancy rate, the effect being more pronounced in the case of IVP embryos (54.3-5.0%, P<0.001) compared with in vivo embryos (90.0-50.0%), although the absolute change was similar (49.3% versus 40.0%). Transfer of fresh IVP blastocysts resulted in a higher proportion of single (78.9% versus 33.3%) and lower proportion of twin (21.1% versus 66.7%) pregnancies than those produced in vivo. This was reflected in a significant difference in embryo survival rate (fresh: 32.8% versus 75.0%, P<0.01; vitrified: 2.5% versus 35.0%, P<0.001, for IVP and in vivo blastocysts, respectively). Similarly, all pregnancies resulting from the transfer of vitrified/warmed IVP blastocysts were single pregnancies, while 40% of those from vitrified/warmed in vivo blastocysts were twin pregnancies; this was reflected in an embryo survival rate of 35.0% versus 75.0%, respectively. There was a significant effect (P=0.0184) of litter size on foetal weight but not on foetal length (P=0.3304). Foetuses derived from the fresh transfer of IVP blastocysts were heavier (6.4+/-0.2g versus 5.8+/-0.2g, respectively, P<0.05) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.01) than those derived from fresh in vivo blastocysts. There was no difference in these parameters as a consequence of vitrification of IVP embryos. However, in vivo blastocysts subjected to vitrification resulted in heavier (6.6+/-0.3g versus 5.8+/-0.2g, respectively, P=0.055) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.05) foetuses than their counterparts transferred fresh.

Animals↗
PubMed2001

Development of blastocyst-stage embryos after round spermatid injection in patients with complete spermiogenesis failure.

PURPOSE: Our purpose was to evaluate the progression of embryos derived from round spermatid injection to the blastocyst stage and compare the results with those obtained by the use of testicular or epididymal spermatozoa. METHODS: Thirty-eight patients with azoospermia enrolled in this study. In 29 patients with obstructive or nonobstructive azoospermia, spermatozoa were recovered from epididymis or testis. In the remaining nine cases with nonobstructive azoospermia, only round spermatids were found in seven, whereas in two of the patients, there were no elongated or round spermatids. Six of these cases underwent round spermatid injection. RESULTS: Twenty-one of 29 patients with injection of spermatozoa underwent embryo transfer on day 3, and 10 pregnancies (47.6%) were obtained. In eight cycles, embryos were further cultured for delayed transfer. In six cases undergoing round spermatid injection, no transfer was performed on day 3 and extended culture with delayed embryo transfer was applied. The mean number of fertilized oocytes and mean number of embryos on day 3 and also the fertilization rate and mean number of good-quality embryos on day 3, mainly grade 1 or 2, were statistically significantly higher in the spermatozoa group than the round spermatid injection group. Compared to the spermatozoa group, the number of arrested embryos was significantly higher and the number of blastocyst-stage embryos and number of good-quality blastocysts were significantly lower in the spermatid injection group. No blastocysts developed in two spermatid cycles and embryo transfer was not possible, and in the remaining four cycles, after at least one blastocyst transfer, no pregnancies were achieved. However, in eight cycles with extended culture in the spermatozoa group, embryo transfers were achieved in all and three pregnancies, for a pregnancy rate of 37.5%, were obtained after blastocyst transfer. CONCLUSIONS: Our preliminary results showed that round spermatid injection was associated with a significantly lower fertilization and embryo development rate and a significantly higher developmental arrest rate compared with the injection of spermatozoa. Extended culture and delayed embryo transfer did not improve the clinical outcome after round spermatid injection, and these results suggested a developmental failure in embryos preventing successful implantation after round spermatid injection.

Adult↗
PubMed2003

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos.

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which 2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos, which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is often assumed that the block of early development is due to the failure of zygotic gene activation (ZGA) in cultured embryos. In this study we examined protein synthesis patterns by two-dimensional gel electrophoresis of [35S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strain were compared in their development both in vitro and in vivo. The detection of TRC expression, a marker of ZGA, at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even in the 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as compared with normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG, TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo. But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA, but to a delay of ZGA.

Animals↗
PubMed1996

Nutrient uptake and culture of Sminthopsis macroura (stripe-faced dunnart) embryos.

Glucose and pyruvate uptake by individual embryos were measured in a marsupial species (stripe-faced dunnart) and a eutherian species (mouse). At each stage of development, nutrient uptake by the dunnart embryo was around an order of magnitude greater than that of the mouse embryo. The pattern of glucose uptake by the dunnart embryo was not like that for any eutherian embryo, all of which have a low glucose uptake before the blastocyst stage. Rather, in the dunnart embryo there was a significant increase in glucose uptake after the third cleavage division, increasing from 13.6 pmol embryo h-1 at the 4-cell stage to 34.9 pmol embryo h-1 by the 8-cell stage. This increase in glucose uptake before blastocyst formation may be attributed to an increased energy demand associated with the movement of cells within the dunnart embryo. Using a new culture system, it was possible to culture 66% of dunnart embryos at the 2-4-cell stage and 80% of those at the 8-16-cell stage to the unilaminar blastocyst stage. Embryos cultured from the 2-cell to the 4-cell stage were retarded by around 12 h when they reached the blastocyst stage. Developmental retardation was also reflected in the pattern of nutrient uptake, which lagged behind that of embryos developed in vivo. The present study has shown that it is possible to culture the early marsupial embryo to the blastocyst stage in a serum-free culture system, while concomitantly quantifying embryonic nutrient requirements. Such an approach is essential for species where there is a paucity of material for study.

Animals↗
PubMed2002

Transmyometrial embryo transfer and junctional zone contractions.

BACKGROUND: Embryo transfer is a crucial step in IVF-embryo transfer cycles. Several studies have explored transmyometrial embryo transfer, but although this procedure has several favourable characteristics, its role in assisted reproduction has not yet been established. Junctional zone (JZ) contractions during embryo transfer are associated with a negative outcome and factors which increase JZ contractions should be avoided. METHODS: In this study, we have investigated the effect of transmyometrial embryo transfer on JZ contractions. Ten patients with a previously difficult embryo transfer, or a difficult mock embryo transfer, underwent transmyometrial embryo transfer. Before and after this procedure a transvaginal ultrasound scan was performed and this was recorded on videotape for 5 min. The recordings were digitized and then analysed for JZ contractions. RESULTS: Transmyometrial embryo transfer causes a significant increase in JZ contractions. CONCLUSION: The increase in JZ contractions after transmyometrial embryo transfer forms a theoretical objection to this procedure. However, its alternative, a difficult transcervical embryo transfer, is also associated with an increase in JZ contractions. We therefore suggest a large prospective study to investigate the most effective method of embryo transfer in cases where a difficult transcervical embryo transfer is anticipated due to cervical factors.

Adult↗
PubMed2003

Embryo donation for medical research: attitudes and concerns of potential donors.

BACKGROUND: The recent derivation of embryonic stem cell lines from human blastocysts and related implications for regenerative medicine has intensified a longstanding debate about the use of human embryos for research purposes. However, studies have shown that few couples with stored embryos opt to donate them for research. Herein, the attitudes and concerns of potential embryo donors to donation of surplus embryos for medical research were examined. METHODS: From a total of 509 couples who had stored frozen embryos and who had received a questionnaire about embryo donations for medical research, 152 women (30%) and 123 male partners (24%) responded. Embryos had been stored for a mean of 2.25 years (range 3 months to 12 years). RESULTS: Some 10% of respondents indicated it probable, and 34% possible, that they would donate their surplus embryos for research in the future. Women respondents whose embryos had been stored longer, and those committed to the practice of a religion, were more worried about their embryos. Respondents positively disposed to donation commented on their desire not to waste embryos, a desire to help infertile couples, and/or to advance scientific knowledge. Those with negative views commented on the embryo as a potential child and expressed concerns about a perceived lack of control over the type of research to be carried out. CONCLUSIONS: Findings indicate a need for tailored education and counselling about embryo donation for medical research.

Adult↗
PubMed2003

Quantitative RT-PCR reveals tuberous sclerosis gene, TSC2, mRNA degradation following cryopreservation in the human preimplantation embryo.

The use of cryopreserved human embryos in gene expression studies provides an additional source to the scarce embryos available for research. To validate their use we have implemented a quantitative RT-PCR to characterize the levels of the tuberous sclerosis, TSC2 gene in fresh and frozen-thawed human embryos. Frozen embryos were thawed using two different clinical protocols. In fresh embryos 9.95 fg of TSC2 cDNA was present in the unfertilized oocyte, which was comparable to the level on day 2 of preimplantation development. On day 3 there was a significant drop (P<0.001) to 6.8 fg, followed by an increase in cDNA levels to 10.8 fg (P<0.01) on day 6 at the expanded blastocyst stage. Day 2 frozen embryos possessed 50% less (P<0.001) TSC2 mRNA in comparison to the fresh embryos using thawing protocol one (from frozen to 37 degrees C) and 25% less TSC2 mRNA (P<0.01) with thawing protocol 2 (from frozen to room temperature). After culturing day 2 frozen embryos for an additional day they showed mRNA levels comparable with fresh day 3 embryos. There was no significant difference in the levels of TSC2 mRNA between fresh and frozen day 3 human embryos with either thawing protocol. This study demonstrates that cryopreservation does affect the normal pattern of gene expression during human preimplantation development, and that intact frozen-thawed embryos are not equivalent to their non-frozen counterparts. Furthermore human embryos frozen on day 2 appear to be more susceptible to temperature change than embryos frozen on day 3.

Cellular Senescence↗
PubMed1995

One year's experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes.

High incidences of multiple pregnancies, after transferring a maximum of three embryos, were observed after in-vitro fertilization (IVF) treatment. In a randomized study, it was demonstrated that, after taking into account embryo quality and other positively interfering parameters, an elective transfer of two good quality embryos does not significantly influence the pregnancy rate. The intracytoplasmic sperm injection (ICSI) technique was successfully developed in the meantime and high incidences of multiple pregnancies were also obtained after ICSI. The question arose whether after ICSI there was also room for elective double embryo transfer in a well-defined patient group. This report covers 1 year of IVF and ICSI treatment and the results are presented in relation to the number of embryos transferred. The embryo development is similar for zygotes obtained after IVF and ICSI; for both techniques 63% of the zygotes develop to type A-B embryos and 13% to type C embryos. There is also no difference in the pregnancy rate after ICSI or IVF. Globally, after IVF, 307 out of the 766 double and triple transfers (40.1%) and 317 out of 774 double and triple transfers (40.9%) after ICSI resulted in a positive HCG. After IVF, 73.9% (227) and after ICSI 76.3% (242) of the pregnancies were evolutive. Neither was there any difference between the two techniques as regards the implantation rate per transferred embryo. After IVF, 22.8% of the transferred embryos implanted compared with 21.8% after ICSI. When the elective double embryo transfers were compared, no difference was found between IVF and ICSI. After IVF, 102 of the 211 elective double transfers (48.1%) resulted in a pregnancy versus 93 out of 225 (41.3%) after ICSI [not significant (NS)]. A high implantation rate per transferred embryo (IVF: 33.2%; ICSI: 26.9%, NS) was obtained in this elective double transfer category, as was also reported in the randomized study. These data confirm the results obtained in our randomized study and the effectiveness of the elective double embryo transfer for IVF as well as for ICSI.

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