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PubMed2002

Effects of oocyte quality on development and transcriptional activity in early bovine embryos.

The objective of this study was to evaluate the effects of oocyte quality on in vitro development and the level of transcriptional activity in early bovine embryos. Cumulus-oocyte complexes (COC) were divided into six classes based on their cumulus investment and on the texture of the ooplasm. Embryos originating from oocytes with more than five layers of cumulus cells and with slight expansion of the cumulus and/or granulation in the ooplasm (class II) developed to the blastocyst stage as frequently as embryos originating from oocytes of class I which showed no signs of atresia (13.9 and 13.7% for classes I and II, respectively). Oocytes with fewer than five layers of cumulus cells and homogeneous ooplasm (class III) had lower cleavage (63.1%) than oocytes with more than five layers of cumulus cells (77.2 and 83.6% for classes I and II, respectively); however, development to the blastocyst stage was similar (12.7%). More advanced atresia such as the presence of granulations in oocytes with less than five layers of cumulus cells (class IV), the absence of cumulus (class V), or the presence of expanded cumulus with dark clumps (class VI) reduced cleavage (57.4, 35.9 and 56.3% for classes IV-VI, respectively) and blastocyst formation (3.5, 0.5 and 1.9% for classes IV-VI, respectively). We examined the effects of oocyte quality on the level of transcriptional activity in in vitro produced embryos at the 2-, 4-, 8-, 16-cell stage and in embryos remaining at the 8-cell stage while the majority had progressed to the 16-cell stage (8-cell delayed embryos) by labeling with 3H-uridine followed by RNA precipitation and scintillation counting. For each developmental stage, there was no significant effect of oocyte class on uptake and incorporation of 3H-uridine into embryos, with the exception of uptake at the 8-cell stage which was higher (P<0.05) in embryos from class V-VI oocytes. Labeled uridine uptake (in embryos from classes I-II and III oocytes) and incorporation (in embryos from oocytes of all classes) increased significantly at the 16-cell stage compared to earlier stages. Eight-cell delayed embryos originating from classes I to IV oocytes incorporated significantly more 3H-uridine than normally developing 8-cell embryos. In conclusion, these results expand on previous work showing that oocytes with early signs of atresia have good development potential. No differences in transcriptional activity in embryos originating from different classes were detected. However, the results obtained with 8-cell delayed embryos indicated that transcriptional activity was determined by the interval after fertilization rather than the number of cell cycles.

Animals↗
PubMed2000

Chromosome abnormalities in 1255 cleavage-stage human embryos.

The relationship was examined between chromosome abnormalities in cleavage stage human embryos and maternal age, embryo morphology and development rate. Embryos that were classified as suboptimal for transfer from patients undergoing IVF treatment were disaggregated, and all or most of their cells were fixed for analysis by fluorescence in-situ hybridization. Chromosomes X, Y, 13, 18 and 21, and in some instances 16 were examined. A total of 731 non-viable embryos was analysed. An increase in chromosome abnormalities with decreasing embryo competence and increasing maternal age was shown. Compared with an earlier study, the major difference was that polyploidy (P<00.01) and aneuploidy were previously more common. After pooling results, it was found that aneuploidy increased with maternal age, from 3.1% in embryos from 20-34 years old patients to 17% in patients 40 years or older. Also, aneuploidy occurred more frequently in embryos with good morphology and development rate than in embryos developing poorly. In contrast, dysmorphic and slowly developing or arrested embryos had significantly more polyploidy and mosaicism than normally developing embryos. Clear associations between maternal age and aneuploidy, and between cleavage anomalies and mosaicism have been established in non-viable embryos. Arrested embryos were mostly polyploid. Moreover, polyploidy was found more frequently in embryos analysed on day 4, suggesting that developmentally compromised embryos became arrested in extended culture. A slightly higher aneuploidy rate in the earlier study may be attributed to differences in hormonal stimulation, which also resulted in different numbers of oocytes recruited and matured.

Journal Article↗
PubMed2005

Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca).

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.

Germination↗
PubMed2006

Hypoxia interferes with ABA metabolism and increases ABA sensitivity in embryos of dormant barley grains.

Two mechanisms have been suggested as being responsible for dormancy in barley grain: (i) ABA in the embryo, and (ii) limitation of oxygen supply to the embryo by oxygen fixation as a result of the oxidation of phenolic compounds in the glumellae. The aim of the present work was to investigate whether hypoxia imposed by the glumellae interferes with ABA metabolism in the embryo, thus resulting in dormancy. In dormant and non-dormant grains incubated at 20 degrees C and in non-dormant grains incubated at 30 degrees C (i.e. when dormancy is not expressed), ABA content in the embryo decreased dramatically during the first 5 h of incubation before germination was detected. By contrast, germination of dormant grains was less than 2% within 48 h at 30 degrees C and embryo ABA content increased during the first hours of incubation and then remained 2-4 times higher than in embryos from grains in which dormancy was not expressed. Removal of the glumellae allowed germination of dormant grains at 30 degrees C and the embryos did not display the initial increase in ABA content. Incubation of de-hulled grains under 5% oxygen to mimic the effect of glumellae, restored the initial increase ABA in content and completely inhibited germination. Incubation of embryos isolated from dormant grains, in the presence of a wide range of ABA concentrations and under various oxygen tensions, revealed that hypoxia increased embryo sensitivity to ABA by 2-fold. This effect was more pronounced at 30 degrees C than at 20 degrees C. Furthermore, when embryos from dormant grains were incubated at 30 degrees C in the presence of 10 microM ABA, their endogenous ABA content remained constant after 48 h of incubation under air, while it increased dramatically in embryos incubated under hypoxia, indicating that the apparent increase in embryo ABA responsiveness induced by hypoxia was, in part, mediated by an inability of the embryo to inactivate ABA. Taken together these results suggest that hypoxia, either imposed artificially or by the glumellae, increases embryo sensitivity to ABA and interferes with ABA metabolism.

Abscisic Acid↗
PubMed1991

Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage.

Energy substrate requirements (pyruvate, lactate and amino acids) were determined for in-vitro development of hamster 1- and 2-cell embryos to blastocysts, using a chemically defined, protein free medium (hamster embryo culture medium, HECM). One-cell embryos were very sensitive to energy substrate type and concentration. Pyruvate alone could not support development of 1-cell embryos to greater than 4-cells, whereas lactate as sole energy substrate supported 14% development into morulae/blastocysts. Pyruvate, with lactate and 20 amino acids, inhibited 1-cell embryo development into blastocysts relative to lactate and 20 amino acids. The highest development of 1-cell embryos to blastocysts (up to 27%) occurred with reduced lactate concentration (less than 10 mM) and either 20 amino acids or 0.2 mM glutamine. Hamster 2-cell embryos were much less sensitive to energy substrates, requiring only lactate for development to blastocysts (53%). Lactate with 20 amino acids supported 70-75% of 2-cell embryos to blastocysts. Glutamine as sole energy and nitrogen source supported development to morulae and blastocysts of some 2-cell, but not 1-cell, embryos. Pyruvate did not enhance development of 2-cell embryos. We conclude that (i) altering the types and concentrations of available energy substrates drastically changes the developmental responses of 1-cell hamster embryos in vitro and (ii) energy substrate requirements for hamster embryo development in vitro are markedly different from those of mouse embryos, the standard model for studies on preimplantation development. This is the first report of successful in-vitro culture of hamster 1-cell embryos to the blastocyst stage.

Amino Acids↗
PubMed1992

Contrasting Storage Protein Synthesis and Messenger RNA Accumulation during Development of Zygotic and Somatic Embryos of Alfalfa (Medicago sativa L.).

During development on hormone-free media, somatic embryos pass through distinct morphological stages that superficially resemble those of zygotic embryo development (globular, heart, torpedo, cotyledonary stages). Despite these similarities, they differ from zygotic embryos in the extent of cotyledonary development and the patterns of synthesis and quantitative expression of seed-specific storage proteins (7S, 11S, and 2S proteins). Alfin (7S) is the first storage protein synthesized in developing zygotic embryos (stage IV). The 11S (medicagin) and 2S (Low Molecular Weight, LMW) storage proteins are not detectable until the following stage of development (stage V), although all three are present before the completion of embryo enlargement. Likewise, the 7S storage protein is the first to be synthesized in developing somatic embryos (day 5). Medicagin is evident by day 7 and the LMW protein by day 10. In contrast to zygotic embryos, alfin remains the predominant storage protein in somatic embryos throughout development. Not only are the relative amounts of medicagin and the LMW protein reduced in somatic embryos but the LMW protein is accumulated much later than the other proteins. Quantification of the storage protein mRNAs (7S, 11S, and 2S) by northern blot analysis confirms that there are substantial differences in the patterns of message accumulation in zygotic and somatic embryos of alfalfa (Medicago sativa). In zygotic embryos, the 7S, 11S, and 2S storage protein mRNAs are abundant during maturation and, in particular, during the stages of maximum protein synthesis (alfin, stages VI and VII; medicagin, stage VII; LMW, stage VII). In somatic embryos, the predominance of the 7S storage protein is correlated with increased accumulation of its mRNA, whereas the limited synthesis of the 11S storage protein is associated with much lower steady-state levels of its message. The mRNA for the LMW protein is present already by 3 days after transfer to hormone-free media, yet that protein is not evident on stained gels until day 10. Thus, both transcriptional and posttranscriptional events appear to be important in determining the protein complement of these seed tissues. On the basis of storage protein and mRNA accumulation, mature (14 days) somatic embryos most closely resemble stage VI zygotic embryos. The results of the developmental comparison also suggest that the patterns of synthesis of the individual storage proteins (7S, 11S, or 2S) are regulated independently of each other during embryogenesis in alfalfa.

Journal Article↗
PubMed2004

Early morphological nuclear events and developmental capacity of embryos reconstructed with fetal fibroblasts at the M or G1 phase after intracytoplasmic nuclear injection in cattle.

We examined morphological nuclear events during the first cell cycle of bovine embryos reconstructed with somatic cells at the M and G1 phases (M-embryos and G1-embryos, respectively) by intracytoplasmic nuclear injection, and the subsequent development of these embryos in vitro and in vivo. Bovine fetal fibroblasts (BFFs) at the M or G1 phase were directly injected into enucleated oocytes, and activated immediately. Only half (48%) of the M-embryos extruded polar body-like cells (PBCs) at 6 h post injection (hpi). At 15 to 19 hpi, 54% of the M-embryos formed a single pronucleus-like nucleus. Nuclear envelope-breakdown, premature chromosome condensation and single nuclear clusters were observed in most of the G1-embryos (88%) within 30 min following the nuclear injection. At 15 to 19 hpi, single pronucleus-like nuclei were formed in most G1-embryos (83%). The potential of G1-embryos to develop to blastocysts was significantly higher than that of M-embryos (31% vs 16%). Three of five recipients following transfer of blastocysts derived from the G1-embryos became pregnant on Day 30, and one recipient delivered a calf. Our results indicate that almost a half of the M-embryos failed to extrude PBCs and that the G1-embryos developed to blastocysts at a higher rate than the M-embryos.

Animals↗
PubMed1999

Effects of nutrient intake and number of oestrous cycles on in vitro development of preimplantation pig embryos.

The effects of nutrient intake and insemination of gilts at first versus third oestrus on the in vitro development of preimplantation pig embryos were investigated. Standard swine management involves ad libitum feeding of gilts at first oestrus and restricted feeding of gilts at third oestrus. According to previous research, gilts inseminated at first oestrus demonstrate greater embryonic mortality than gilts inseminated at third oestrus, and it is possible that differences in nutrient intake between gilts inseminated at first versus third oestrus affect the viability of eggs or embryos. In the present study, experimental gilts were assigned to three treatments: animals designated 1A were inseminated at first oestrus and fed ad libitum; animals designated 3R were inseminated at third oestrus and were fed a restricted diet; and 3A animals were inseminated at third oestrus and fed ad libitum. Embryos collected from each treatment group were cultured in vitro, and data were evaluated according to cell stage at collection. Comparison of treatments 1A and 3R supported the contention of increased embryo mortality in gilts inseminated at first oestrus under normal management conditions. When cultures were initiated at the one- to two-cell or two- to four-cell stages, the percentage of 1A embryos developing to the morula stage (50.9%, 68.0%) was significantly lower than that of 3R embryos (88.9%, 90.9%; P < 0.05). Comparison of treatments 1A and 3A addressed effects due to the number of oestrous cycles. Significantly more two- to four-cell embryos from gilts inseminated at third oestrus and fed ad libitum reached the morula and expanded blastocyst stages of development (87.0%, 41.3%) compared with embryos from gilts inseminated at first oestrus and fed ad libitum (68.0%, 20.3%; P < 0.05). Finally, the effects of ad libitum feeding were determined by comparing treatments 3A and 3R. These data were inconclusive, as both positive and negative effects were observed. More one- to two-cell embryos from treatment 3R developed to the morula stage (88.9%) compared with 3A embryos collected at the same stage (64.7%), whereas a greater number of 3A embryos in the two- to four-cell category reached the expanded blastocyst stage (41.3%) than 3R embryos (21.2%; P < 0.05). These results support the hypothesis of lower in vitro developmental capacity for embryos collected from gilts inseminated at first oestrus. Furthermore, the findings indicate that differences in embryo viability between gilts inseminated at first versus third oestrus are related to the number of oestrous cycles and possibly to differential nutrition.

Animal Nutritional Physiological Phenomena↗
PubMed2001

Morphologic stages of the equine embryo proper on days 17 to 40 after ovulation.

OBJECTIVE: To describe the gross and histologic changes that develop in the equine embryo proper (ie, the portion of the embryo that becomes the fetus) from days 17 to 40 after ovulation and to compare the external features of equine embryos with those of porcine, ovine, and human embryos. SAMPLE POPULATION: 34 embryos collected from mixed-breed pony mares. PROCEDURE: External features for each embryo proper, including length, number of branchial arches, growth of appendages, face and head features, and body features, were examined, using a dissecting microscope, for embryos collected on days 17 to 40. Internal features were histologically examined by serially sectioning embryos collected on days 20 to 35. RESULTS: Number of embryos recovered for each day ranged from 1 to 5. The initial detection of features was not related closely to age; typically, the first attainment of a given body length or characteristic varied over a 3-day period among embryos. Similarly, the period during which individual characteristics for a given Carnegie stage were attained ranged from 3 to 6 days. Age at first appearance of a characteristic was greater for equine embryos than ages reported for ovine and porcine embryos but less than for human embryos. Indicators of age included number of pairs of branchial arches, all limb buds present, retinal pigmentation, and prominence of the pontine flexure. CONCLUSIONS: No embryologic structures or changes were found that could be considered unique to equine embryos on days 17 to 40 after ovulation.

Animals↗
PubMed2000

[Preliminary analysis of chromosome mosaicism in preimplantation embryos].

OBJECTIVE: Using fluorescence in situ hybridization to analyze chromosome mosaicism in human preimplantation embryos and access the influence of mosaicism on in vitro fertilization-embryo transfer (IVF-ET) and preimplantation genetic diagnosis. METHODS: Normal fertilized embryos, which were not suitable for embryo transfer and cryopreservation, and male embryos in preimplantation gender diagnosis were analyzed by fluorescence in situ hybridization. RESULTS: Two hundred and ninety three nucleuses were found among 38 embryos. The hybridization rate was 96.2%. Twelve embryos (31.6%) were normal. Twenty-eight embryos (73.7%) were diploid embryos, which included normal diploid embryos and embryos with diploid moscaicism. In addition, 19 (50.0%) embryos were considered to be chromosomal mosaics due to diploid mosaicism and abnormal mosaicsm. The frequency of mosaicism increased from 18.2% in < or = 4 cell-stage embryos to 68.4% and 50.0% in 5-8 cell-stage and > or = 9 cell-stage embryos respectively. CONCLUSION: The frequency of Mosaicism increases with successive cleavage divisions. Moscaicism may be one of the important factors affecting the success rates in IVF-ET. Identification of sex by analysis of a single cleavage cell is accurate; however, it would be better to biopsy 2 cells to reduce errors in preimplantation genetic diagnosis of single gene diseases.

Blastocyst↗
PubMed2002

Modulation of nitric oxide concentration and lipid metabolism by 15-deoxy Delta12,14prostaglandin J2 in embryos from control and diabetic rats during early organogenesis.

The concentration of 15-deoxy Delta(12,14)PGJ(2) (15dPGJ(2)) and its effects on nitric oxide generation and neutral lipid in embryos from control and neonatal streptozotocin-induced (n-stz) diabetic rats during organogenesis were investigated. 15dPGJ(2) is produced in embryos during organogenesis, and its production is lower in embryos of n-stz diabetic rats than in embryos from control rats. Nitrate and nitrite concentrations were higher in embryos from n-stz diabetic rats and were reduced in the presence of 15dPGJ(2) both in embryos from control and diabetic rats. Thus, decreased 15dPGJ(2) concentrations in embryos from n-stz diabetic rats may be related to the high nitric oxide concentrations found in those embryos. Exogenous 15dPGJ(2) decreased cholesterol and cholesteryl ester concentrations in embryos from control and n-stz diabetic rats, and reduced triacylglycerol concentrations in control embryos. Incorporation of [(14)C]acetate into lipids showed decreased de novo synthesis of cholesteryl ester and triacylglycerides in embryos from n-stz diabetic rats compared with controls. Exogenous 15dPGJ(2) reduced the incorporation of [(14)C]acetate into triacylglycerides, cholesterol and cholesteryl ester in embryos from both control and n-stz diabetic rats. 15dPGJ(2) is present in embryos during organogenesis, and reduces embryonic nitric oxide production and lipid synthesis. The lower 15dPGJ(2) concentration in embryos from n-stz diabetic rats may result in developmental alterations in this diabetic model.

Acetates↗
PubMed2004

[Efficiency of cryoembryotransfer using embryos frozen in the pronuclear stage].

OBJECTIVE: To evaluate results of cryoembryotransfers in cycles with estrogen-progestin preparation of the endometrium. DESIGN: Prospective study. SETTING: Centre of Assisted Reproduction, Dept. of Obstetrics and Gynecology, Palacký University Medical School, Olomouc. METHODS: The study included 51 patients in whom in the period of January to August 2003 altogether 42 cycles the embryotransfer with frozen and thawed embryos were performed. The embryos for cryopreservation were obtained in the classical IVF (n = 21) or ICSI (n = 30) cycles and were cryopreserved in the pronuclear stage (PN). The storage interval varied from 3 months to 5 years. The endometrial preparation was performed in all cycles with oral estrogen--progestin substitution. Embryos in the PN stage were examined for survival (rate of intact embryos after thawing) and progress of their further development. Endometrial thickness, number of transferred embryos, pregnancy rate (PR), implantation rate (IR) and abortions (AB) were evaluated as outcome parameters. RESULTS: A total of 210 thawed embryos were evaluated. Survival was 79% without regard to the method of fertilization. For 42 cryoembryotransfers 131 embryos (62%) in 6 to 10 blastomeres after 48 hours cultivation in G 1.2 and G 2.2 fy Vitrolife were selected. Further development was more regular in embryos obtained in classical IVF cycles, where 41% of the embryos were in 4 cells stage after 24 hours of cultivation and 36% of the embryos were in 8 cells stage after 48 hours while after ICSI 32% 4 cells embryos and 26% 8 cells embryos respectively. Endometrial thickness in the day of transfer in all patients was 10.9 +/- 1.3 mm, the average number of transferred embryos was 2.4 embryos/transfer, PR was 28.5 (IVF 42.9% and ICSI 20.7%), IR 12.0% (IVF 13.0% and ICSI 11.3%) and AB 41.6% (IVF 33.3% and ICSI 50.0%). CONCLUSION: Improvement of cryopreservation procedures, freezing of embryos in the pronuclear stage and last but not least the optimal endometrial preparation can positively influence the cumulative efficiency of assisted reproduction.

Cryopreservation↗
PubMed1996

Clinical factors for successful cryopreserved-thawed embryo transfer.

PURPOSE: To study how clinical factors such as embryo quality, cell stage of embryo at cryopreservation, and synchronization of developmental stages between embryo and endometrium at thawing affect the implantation rate after cryopreserved-thawed embryo transfer (ET), these factors were examined in 106 cryopreserved-thawed ETs including 204 embryos. RESULTS: In 86 embryos graded as high quality before transfer by morphological evaluation, 31 implantations were successful, while the other, low-quality embryos did not implant at all. High-quality embryos received less cryoinjury during cryopreservation compared to the injuries sustained by embryos in moderate and poor quality. When cyopreservation was performed at the 1-, 2-, and 3-day cell stages, pregnancies were similiarly achieved among most of the embryos at all cell stages. At thawed ETs in natural ovulation cycles, there were some differences among the developmental stages between thawed embryo and endometrium that received the thawed transferred embryos. Although the transfer timelag ranged a day or more, asynchronism of endometrium growth to the cell stage did not reduce pregnancy rates. CONCLUSIONS: These results indicated that embryo quality evaluated morphologically was the most important clinical factor for successful implantation of cryopreserved-thawed ET.

Blastocyst↗
PubMed1995

Sex ratio of babies is unchanged after transfer of fast- versus slow-cleaving embryos.

PURPOSE: A higher proportion of male offspring has been observed after transferring faster-developing embryos in a number of animal species. Therefore, we evaluated the correlation between the sex ratio of delivered babies and the cleavage stage of transferred embryos in a human IVF-ET program. METHODS: The sex of infants born (n = 104) after transfer of exclusively slower-cleaving < or = 3 cell (n = 41) versus exclusively faster-cleaving > or = 4 cell (n = 63) embryos was compared. Furthermore, all boys and girls resulting from IVF-ET (n = 213) were compared with respect to: the average number of cells in the embryos that were transferred, the embryo with the greatest number of cells in the cohort transferred and the percentage of embryos that were faster cleaving. RESULTS: Thirty seven percent (15/41) of infants resulting from the transfer of exclusively slower-growing embryos were girls and 38% (24/36) of the infants from the faster-growing embryos were girls (NS). The analysis all 213 babies born after 145 embryo transfer procedures did not suggest any differences in embryo cleavage rates in embryo transfers leading to male versus female infants. CONCLUSIONS: A greater number of boys born was not observed after transfer of faster-cleaving embryos as has been described in other animal species. The race to be male may not occur until later cleavage divisions or may not occur in the human embryo.

Cell Division↗
PubMed2004

Cumulative pregnancy rates after transfer of fresh and thawed embryos.

Frozen-thawed embryo transfer is an effective procedure that allows further possibilities of pregnancy in addition to those obtained after the fresh in vitro fertilization (IVF). In our follow-up study we analysed all fresh embryo transfer procedures and every frozen-thawed embryo transfer performed from January 2000 to December 2001 evaluating the cumulative pregnancy rates. The study population was divided into two groups according to the female age: <38 years (group I) and >38 years (group II). All the best embryos were chosen for transfer and all the supernumerary good quality embryos were cryopreserved on the day of transfer. The embryos were then thawed and manipulated using a new technique. In group I, 527 patients (619 cycles) underwent fresh embryo transfer and in 232 of them (238 cycles) the embryos were frozen (44% per patients and 38.4% per cycle). In group II, 156 patients (193 cycles) underwent fresh embryo transfer and in 14 of them (15 cycles) the embryos were frozen (9% per patient and 7.8% per cycle). The pregnancy rate of group I patients that had their supernumerary embryos frozen (232 patients and 238 cycles) was 47.4% per cycle and 48.7% per patient whereas in the same population of group II patients (14 patients and 15 cycles) the clinical pregnancy rate was 35.7% per cycle and 38.5% per patients. The cumulative clinical pregnancy rate after transfer of fresh and thawed embryos was: group I, 74% per cycle and 76% per patients; group II, 42.8% per cycle and 46.1% per patient. Frozen-thawed embryo transfer is a cost-effective practice.

Adult↗
PubMed2005

In vitro comparisons of two cryopreservation techniques for equine embryos: slow-cooling and open pulled straw (OPS) vitrification.

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.

Animals↗
PubMed2003

Pronuclear morphology evaluation with subsequent evaluation of embryo morphology significantly increases implantation rates.

OBJECTIVE: To elucidate the relative predictive value of implantation markers at different stages of preimplantation development. DESIGN: Correlation of pronuclear morphology with embryo morphology and implantation rates in retrospective and prospective analysis of in vitro fertilization/intracytoplasmic sperm injection (IVF-ICSI) treatment cycles. SETTING: Private infertility center. PATIENT(S): A total of 441 couples undergoing infertility treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Size of pronuclei and distance between them, the number and polarization of nucleolus precursor bodies (NPB) at the one-cell stage, embryo cleavage and fragmentation rates on days 2 and 3, and pregnancy and implantation rates. RESULT(S): Polarization of the NPB in both pronuclei had a statistically significant correlation with normal membrane breakage during ICSI (40%, compared with 33% easy, and 31% difficult membrane breakage) and also with faster cleavage and lower fragmentation rates of embryos. Sixty-one percent of implanting embryos had polarization of the NPB in both pronuclei compared with 37% for all embryos. Larger distance between pronuclei and their unequal size had a statistically significant correlation with slower cleavage and inferior embryo quality. Embryo selection based on only pronuclear morphology or on only day-3 embryo morphology yielded implantation rates of 15.1% and 12.1%, respectively. Embryo selection based on sequential evaluation of both pronuclear morphology and embryo morphology on day 3 resulted in a 21.1% implantation rate. CONCLUSION(S): Polarization of NPB in both pronuclei is as reliable marker of implantation as embryo morphology on day 3. However, pronuclear morphology assessment improves embryo selection only when it is combined with embryo morphology evaluation on day 3.

Adult↗
PubMed2000

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer.

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.

Acepromazine↗