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PubMed2000

Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models.

The efficiency of embryo banking for rat and mouse models of human disease and normal biological processes depends on the ease of obtaining embryos. Authors report on the effect of genotype on embryo production and rederivation. In an effort to establish banks of cryopreserved embryos, they provide two databases for comparing banking efficiency: one that contains the embryo collection results from approximately 11,000 rat embryo donors (111 models) and another that contains the embryo collection results from 4,023 mouse embryo donors (57 induced mutant models). The genotype of donor females affected the efficiency of embryo collection in two ways. First, the proportion of females yielding embryos varied markedly among genotypes (rats: 16-100 %, mean =71 %; mice: 24-95 %, mean =65 %). Second, the mean number of embryos recovered from females yielding embryos varied considerably (rats: 4-10.6, mean =7.8; mice 5.3-32.2, mean =13.7). Genotype also affected the efficiency of rederivation of banked rat and mouse embryos models by embryo transfer. For rats, thawed embryos (n =684) from 33 genotypes were transferred into 66 recipient females (pregnancy rate, 78 %). The average rate of developing live newborns for individual rat genotypes was 30 % with a range of 10 to 58 %. For mice, thawed embryos (n =2,064) from 59 genotypes were transferred into 119 pseudopregnant females (pregnancy rate: 76 %). The average rate of development of individual mouse genotypes was 33 % with a range of 11 to 53 %. This analysis demonstrates that genotype is an important consideration when planning embryo banking programs.

Animals↗
PubMed1999

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo.

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (</= 8-cell stage) collected 12-48 h after IVF were separately fixed, stained by orcein, and examined under phase contrast microscopy. It was found that 27% of 2-cell, 74% of 3-cell, 51% of 4-cell, and 74% of 5- to 8-cell-stage embryos were abnormal in morphology. Morphological anomalies included fragmentation (no nucleus in one or more than one blastomere) and/or binucleation (two nuclei in one or more than one blastomere). In experiment 2, actin filament distribution of the embryos at 2-cell to blastocyst stages that were produced in vivo and in vitro were stained by rhodamine-phalloidin and examined by confocal microscopy. Actin filaments were distributed in all in vivo-derived embryos at the cell cortex, and at the joints of cells and perinucleus in 2- to 8-cell-stage embryos and in some cells of morulae and blastocysts. Actin filaments were also distributed in the cortex and at the joints of cells of all in vitro-produced embryos. However, only 20% of in vitro-produced embryos at 2- to 8-cell stages had perinuclear actin filaments in all blastomeres. Most in vitro-produced embryos had fewer perinuclear actin filaments or did not have perinuclear actin filaments in some blastomeres. Fragmentation and binucleate blastomeres were not observed in in vivo-derived embryos. In vivo-derived Day 5 (136.5 +/- 60.4 nuclei per blastocyst) and Day 6 (164.5 +/- 51.9 nuclei per blastocyst) blastocysts had significantly (p < 0.001) more cells than in vitro-produced Day 6 blastocysts (37. 3 +/- 11.7 nuclei per blastocyst). In experiment 3, when cytochalasin D, an inhibitor of microfilament polymerization, was included in the culture medium, it prevented 2- to 4-cell-stage embryos from developing to the blastocyst stage. These results indicate that abnormal actin filament distribution is one possible reason for abnormal embryo cleavage and small cell numbers in pig embryos produced in vitro. Culture conditions that mediate normal actin filament distribution may result in an improvement in embryo quality.

Actins↗
PubMed2003

Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture.

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.

Animals↗
PubMed2000

Microscopic assessment of pronuclear embryos is not definitive.

The microscopic classification of embryos, especially unipronuclear embryos, is not very precise. A number of undocumented and unipronuclear embryos were determined to be diploid following karyotyping and fluorescence in situ hybridization (FISH). Accelerated and asynchronous pronuclear dismantling at the time of checking for embryo fertilization accounts for this disparity. Diploid embryos were also observed among tripronuclear embryos. However, not all embryos ascertained as diploid by FISH were karyotypically normal following full karyotype analysis. By taking into account the "background" abnormality rate, the rate of diploid embryo wastage was estimated to be about 40% among undocumented embryos and about 58% in total. A high percentage of misclassification infers an unintended loss of otherwise transferable embryos. Such a discrepancy is particularly important to older women who have fewer embryos. If these are a woman's only embryos, preimplantation genetic diagnosis might be applicable in determining those that are diploid and suitable for transfer. This could potentially reduce the number of wasted embryos and cycles. The present study has also shown that mosaicism is common but it is still unclear whether mosaicism is indicative of embryonic abnormality or is a fairly common phenomenon among healthy embryos. Bipronuclear embryos that present with abnormal or delayed cleavage are often chaotic in their chromosomal constitution. Such embryos should not be transferred.

Adult↗
PubMed2000

Refreezing of murine intact and biopsied embryos by rapid-freezing procedure.

The in-vivo development of murine morula stage embryos frozen-thawed once or twice and embryos biopsied after one freezing cycle and refrozen was studied. Embryos (n = 860) were cryopreserved using a rapid-freezing procedure. At least 24 h after freezing, embryos were thawed and cultured in vitro for 3 h. In experiment I, morphologically intact embryos were either transferred (n = 180) into recipients or refrozen (n = 160). Unfrozen embryos (control group, n = 180) and refrozen embryos stored for at least 24 h and then thawed, were transferred into recipients. In experiment II, embryos frozen once were thawed and biopsied or sham-biopsied (n = 230 and 180 respectively) and refrozen (n = 226 and 179 respectively). They were thawed and transferred (n = 192 and 160 respectively) into recipients. Recipient mice were either killed on day 15 after embryo transfer and number of implantation sites and live fetuses recorded or pregnant recipients (n = 6, experiment II) were allowed to carry the fetuses to term. There was no difference in the survival rate of embryos at thawing between those frozen once or twice (91 versus 93%). The implantation rate and number of live fetuses in the pregnant recipients at necropsy among those transferred with unfrozen embryos (57% and 51%; 8/9), embryos frozen once (55% and 45%; 8/9) or twice (51% and 48%; 6/8) was not different. There was no difference in the survival rate of refrozen embryos biopsied or sham-biopsied after one freezing cycle (89 versus 87%). The implantation rate and number of live fetuses in pregnant animals transferred with biopsied or sham-biopsied embryos was not different (64 and 41% versus 57 and 37% respectively). All six pregnant animals allowed to carry the fetuses to term delivered normal live fetuses (n = 39). On mating 12 females with six males of the progeny born out of biopsied embryos, all became pregnant and delivered live fetuses. It may be concluded that murine biopsied and intact embryos can be successfully refrozen by rapid-freezing procedure.

Animals↗
PubMed2001

A prospective, randomized study comparing day 2 and day 3 embryo transfer in human IVF.

It is believed that delayed transfer of embryos after IVF allows for a better selection of good quality embryos. Hence, the number of embryos and all other prognostic factors being equal, transfer of day 3 embryos should be associated with higher implantation and pregnancy rates than transfer of day 2 embryos. To investigate this hypothesis, a prospective randomized study was carried out to compare implantation and pregnancy rates between day 2 and day 3 transfers. The relationship between the embryo quality score of day 2 and day 3 embryos and their respective implantation rates was also analysed. In a 2 year period all patients undergoing infertility treatment and in whom at least seven normally fertilized oocytes were obtained were included in the study. A minimization procedure was performed taking into account the patient's age and the method of fertilization (IVF or intracytoplasmic sperm injection). By using a uniform policy of embryo transfer, the number of embryos transferred was similar in both groups. The outcome parameters were embryo quality, implantation and pregnancy rates. No difference was observed in implantation and pregnancy rates between transfers on day 2 versus day 3 (23.8 versus 23.8% and 47.9 versus 46.8% respectively). The incidence of embryos of moderate to poor quality was higher in embryos cultured for 3 days compared with those cultured for 2 days. It is concluded that the outcomes of embryo transfer in terms of implantation and pregnancy rates are comparable for day 2 and day 3 embryos, although the overall embryo quality score decreases when embryos are kept in culture till day 3.

Embryo Implantation↗
PubMed1999

Nucleolar ultrastructure in bovine nuclear transfer embryos.

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.

Animals↗
PubMed1988

Development of golden hamster embryos through the two-cell block in chemically defined medium.

The effect of increasing the embryo:medium volume ratio on overcoming the hamster two-cell block was examined. Two-cell golden hamster embryos from each superovulated female were cultured in microdrops (estimated at 0.75 microliter) or 100 microliter macrodrops of chemically defined medium (modified Tyrode's solution [TLP] plus glutamine, isoleucine, methionine, phenylalanine, and taurine). In 11 trials (i.e., with embryos from 11 donors), 28.6% of 269 embryos developed to the four-cell stage in microdrops, whereas only 2 (0.7%) embryos developed in the macrodrops. When two microdrops were used to culture the two-cell embryos from each donor (n = 8), 17.8% of 304 embryos developed to four cells. Increasing the embryo:medium volume ratio further by culturing all of the embryos from each donor (n = 10) in single microdrops resulted in 53.1% of 397 embryos developing to four cells. Conditioning of the culture medium by these embryos could not be demonstrated. Increasing the embryo:medium volume ratio may protect against loss of some intracellular component essential for growth of early-stage hamster embryos. Alternatively, increasing this ratio may permit embryos to reduce the concentration of a substance detrimental to their growth. This work represents the first report of cleavage of hamster two-cell embryos in vitro. These findings are a significant step towards our goal of obtaining complete preimplantation developmental of hamster embryos in vitro and may be helpful for solving the in vitro developmental blocks in embryos from other species.

Animals↗
PubMed1988

Development of screening systems for evaluation of materials used in mammalian embryo transfer.

Embryo transfer units use a wide variety of materials that come in contact with embryos. Studies were conducted to evaluate procedures that could be utilized to determine the toxicity of some commonly used materials in embryo collection, culture and transfer. Forty-five female mice were sacrificed on Day 3 or 4 of gestation (Day 1 = vaginal plug), and the uterus and oviducts were removed and minced. A total of 522 embryos was collected (4-cell to blastocyst stages). Four to 16 cell embryos were cultured in Phosphate Buffered Saline (PBS) plus 20% fetal bovine serum. Morula to blastocyst stage embryos were cultured in Nutrient Mixture F10 (HAM) plus 20% fetal bovine serum gassed with 5% CO(2), 5% O(2) and 90% N(2). In Experiment I, embryos and culture media were placed in a covered embryological watch glass (EWG, control) or sealed in the lumen of a siliconized Foley catheter or a section of 1) latex tubing, 2) tygon tubing or 3) silastic tubing. In Experiment II, embryos were placed in EWG and cultured alone (control) or cocultured with sections of 1) tygon tubing, 2) silastic tubing or 3) latex tubing. In Experiment III, embryos were cultured in covered plastic petri dishes containing 15 ml of media, alone (control) or co-cultured with two new plunger tips from sterile Monoject syringes. All embryos were cultured at 32 to 34 degrees C for 24 h. The Criterion used for development was two or more cellular divisions within the 24-h period. Embryo development in Experiment I was lower (P<0.05) in latex (0%) and tygon (24%) tubing and in the siliconized Foley catheter (2%) than in silastic tubing (51%) and the EWG (46%), which did not differ. Experiment II embryos that were co-cultured with latex tubing (5%) showed very little development as compared with those co-cultured with tygon tubing (76%), silastic tubing (76%) and EWG (93%), the last of which were not significantly different. Embryos co-cultured with Monoject syringe plunger tips had a reduced embryo development rate compared to embryos in the control group (0% vs 52%). Although the embryos did not remain in contact with these seemingly toxic materials for prolonged periods, our results indicate that a significant reduction in embryo viability may occur due to this exposure.

Journal Article↗
PubMed1996

Survival rates and sex ratio of bovine IVE embryos frozen at different developmental stages on day 7.

Two experiments were designed to determine the effects of stage of development on Day 7 of in vitro-produced bovine embryos on survival after deep freezing and on sex ratio. Bovine IVF embryos and bovine oviductal epithelial cells (BOEC) were co-cultured in TCM-199 and, on Day 7 after insemination (Day 0), were morphologically evaluated and divided into groups by developmental stage. In Experiment 1, embryos classified as early blastocysts, blastocysts and full-expanding blastocysts were randomly subdivided into 2 groups by replicate: 50% of the embryos were placed immediately in a new BOEC co-culture (fresh group), while the other 50% were frozen, thawed and placed in a new BOEC co-culture (frozen/thawed group). Embryos were frozen in 1.5 M glycerol using a standard slow cooling technique. Fresh and frozen/thawed embryos were compared for survival rate (embryos hatching/hatched) in BOEC co-culture over the following 3 d (i.e., Days 7 to 10). The overall survival of the 425 embryos (early to full-expanding blastocysts) was 33% and was not different between fresh (35%) and frozen/thawed (30%) embryos. Survival of embryos cultured fresh or after freezing/thawing was higher for full-expanding blastocysts than for early blastocysts or for blastocysts, both of which were not different. In Experiment 2, all frozen/thawed embryos used in Experiment 1 plus all morulae and hatched blastocysts collected and frozen on Day 7 without regard to survival were sexed utilizing the polymerase chain reaction (PCR) technique. Sex of the embryos, by stage of development on Day 7, was determined in order to compare the rate of development in BOEC co-culture with the sex ratio (percentage of males). A total of 235 embryos was sex-determined with an overall percentage of males of 51%, which was not different from the expected 1:1 sex ratio. Both full-expanding blastocysts and hatched blastocysts had a significantly higher (P < 0.05) proportion of males (68 and 100%, respectively), while morulae had a significantly lower proportion of males (24%). Early blastocysts and blastocysts did not differ from a 1:1 sex ratio. The results indicate that male embryos develop faster in vitro than female embryos. The higher survival rate of full-expanding blastocysts after freezing/thawing, and the production of a higher number of males than females among embryos of this developmental stage suggest that a greater number of male fetuses may result from the successful freezing and transfer of in vitro-produced bovine embryos.

Journal Article↗
PubMed2001

Effect of cooling rate and partial removal of yolk on the chilling injury in zebrafish (Danio rerio) embryos.

High chilling sensitivity is one of the main obstacles to successful cryopreservation of zebrafish embryos. So far the nature of the chilling injury in fish embryos has not been clear. The aim of this study is to investigate the effect of cooling rate and partial removal of yolk on chilling injury in zebrafish embryos. Zebrafish embryos at 64-cell, 50%-epiboly, 6-somite and prim-6 stages were cooled to either 0 degrees C or -5 degrees C at three different cooling rates: slow (0.3 degrees C/min or 1 degree C/min), moderate (30 degrees C/min), and rapid (approximately 300 degrees C/min). After chilling, embryos were warmed in a 26 degrees C water bath, followed by 3-day culturing in EM at 26 +/- 1 degrees C for survival assessment. When embryos were cooled to 0 degrees C for up to 30 min, 64-cell embryos had higher survival after rapid cooling than when they were cooled at a slower rate. When 64-cell embryos were held at -5 degrees C for 1 min, their survival decreased greatly after both slow and rapid cooling. The effect of cooling rate on the survival of 50%-epiboly and 6-somite embryos was not significant after 1 h exposure at 0 degrees C and 1 min exposure at -5 degrees C. However, rapid cooling resulted in significantly lower embryo survival than a cooling rate of 30 degrees C/min or 1 degree C/min after 1 h exposure to 0 degrees C for prim-6 stage or 1 h exposure to -5 degrees C for all stages. Chilling injury in 64-cell embryos appears to be a consequence of exposure time at low temperatures rather than a consequence of rapid cooling. Results also indicate that chilling injury in later stage embryos (50%-epiboly, 6-somite and prim-6) is a consequence of the combination of rapid cooling and exposure time at low temperatures. Dechorionated prim-6 embryos were punctured and about half of yolk was removed. After 24 h culture at 26 +/- 1 degrees C after removal of yolk, the yolk-reduced embryos showed higher embryo survival than did control embryos after rapid cooling to -5 degrees C for 10 to 60 min. Results suggest that cold shock injury after rapid cooling can be mitigated after partial removal of yolk at the prim-6 stage. These findings help us to understand the nature of chilling sensitivity of fish embryos and to develop protocols for their cryopreservation.

Animals↗
PubMed2004

[Cloning of compaction-related gene Crg1 of mouse preimplantation embryos].

A total of 181 8-cell embryos and 241 8-cell compacted embryos were collected respectively from C57BL/6 mouse and their cDNA was synthesized by SMART-PCR. Then PCR productions after SSH were cloned into pUCm-T vector according to the size after isolated and purified. Choice the positive clones for sequencing after being confirmed with PCR. All fragment cloned were blastered matching in GenBank for homology analysis. On the base of this work, we select two ESTs (GenBank accession No.: BQ740263 and BQ740251), which were reverse complementary to each other, assemble into a cDNA fragment, which include a whole open read frame (ORF) and submitted to GenBank with an accession number, AY134859. The target fragment were amplified from cDNA of mouse compacted embryos using primers designed according to the ORF and cloned into pUCm-T vector and sequenced. It's confirmed that the gene, Crg1, was the same as AY134859. Crg1 were also blastered matching in GenBank for homology analysis and mapped by database analyses. RT-PCR analysis of Crg1 were done in series mouse embryos, mouse embryo stem cell and mouse embryo fibroblast. We analyzed the expression of Crg1 in 8-cell embryos and 8-cell compacted embryos using semi-quantity RT-PCR. Northern-blot analysis of Crg1 expression in some adult mouse tissues was done too. The results showed that the length of Crg1 is 810 bp. It include only one exon and code a 150 amino acid protein with a theoretical molecular weight of 17670.34 Dalton. The protein is similar to a protein encoded by a known gene, Stella. Crg1 was mapped to chromosome 14 by database analyses. RT-PCR analysis shows that Crg1 expressed in series mouse embryos (2-cell embryos, 4-cell embryos, 8-cell embryos, compacted embryos and blastocyst), and expressed little higher in compacted embryos. Crg1 also expressed in mouse embryo stem cell but not expressed in mouse embryo fibroblast. The gene only expressed weakly in adult mouse ovary, but not expressed in other adult mouse tissues (brain, spleen, heart, skeletal muscle, kidney, testis, epididymis, liver and lung). So, Crg1 may related to compaction in compacted embryos and maintaining cell's pluripotentiality.

Amino Acid Sequence↗
PubMed1997

Multiple pregnancy rate and embryo number transferred during in vitro fertilization.

OBJECTIVE: Our goal was to achieve a good pregnancy rate after in vitro fertilization; more than one embryo, if available, is transferred to the uterine cavity. This is a recognition of the low implantation rates of embryos from in vitro fertilization. A consequence of this can be high-order multiple implantation with obstetric complications. STUDY DESIGN: Retrospectively, we reviewed 42 months' in vitro fertilization experience; we related the number of embryos transferred and the pregnancy outcome. During this period 2173 fresh and frozen-thawed embryo transfers were performed. One to six embryos were transferred to women whose average age was 34.4 years (range 21 to 49). RESULTS: A total of 734 delivered pregnancies (33.8% per embryo transfer) was analyzed according to whether they were single or multiple, and this was related to the original number of embryos transferred. The overall multiple pregnancy rate was 31.3% (24.7% twins, 5.8% triplets, 0.8% quadruplets). CONCLUSIONS: There was a trend toward a higher pregnancy rate with more embryos transferred. The embryonic implantation rate, which reflects the number of embryos that implant per total transferred, was not significantly different in any one group, except in older women in whom more than one embryo was transferred. Whereas greater numbers of embryos (more than three) were transferred in couples with a poorer prognosis for successful in vitro fertilization (e.g., older women [> 36 years old], previous failure of in vitro fertilization, poor embryo quality, or severe male factor causing infertility), there still remained a significant trend toward a higher pregnancy rate when more embryos were transferred. The embryonic implantation rate did not decline in the poorer-prognosis groups (more than three embryos transferred), yet the multiple pregnancy rate was increased. Technologic procedures such as embryo biopsy for aneuploidy screening are proposed as one means to reduce embryo numbers transferred without decreasing the overall pregnancy rate.

Adult↗
PubMed2004

Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions.

For practical applications of porcine embryo transfer (ET) it is important to develop feasible embryo storage conditions. The aim of the present study was to evaluate the effect of short-term storage (24 h) on the quality of in vivo produced porcine embryos. Three temperatures 18, 25 and 38 degrees C and three different media: Dulbecco's phosphate buffered saline (DPBS), TCM199 and Emcare, were tested for two different embryo ages: D4 embryos (collected 144 h after hCG treatment) and D5 embryos (collected 168 h after hCG). After slaughter of the donor gilts, embryos were collected and transported at 25 degrees C to the lab where morulas and blastocyst were selected (D4 n = 222; D5 n = 167) and randomly used as controls or distributed over the treatment groups. Developmental stage and embryo diameter were assessed by normal light microscopy, while total number of cells and incidence of apoptosis were assessed using a fluorescent embryo quality staining technique that combines three different dyes: Ethidium Homodimer (EthD-1), TUNEL and Hoechst 33342. Following 24 h storage, D5 embryos had higher rates of hatching (24%) and degeneration (9%) compared to D4 embryos (10 and 4%, respectively; P < 0.05). Embryos stored at 38 degrees C had higher rates of hatching (37%) compared to those ones stored at 25 degrees C (13%) or 18 degrees C (0%; P < 0.01). More embryos hatched when stored in medium Dulbecco's phosphate buffered saline (DPBS) or in TCM199 compared to those stored in Emcare (P < 0.05). A higher percentage of embryos stored at 18 degrees C degenerated compared to those stored at 25 or 38 degrees C (P < 0.01). No significant increase in apoptosis was observed after storage compared to the rates of apoptosis at 0 h (controls) or between the different storage groups. Based on the results we conclude that D4 porcine embryos produced in vivo, selected under normal light microscopy and stored at 25 degrees C in a serum free medium for 24 h will have a suitable developmental stage for ET and a high embryo quality.

Animals↗
PubMed1990

Effect on embryo survival of short-term exposure to the uterine environment of two selected lines of mice.

Two genetic lines of mice (Mus musculus), one selected for high embryo survival (Line E) and the other for small litter size (Line CN-), were used as models to study preimplantation embryo survival. The two lines displayed similar ovulation rates, but significantly lower embryo survival to implantation was observed in Line CN- females (P less than 0.01). The effect of the uterine environment on embryo survival was examined by incubating embryos in pseudopregnant host females for 26 hours beginning at 1500 hours on day 2 (day of copulatory plug = day 0). Embryos were then removed from the host and transferred to recipient uteri of the same genetic line as the embryos for development to term. Survival of embryos was significantly greater after exposure to Line E hosts (P less than 0.01). Survival of Line E embryos was reduced from 71% in Line E hosts to 28% in Line CN- hosts. Survival of Line CN- embryos increased from 17% in Line CN- hosts to 70% in Line E hosts. Embryos also were cultured in vitro in uterine flushings collected from each line. More Line E embryos developed to the blastocyst stage than did Line CN- embryos (P less than 0.01). Development of Line CN- (P less than 0.01) embryos, but not Line E embryos, was affected by the line from which uterine flushings were collected. These lines of mice provide a useful model for the study of the physiological basis for differences in embryo survival due to genetic selection.

Animals↗
PubMed2002

Embryo freezing for preventing Ovarian Hyperstimulation Syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic potentially life threatening condition resulting from an excessive ovarian stimulation. Its reported incidence varies from 1% to 10% of in vitro fertilization (IVF) cycles. The factors leading to this syndrome have not been completely explained. It seems likely that the release of vasoactive substances, secreted by the ovaries under human chorionic gonadotropin (hCG) stimulation plays a key role in triggering this syndrome. The hallmark of this condition, is a massive shift of fluid from the intra-vascular compartment to the third space resulting in profound intra-vascular depletion and hemoconcentration. OBJECTIVES: To evaluate (i) the effectiveness of cryopreservation (embryo freezing) for the prevention of OHSS when compared with human intra-venous albumin infusion and (ii) the effectiveness of the elective cryopreservation (embryo freezing ) of all embryos for the prevention of OHSS when compared with fresh embryo transfer. SEARCH STRATEGY: Publications in the literature that describe or may describe randomised controlled trials of both human intra-venous albumin and freezing of all embryos in the management of OHSS as consequence of the superovulation in assisted reproduction techniques (ART) cycles were searched. The Cochrane Menstrual Disorders and Subfertility Review Group specialised register of controlled trials was searched. In addition, MEDLINE (PUBMED 1985 to 2001), EMBASE (1985 to 2001), CINHAL (1985 to 2001) and the National Research Register were searched SELECTION CRITERIA: Randomised controlled trials (RCTs) in which either human intra-venous albumin or cryopreservation of all embryos were used as a therapeutic approach to OHSS were included. The participants were women of reproductive age who were down regulated by gonadotrophin-releasing hormone-analogue (GnRH-a), undergoing superovulation in in vitro fertilization/intra-cytoplasmic sperm injection (IVF/ICSI) cycles. DATA COLLECTION AND ANALYSIS: Two reviewers, Mr N.N. Amso (NNA) and Dr A. D'Angelo (ADA) scanned the titles and the abstracts of the reports identified by electronic searching in order to find relevant papers. One reviewer (ADA) obtained copies of the full text articles and made copies for the other reviewer (NNA) in which details of authors, institution, results and discussion were removed in order to assess their eligibility for inclusion. Then, both reviewers extracted data independently using forms designed according to Cochrane guidelines. Disagreements were resolved by discussion. Additional information on the trial methodology or data were requested by writing to the corresponding authors directly. The interventions compared were cryopreservation (embryo freezing) versus intra-venous human albumin administration and elective cryopreservation of all embryos versus fresh embryo transfer. The primary outcomes were: incidence of moderate and severe OHSS versus nil/mild OHSS, clinical pregnancies/woman. The secondary outcomes were: number of oocytes retrieved, number of oocytes fertilized, number of embryos transferred, number of embryos frozen, multiple pregnancy rate, live birth rate, number of women admitted to the hospital as inpatient or outpatient and time to the next menstrual period (resolution time). Statistical analysis was performed in accordance with the Cochrane Menstrual Disorders and Subfertility Group guidelines. MAIN RESULTS: Seventeen studies were identified, two of which met our inclusion criteria. One study was included where cryopreservation (embryo freezing) was compared with intra-venous human albumin administration (Shaker 1996) and one study was included where elective cryopreservation of all embryos was compared with fresh embryo transfer (Ferraretti 1999). When cryopreservation was compared with intra-venous human albumin administration no difference was found in all the outcomes examined between the two groups. When elective cryopreservation of all embryos was compared with fresh embryo transfer no difference was found in all the outcomes examined between the two groups. REVIEWER'S CONCLUSIONS: This review has showed that there is insufficient evidence to support routine cryopreservation and insufficient evidence for the relative merits of intra-venous albumin versus cryopreservation.

Albumins↗
PubMed2007

Morphologic and histologic comparisons between in vivo and nuclear transfer derived porcine embryos.

Nuclear transfer (NT) is an inefficient but invaluable tool of the biotechnology industry. This study looked at abnormalities associated with peri-implantation NT porcine embryos. Four experimental groups were examined: nonpregnant animals, in vivo pregnant animals, NT recipients, and manipulation control embryos (MC). Embryos (Day 10, 12, or 14) were evaluated for embryonic disc diameter, gross morphology, nucleoli density, and mitotic figure index. Day 12 (P < or = 0.03) and Day 14 (P < or = 0.01) NT embryos had increased numbers of nucleoli, and Day 14 NT embryos had an increased (P < or = 0.03) mitotic index compared to in vivo and MC embryos. In vivo produced Day 14 embryos had increased (P < or = 0.01) disk diameters when compared to other embryos except for MC Day 14, which also showed increases (P < or = 0.01) in disk diameter except when compared to in vivo produced Day 12 and Day 14 embryos. In vivo produced Day 12 had greater (P < or = 0.03) disk diameters when compared to NT and MC embryos except for MC Day 14, and in vivo produced Day 14 embryos, which had a significantly increased (P < or = 0.01) disk diameter. In vivo produced Day 14 embryos were morphologically more advanced (P < or = 0.01) than Day 14 NT and MC counterparts. NT embryos develop at a slower rate than their in vivo produced counterparts. The increase in nucleoli and mitotic index of NT embryos suggest the cell cycle may be affected or the NT embryos are employing other means to compensate for slow development. The techniques used during NT also appear to compromise embryo development.

Animals↗
PubMed2003

Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine.

Cryopreservation can cause changes to the genetic material of cells, but the mechanism and significance of these changes are still unknown. It has been suggested that some damage to the sperm genome could be repaired by the DNA repair system of the oocyte after fertilisation. Caffeine has been reported to be an inhibitor of such repair processes. In this study the effect of caffeine on the repair system of Loach (Misgurnus fossilis) oocytes was investigated. Loach eggs were fertilised using cryopreserved sperm. Embryos derived from cryopreserved sperm were exposed to 2.6mM caffeine for 1h after fertilisation. The experiments were carried out using 32313 embryos from four females and eight males. Embryo survival was evaluated for 46 h until the hatching stage. Reduction in embryo survival after 20th stage is generally believed to result from the failure in the genome function of embryos. Cryopreservation of sperm significantly decreased embryo survival (53.4+/-2.8% compared to 68.4+/-2.8% of control) after the 20th stage. However, the addition of caffeine to the embryos derived from cryopreserved sperm, in contrast to our expectation, significantly increased survival of loach embryos (70.9+/-2.8% compared to 53.4+/-2.8% of embryos derived from cryopreserved sperm in the absence of caffeine). The effect of individual donors of sperm and eggs on overall embryo survival was also studied. Whilst no significant differences were observed between males, the effect of individual females on embryo survival was significant. The analysis of embryo survival at different developmental stages showed that embryo survival both before and after 20th stage decreased with embryo development. When fresh sperm were used the decline of embryo survival with development was more pronounced compared with those embryos derived from cryopreserved sperm. Possible explanations of these effects are presented.

Animals↗