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PubMed2006

Oxidative stress status and development of late organogenesis stage rat whole embryos cultured from gestational days 13.5 to 14.5.

A new method for culturing rodent whole embryos at the late organogenesis stage was developed using a roller-bottle system with intermittent gassing. Rat embryos were cultured for 24 h from gestational day (GD) 13.5 to 14.5. Growth and metabolic comparisons were made between in vivo embryos and embryos of the same GD cultured under various media and conditions. Crown-rump length, head length and protein content were used as growth indicators. Biologic markers such as embryonic tissue concentration of glutathione (GSH), glutathione disulfide (GSSG) and lipid peroxidation were used as assessments of metabolic activity in terms of oxidative stress. Embryos cultured with media consisting of either 15% or 20% male rat serum and balanced with Dulbecco's Modified Eagle Medium (DMEM) were found to most closely match in vivo embryos. 6 h gassing intervals and 5 mL medium volume/embryo provided optimal conditions for cultured embryos. By shortening the 24 h embryo culture period to 12 h, embryonic haemorrhaging was avoided. Moreover, the 12-h cultured embryos showed similar redox GSH/GSSG ratios and similar GSH content to the in vivo embryos, which was not observed in the embryos cultured under 24 h culture conditions. The present work demonstrates the utility of late organogenesis stage embryo culture as a model for the assessment of in vivo embryonic growth and oxidative stress indices.

Animals↗
PubMed2002

Improvement in early human embryo development using new formulation sequential stage-specific culture media.

OBJECTIVE: To determine whether altering selected components of sequential culture media can improve early development variables of human embryos. DESIGN: Prospective, randomized, sibling oocyte split trial. SETTING: Private ART center. PATIENT(S): Two hundred eight undergoing treatment with in vitro fertilization or microinjection. INTERVENTION(S): Oocytes from each patient were randomly allocated to fertilization and cleavage media of a control and a trial culture medium formulation. MAIN OUTCOME MEASURE(S): Rates of fertilization, cleavage, and uncontrolled division; average embryo morphology score; blastomeres per embryo; embryo score parameter (number of blastomeres x embryo morphology grade); and embryo utilization. The trial media resulted in a higher fertilization rate, higher cleavage rate, lower rate of uncontrolled division, higher number of blastomeres per embryo, higher average embryo morphology score, a higher embryo score parameter, and higher embryo utilization rate compared to the control media. All differences were statistically significant. CONCLUSION(S): Improved sequential stage-specific culture media can reduce the occurrence of severe human embryo fragmentation and improve developmental variables in early IVF- and ICSI-generated embryos.

Adult↗
PubMed1988

Implantation potential of each pre-embryo in multiple pregnancies obtained by in vitro fertilization seems to be different.

Endometrial receptivity and pre-embryo quality are the main factors determining embryo implantation in in vitro fertilization. If one embryo implants normally and goes to term, the endometrium can be assumed to be normal. Eighty-one patients (March 1981 to 1987) had transfers of multiple pre-embryos, resulting in multiple pregnancies with at least one reaching term. The number of gestational sacs at 8 to 12 weeks of pregnancy, at 20 weeks, and the number of term pregnancies were studied longitudinally. Twelve patients (14.8%) had 2 pre-embryos transferred, 19 (23.5%) had 3, 21 (25.9%) had 4, 17 (21.0%) had 5, 9 (11.1%) had 6, and 3 (3.7%) had 7. At 20 weeks 18 patients (22.2%) had a single viable fetus, 56 (69.1%) had twins, 6 (7.4%) had triplets, and 1 (1.2%) had quadruplets. A total of 325 pre-embryos were transferred; 178 gestational sacs were observed at 8 to 12 weeks, but only 153 showed fetal heartbeat. Total embryo loss at 8 to 12 weeks was 53.0%; when the "index" embryo was excluded, it reached 61%. Regardless of the number of pre-embryos transferred, only 2.2 embryos on average were able to establish a normal pregnancy. (A)synchronism of the pre-embryos did not affect outcome. Results suggest that with normal endometrial receptivity, the implantation potential of each pre-embryo is different.

Embryo Transfer↗
PubMed2003

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro.

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.

Animals↗
PubMed2003

Effect of recombinant bovine somatotropin on superovulatory response and recipient pregnancy rates in a commercial embryo transfer program.

Recombinant bovine somatotropin (rbST) has been shown to increase follicular growth in cattle and some studies have demonstrated an increase in superovulatory response for rbST-treated cows. Pregnancy rates have also been shown to increase when rbST was administered around the time of insemination or prior to embryo transfer. The application of rbST for the purpose of increasing superovulatory responses of donor cows and increasing pregnancy rates of recipient heifers was tested in a commercial embryo transfer program. In Experiment 1, embryo donor cows (n = 56) underwent three cycles of control superovulation (two before and one after weaning) and subsequently underwent up to four additional superovulations while being treated with either rbST (500 mg sustained-release rbST; Posilac, Monsanto, St. Louis, MO; n = 28) or excipient (control; n = 28) once every 14 days. In Experiment 2, lactating embryo donor cows (n = 37) underwent a control superovulation and then underwent a superovulation while lactating and being treated with either rbST (n = 16) or excipient (n = 21). In Experiment 3, embryo recipient heifers that were being implanted with either in vitro or in vivo produced embryos were treated with either rbST (n = 146) or excipient (n = 143) at the time of embryo transfer. Treatment of non-lactating (Experiment 1) or lactating (Experiment 2) donor cows with rbST during repeated superovulation did not affect the number of corpora lutea, the sum of transferable embryos, degenerate embryos, and unfertilized oocytes, or the number of transferable embryos. Treatment of recipient heifers with rbST (Experiment 3) did not affect pregnancy rates for either in vitro or in vivo produced embryos. We conclude that superovulatory response and pregnancy rates (respectively) are similar to control for rbST-treated cows undergoing repeated superovulations and rbST-treated recipient heifers treated at the time of embryo transfer.

Animals↗
PubMed2003

Development of in vitro matured, in vitro fertilized domestic cat embryos following cryopreservation, culture and transfer.

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 degrees C in 5% O(2), 5% CO(2), 90% N(2) for 24h. In Experiment 1, after IVM/IVF, on Day 2 (n=56), Day 4 (n=48) and Day 5 (n=42) of IVC, embryos were equilibrated for 10 min at 22 degrees C in HEPES (15m M) Tyrode's (HeTy) with 1.4M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 degrees C/min to -6.0 degrees C and held for 10 min. After seeding, cooling resumed at 0.3 degrees C/min to -30 degrees C and after a 10 min hold, straws were plunged into liquid nitrogen (LN(2)). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (+/-S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152+/-19, Day 4=124+/-20, Day 5=121+/-24) than in controls (Day 2=141+/-25, Day 4=169+/-21, Day 5=172+/-19). In Experiment 2, embryos frozen on Day 2 (n=68), Day 4 (n=49) or Day 5 (n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6+/-2.4), 4 (mean=12.2+/-3.7) and 6 (mean=12.0+/-1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.

Animals↗
PubMed2003

Chromosomal abnormalities in Day-6, in vitro-produced pig embryos.

A cytogenetic study was undertaken to quantify, by chromosomal karyotyping, the incidence and type of chromosomal abnormalities present in Day-6 in vitro-produced (IVP) porcine embryos. Morphologically normal Day-6 blastocysts (n=318) were fixed and grouped into six classes according to the number of total cells (from < or =20 to 61-70). Of 248 embryos suitable for analysis, 97 (39.1%) displayed chromosomal abnormalities. The abnormalities included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%). Within polyploid embryos, triploidy and tetraploidy showed the highest incidence (56.5 and 27.5%, respectively); among mixoploid embryos, diploid-triploid embryos (2n/3n) were prevalent (36.8%). Overall, the mean cell number was 34.3 +/- 12.1 and the mitotic index was 8.6 +/- 6.1. Chromosomally abnormal embryos had fewer (P<0.01) total cells compared to normal (2n) embryos (31.8 +/- 1.3 versus 35.9 +/- 1.0). In addition, the incidence of polyploidy decreased as the number of cells increased, while that of mixoploidy did not differ. These data indicate that polyploidy affects a large percentage of IVP porcine embryos capable of developing to blastocysts and the incidence of chromosomal abnormalities is much higher than that reported previously in in vivo embryos in this species. Given the ability of morphologically normal embryos with an abnormal chromosome complement to undergo preimplantation development in vitro, and the inability to identify blastocysts with abnormal karyotype without cytogenetic analysis, careful consideration should be given to factors affecting ploidy of IVP embryos, especially the incidence of polyspermic fertilization, when evaluating criteria of a porcine in vitro embryo production scheme.

Animals↗
PubMed2002

Vitrification of in vitro produced bovine embryos: in vitro and in vivo evaluations.

The efficacy of different vitrification solutions to cryopreserve in vitro produced bovine blastocysts was evaluated based upon in vitro development of embryos in culture and on in vivo development of embryos transferred into recipients. In the first experiment, ethylene glycol + glycerol (Eg + Gly) + different sucrose concentrations were evaluated. There were no significant differences in development rates among solutions. As for hatching, the Eg + Gly + 0.1 M sucrose group had a greater rate as compared with Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in the evaluations of Day 6, Day 7 and Day 6 + Day 7 embryos; and, Eg + Gly + 0.3 M sucrose group had a greater rate as compared with the Eg + Gly + 0 M sucrose and Eg + Gly + 0.5 M sucrose groups in evaluations of Day 6 and Day 6 + Day 7 embryos. There were no significant differences in development and hatching rates between Day 6 and 7 in in vitro produced bovine embryos within each treatment group. There were significant differences in nuclei number after vitrification between Eg + Gly + 0.1 M and Eg + Gly + 0 M sucrose groups and the Eg + Gly + 0.5 M sucrose group. Pregnancy after 60 days of transfer and calving rates showed a difference between in vivo produced embryos freshly transferred and in vitro produced embryos vitrified with Eg + Gly + 0.3 M. There were no significant differences in gestation length and sex ratio between treatments. As for birth weight, there were significant differences between fresh in vivo produced embryos and all treatments of in vitro produced embryos. There were significant differences in dystocial parturition between in vivo produced embryos and all treatments with in vitro produced embryos. These results demonstrate that vitrification can be used successfully in the cryopreservation of in vitro produced bovine embryos, and that it might be considered for use in commercial programs.

Animals↗
PubMed2003

The contribution of the male to ovine embryogenesis in an in vitro embryo production system.

The differences in the embryo production potential of four rams used in a commercial embryo transfer program were examined in both in vivo and in vitro embryo production systems. Processing frozen-thawed spermatozoa through Percoll density gradients prior to in vitro insemination eliminated differences in the estimates of sperm viability between the four rams, and yet, differences in embryo production persisted throughout the in vitro culture period. However, there was no effect of ejaculate within ram on embryo production rates. In addition, the timing of the onset of the differences between the rams at each stage of in vitro embryo development were revealed. Ram 2 differed from ram 4 in the proportion of fertilized oocytes at 17 h post-insemination (pi) and by 52 h, ram 3 differed from ram 4 in the proportion of cleaved embryos, and the observed differences between ram 1 and ram 2 in their blastocyst production were initiated prior to activation of the embryonic genome. Once differences in embryo development rates were detected among the four rams, they persisted throughout the in vitro culture period. The reduced in vitro fertilization (IVF) rates from ram 2 compared with the other rams was paralleled in vivo by the significantly lower proportion of embryos recovered from ewes mated to ram 2, and this was further exacerbated by a significantly lower embryo survival rate after transfer. However, the subtle differences observed in the timing of the contribution of each sire to embryo development during in vitro culture were not able to be detected in vivo. However, the higher proportions of transferable quality blastocysts obtained from ewes mated to ram 4 did not result in increased embryo survival throughout the remainder of gestation. Therefore, in this study, the blastocyst production potential for a particular sire, either in vitro or in vivo, does not necessarily reflect the potential for the production of live offspring.

Animals↗
PubMed2005

Gene expression in the preimplantation embryo: in-vitro developmental changes.

The regulation of early embryo development and the mechanism of implantation remains poorly understood, due to the large number of genes and the complexity of the systems involved. The effect of in-vitro culture on embryos also remains unclear, which raises concerns about the safety of assisted reproductive technology. Changes in the expression of several individual genes in cultured embryos have been reported previously, but a large-scale comparison has not yet been performed to investigate the effects of in-vitro culture systems on embryo development. This study investigated established gene expression profiles of more that 20,000 genes from in-vitro cultured mouse embryos at eight different stages (oocytes, zygote, 2-, 4-, 8-cell embryo, compacting embryo, morula and blastocyst) using microarray technology, and compared these profiles with in-vivo embryos. In most stages of development there was little significant difference in overall expression patterns between in-vitro and in-vivo embryos. In addition, the expression patterns of developmentally important genes from several different categories, such as apoptosis, glycolysis, adhesion and methylation, were examined and compared between in-vitro and in-vivo embryos. Among the genes examined, DNA methyltransferase 1 (DNMT1) shows a significantly higher (P < 0.05) expression level in cultured embryos. Cadherin-11 also demonstrates a slightly different pattern, although the difference is not statistically significant. All the other genes have remarkably similar expression patterns between in-vitro and in-vivo embryos throughout preimplantation stages.

Animals↗
PubMed2005

Eligibility of cryopreserved human embryos for stem cell research in Canada.

OBJECTIVE: To determine whether cryopreserved embryos in Canadian in vitro fertilization (IVF) clinics available (in theory) for research use are actually eligible (in practice) for stem cell research in accordance with the Assisted Human Reproduction Act, the Canadian Institutes of Health Research's Human Pluripotent Stem Cell Research: Guidelines for CIHR-Funded Research, and the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans. METHODS: Copies of current and past patient information sheets and consent forms for embryo cryopreservation, the future disposition of cryopreserved embryos, and the donation of cryopreserved embryos for research were requested from Canadian IVF clinics. Fourteen of 24 clinics (response rate 58%) provided one or more of the requested documents. RESULTS: A review of the documents currently in use showed that, as of May 2005, all 14 responding clinics require an advance directive for the eventual disposition of cryopreserved embryos. Ten of these 14 clinics allow the donation of cryopreserved embryos for research. Only three of these 10 clinics, however, satisfy in writing the disclosure requirements for embryo research at the time of initial consent for the cryopreservation and future disposition of embryos. Only one clinic specifically identifies the option of embryonic stem cell research. CONCLUSION: Only three of the responding Canadian IVF clinics provide written details about future donation of cryopreserved embryos for research use and about the need for re-contact in accordance with relevant legislation and guidelines. Two of these three clinics, however, include disposition options that may not be legally available. In the event of death or loss of contact, it will not be possible to get project-specific research consent from the legitimate embryo providers as required by law. These same two clinics also fail to mention embryonic stem cell research as one of the research categories. It follows that we have identified only one Canadian IVF clinic with cryopreserved embryos unequivocally eligible for stem cell research, provided the forms we reviewed were used to obtain consent for the cryopreservation of those embryos.

Canada↗
PubMed1985

In vitro development of zinc-deficient and replete rat embryos.

The development of the zinc-deficient rat embryo has been studied in vitro using embryo culture techniques. Normal 9.5 day embryos cultured for 48 h in serum obtained from zinc-deficient rats grew and developed to the same extent as those cultured in zinc-replete serum. Embryos from dams which had been fed a zinc-deficient diet since mating were also removed for culture. Such zinc-deficient embryos fell into two broad morphological categories. One group appeared identical to the normal embryos, while the others had apparently normal visceral yolk sacs but small embryonic poles and retarded or abnormal embryonic development. Culture of the first group in either zinc-deficient or replete serum produced morphologically normal embryos; however, those which appeared abnormal at day 9.5 were grossly malformed after 48 h incubation in either sera. When embryos were cultured in the presence of 65Zinc, the most severely affected zinc-deficient embryos accumulated as much zinc as the zinc-replete and apparently unaffected zinc-deficient embryos, indicating that the malformations do not arise from an inability of the embryo or yolk sac to accumulate zinc from the surrounding fluid. The results from these studies suggest that the teratogenic effects of zinc deficiency cannot be induced by direct culture of zinc-replete embryos in zinc-deficient serum. Furthermore, it would appear that maternal zinc deficiency can exert its teratological influence prior to day 9.5 of gestation and that these effects are not readily reversible.

Abnormalities, Multiple↗
PubMed2006

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation.

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.

Angiopoietin-1↗
PubMed1992

Metabolic and developmental responses of preimplantation embryos to platelet activating factor (PAF).

Platelet activating factor (PAF) is an ether phospholipid produced by preimplantation embryos of a number of species. Production of PAF by embryos has been measured by detecting thrombocytopenia in a splenectomized mouse bioassay, platelet aggregation bioassays in vitro and a specific radioimmunoassay. Production is highly variable and is adversely affected by culture in vitro. It has, however, been correlated with morphology, development rates in vitro and the pregnancy potential of embryos following transfer. Investigations using PAF-antagonists have established an essential role for PAF in early pregnancy. Together with studies that have shown PAF to have direct effects on embryonic metabolism during culture in vitro, these observations suggest that PAF acts as an embryonic autocoid. Hence, a major site of action for embryo-derived PAF in vivo is the embryo itself. Supplementation of embryo culture media with PAF had no effect on the rate of development in vitro of 2-cell mouse embryos through to the blastocyst stage. However, PAF increased cell numbers of blastocysts cultured from the 2-cell stage and the mitotic index of embryos at both the 8-cell and blastocyst stages. Supplementation of culture media with PAF has also been shown to increase the implantation potential of both mouse and human embryos cultured in vitro. In the mouse, the effect of PAF in enhancing implantation rates was most evident when the developmental potential of untreated embryos was suboptimal. These observations suggest that the production of embryo-derived PAF is one limiting factor in maintaining the viability of embryos cultured in vitro.

Animals↗
PubMed2006

Chromosomal aneuploidy in African Wildcat somatic cells and cloned embryos.

In the present study, we compared the incidence of aneuploidy in in vitro fertilized domestic cat embryos (DSH-IVF) with that of African Wildcat (AWC) cloned embryos reconstructed with AWC fibroblast donor cells from different passages (AWC-NT). Fibroblast cells were cultured to passages 1 (P1), 3 (P3), 4 (P4), and 9 (P9), after which cells at each passage were karyotyped and serum-starved before being frozen for nuclear transfer. AWC-NT embryos were produced by fusion of a single AWC somatic cell at P1, P3, P4, or P9 to enucleated domestic cat cytoplast derived from in vitro matured (IVU) oocytes. DSH-IVF embryos were produced after IVU oocytes were fertilized in vitro with domestic cat spermatozoa. To determine chromosome numbers, embryos (2-4-cell) or fibroblast cells were cultured in medium containing 0.28 microg/mL of Colcemid for 22-24 h or 15-24 h, respectively. Subsequently, embryos and cells were placed in hypotonic solution, fixed, and stained for analysis of chromosome spreads by bright field microscopy. Chromosomal abnormalities in AWC fibroblast cells increased progressively during culture in vitro: P1 (43%), P3 (46%), P4 (62%), and P9 (59%). In fibroblast cells, hypoploidy (94/202, 46%) was the major chromosomal abnormality, and it occurred more frequently than hyperploidy (14/202, 7%; p < 0.05). While the percentage of hyperploid cells remained stable during all passages, the proportion of hypoploidy in fibroblast cells increased significantly after P4. The overall incidence of chromosomal abnormalities in AWC-NT embryos at P1 (45%), P3 (60%), and P4 (50%) was similar to that of the fibroblast cells from which they were derived; however, the incidence was higher for embryos reconstructed with donor fibroblasts at P9 (89%). Hypoploidy was the most common chromosomal abnormality observed in either AWC-NT or DSH-IVF embryos. AWCNT embryos reconstructed with donor cells at early passages (P1, P3, and P4) had similar frequencies of chromosomal diploidy, as did DSH-IVF embryos. Accordingly, based on the present results, for NT we are currently using cat donor cells at early passages, when the percentage of cells with chromosomal abnormalities is low. It is recommended that the chromosomal stability of each cell line be analyzed before use as NT donor cells to reduce the incidence of chromosomal anomalies in reconstructed embryos and to possibly produce a subsequent increase in cloning efficiency.

Aneuploidy↗
PubMed1998

Intracytoplasmic sperm injection, results in women older than 39, according to age and the number of embryos replaced in selective or non-selective transfers.

The aim of this study was to assess the results of intracytoplasmic sperm injection (ICSI) in a large cohort of women older than 39 according to age and to embryo transfer policy. In all, 736 ICSI cycles were analysed retrospectively. In 576 (78.3%) cycles an embryo transfer was carried out. The embryo transfer was defined as non-selective when all the available embryos were transferred, and as selective when fewer than the available number of embryos were replaced. A statistically significant gradual decrease in the number of embryos available for transfer, the number of good or excellent quality embryos available for transfer, the pregnancy rates, the clinical pregnancy rates, the implantation rates and the viable pregnancy rates was found with advancing age. No viable pregnancies ensued in women from 45 years old onwards. There was a statistically significant gradual increase in the pregnancy rates, the clinical pregnancy rates, the implantation rates and the viable pregnancy rates from non-selective to selective transfers. The results were similar in women with five or more embryos available, irrespective of the embryo transfer policy. It seems, therefore, that the ovarian reserve and the chances for a successful pregnancy decrease gradually with advancing age, and it is pointless to treat women from 45 years old onwards. A subgroup of patients with better ovarian response and more embryos available for transfer have higher chances of conception. Conception and implantation rates depend mainly on the quality of the transferred embryos. However, the implantation capacity of the embryos is generally lower irrespective of their good morphology.

Adult↗
PubMed2004

Traditional detection versus computer-controlled multilevel analysis of nuclear structures from donated human embryos.

BACKGROUND: Multinuclearity is known to correlate with decreased implantation and pregnancy rates. Thus, a valid detection of nuclear structures especially among otherwise good quality embryos may be of great importance in order to improve clinical outcome. In this study, we have compared traditional manual microscopic analysis with computer-controlled multilevel morphological assessment for analysis of nuclear status in human embryos. METHODS: In total, 84 donated 2- and 4-cell embryos with < or = 20% fragmentation from patients referred for IVF or ICSI treatment were included. Mono- and multinuclearity was recorded using traditional analysis as well as computer-controlled multilevel analysis of each intact embryo. Subsequently, the embryos were separated into individual blastomeres to assess the number of nuclear structures. All nuclear structures were fixed and stained for DNA. RESULTS: There was no significant difference (P = 1.0) between embryonic nuclear status detected by computer-controlled analysis of the intact embryos and of the separated blastomeres. Additionally, 100% of the fixed nuclear structures contained DNA. However, using traditional morphological analysis, significantly more embryos (26%) had incorrect nuclear status detected (P = 0.002). Further, the presence of <10% embryonic fragmentation had no impact on the correct detection of nuclear structures using the multilevel analysis. For embryos with 11-20% fragmentation, 86% of the nuclear structures detected in the separated blastomeres were found in the intact embryos. The mean diameter of nuclear structures was significantly decreased from 22.1 microm in mononucleate 2-cell embryos to 18.7 microm in mononucleate 4-cell embryos (P < 0.001). CONCLUSION: The results of this study indicate that the use of computer-controlled multilevel morphological analysis can improve the detection of nuclear structures in human embryos.

Adult↗
PubMed1994

The contribution of embryo cryopreservation to in-vitro fertilization/gamete intra-fallopian transfer: 8 years experience.

In this paper, the authors summarized their experience with embryo cryopreservation over an 8-year period. The results, therefore, reflect the long-term benefit of embryo cryopreservation to the overall in-vitro fertilization/gamete intra-Fallopian transfer (IVF/GIFT) programme and to the women who had embryos cryopreserved. The stable survival rate of thawed embryos and pregnancy rate, especially over the past 4 years, suggests that the results can reliably be used to evaluate the efficacy of the embryo cryopreservation programme. The ongoing pregnancy rate of frozen/thawed embryo transfer is 10.9%, comparable with the ongoing pregnancy rate of fresh IVF/embryo transfer in our unit over the same period. In addition to those factors known to affect the pregnancy rate in fresh IVF/GIFT cycles, such as age of the recipients and number of embryos transferred, the major factor affecting the efficacy of the cryopreservation programme is the number of oocytes retrieved in the initial stimulation cycle, and the number of embryos available for cryopreservation. The storage time of cryopreserved embryos will also have a significant effect on the realization of the total potential of embryo cryopreservation. Overall the contribution of cryopreservation to our IVF/GIFT programme is substantial, increasing pregnancy rate by 4%, while the greater net benefit, of course, is for the women who had embryos cryopreserved (pregnancy rate increased by 7%), especially for those who returned for frozen/thawed embryo transfer cycles (pregnancy rate increased by 11%).

Adult↗