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Embryo manipulation and experimentation.

I have argued that early human embryos are not human beings, and do not have normal rights. Like human sperm and ova, they are both alive and biologically human. However, they lack the physiological development necessary to sustain a capacity for sentience. If Ford is right, then they are not yet individual human organisms. But the more important point is that their lack of a capacity for sentience makes them inappropriate candidates for the ascription of moral rights. Thus, research on human embryos produced in vitro is not a wrong against them--at least so long as experimentally manipulated embryos are not returned to the womb, or artificially gestated to a stage at which they might become sentient. Some of the more difficult issues about embryo experimentation involve the rights of women as experimental subject and donors. The consent of both male and female gamete donors should normally be required for the production or experimental use of IVF embryos. (Possible exceptions might include cases in which one or both progenitors have died, and the survivor or other responsible family member wished to donate the (frozen) IVF embryos for research or other uses.) However, it is women's rights that are most apt to be endangered, for example, if the large scale therapeutic or commercial use of human embryos leads to a demand for large numbers of ova. Thus, it is vital that researchers and policy-makers heed feminist concerns about embryo research and the new biomedical technologies it may yield. Given adequate information and appropriate procedural protections, women are capable of making autonomous decisions about donating ova or embryos for biomedical research. But regulatory safeguards are needed to ensure against their being coerced, deceived, or manipulated into becoming ovum or embryo donors. As Daniel Callahan has detailed, biomedical technology has reached the point where we can no longer afford to provide everyone with all of the innovative therapies that might prove beneficial (Callahan, 1989, p 523). Thus, questions about the just and cost-effective distribution of medical resources will become increasingly difficult and important. Among these questions will be whether it is wise or just to invest large sums in the development of innovative methods of assisting human reproduction. We must also consider the interests of future persons who may suffer because of the methods used to bring them into existence.(ABSTRACT TRUNCATED AT 400 WORDS)

Beginning of Human Life↗

Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos.

Chicken embryos were chronically exposed to hypoxia (P(O(2)) approximately 110 mmHg) during development, and assessed for detrimental metabolic and morphological effects. Eggs were incubated in one of four groups: control (i.e. 151 mmHg), or treated with continuous 110 mmHg (15% O(2)) during days 1-6 (H1-6), 6-12 (H6-12), or 12-18 (H12-18) with normoxia during the remaining incubation. Metabolism (V(O(2))), body mass, hemoglobin (Hb) and hematocrit (Hct) were measured in embryos on days 12 and 18 of incubation and in day-old hatchlings. Ability to maintain V(O(2)) was acutely measured during a step-wise decrease in P(O(2)) from normoxia to hypoxia (55 mmHg). On day 12, V(O(2)) of H1-6 eggs were significantly lower than in the control and H6-12 eggs. P(crit) in H6-12 eggs was lower than in control and H1-6 eggs. Body mass of H1-6 and H6-12 embryos on day 12 was significantly lower than in control embryos, while in H6-12 embryos, Hct and Hb were higher. On day 18, H6-12 embryos had significantly lower V(O(2)) than control eggs. Body mass of H6-12 and H12-18 embryos was significantly lower than control embryos. Hct and Hb did not differ between treatments. In hatchlings, mass, Hb and Hct had returned to values statistically identical to controls. However, H6-12 embryos had significantly lower V(O(2)). Long-term hypoxia altered V(O(2)) when hypoxic incubation occurred during the middle third of incubation, but not during earlier or later incubation. Thus, chronic hypoxic exposure during critical periods in development altered the developmental physiological trajectories and modified the phenotypes of the developing embryos.

Animals↗

Attitudes of patients, healthcare professionals and ethicists towards embryonic stem cell research and donation of gametes and embryos in Germany.

Due to the Embryo Protection Act, creation of supernumerary embryos, as well as egg and embryo donation, is prohibited in Germany. Human stem cell research is regulated through the Act on stem cells that came into force in 2002. A cross-sectional survey of 101 IVF couples (n=202) in two fertility centres, and representative samples of healthcare professionals and ethicists (n=879), was carried out, and their attitudes towards embryonic stem cell research and donation of gametes and embryos compared. A clear majority of IVF couples favoured legalization of egg and embryo donation and embryonic stem cell research for various purposes. The willingness of couples to donate was related to purpose and to other independent influences. The majority of physicians voted for legalization of embryonic stem cell production from surplus embryos. Most human geneticists and obstetricians approved egg, but not embryo, donation to other couples. Ethicists and midwives were opposed to every kind of donation and research on surplus embryos. The IVF couples surveyed have positive attitudes towards donation and research using surplus embryos, whereas the healthcare professionals and ethicists are predominantly sceptical about most research activities destroying human embryos. This difference should be considered carefully in legal and ethical discussions on reprogenetics.

Attitude of Health Personnel↗

Outcome of assisted reproductive technologies after different embryo transfer strategies.

The outcomes of 1028 assisted reproductive technology cycles were studied retrospectively, considering two different periods of embryo transfer. In the first period, 262 cycles in women < 36 years old were studied, in which three embryos were transferred, followed by 157 cycles in women > or = 36 years, in which four embryos were transferred. In the second period, 332 cycles were evaluated in women < 36 years and 277 cycles in women > or = 36 years old, reducing the number of embryos transferred to two and three respectively. Embryos were only scored morphologically, and the best embryos were chosen for transfer. In the first period, in women < 36 years old, a clinical pregnancy rate of 55.7% was achieved, compared with 42.5% in the second period (P < 0.01). In women > or = 36 years old, the first period of embryo transfer showed a clinical pregnancy rate of 39.5%, compared with 28.5% in the second period (P < 0.01). The number of twin pregnancies in the three groups of patients in whom one to four embryos were transferred was not significantly different (24.2, 28.4, 24.8%). It is concluded that even with the biases induced by a retrospective study, the reduction in the number of embryos transferred, from three to two in women < 36 years of age, and from four to three in women > or = 36 years of age, without any selection other than pre-transfer morphological score, adversely affects the outcome of treatment, without a significant reduction in twin gestation rate. Other strategies are to be implemented in gametes and embryo selection, and patients must be aware that, even with a reduction in pregnancy rate, the goal is to achieve a high cumulative pregnancy rate, reducing the complications induced by multiple pregnancies.

Adult↗

Day 3 and day 5 morphological predictors of embryo viability.

Controlling multiple pregnancies in patients undergoing artificial reproductive procedures requires consideration of single embryo transfers. Therefore, refinements for embryo evaluation are needed that select for the most developmentally competent embryo. The present study was designed to identify day 3 and day 5 morphological predictors of viability following transfers in which the morphology and fate of each embryo was precisely determined. Assessments on day 3 included cell number, and the extent of fragmentation and asymmetry, and on day 5, the developmental stage. Embryos resulting in a viable fetus at 11 weeks gestation were considered developmentally competent. The relationships among individual and collective embryo morphological characteristics were evaluated. Analysis of the interactions among morphological characteristics of embryos transferred on day 3 enabled identification of a multivariable selection order. Assessment of day 5 embryos revealed that expanding and expanded blastocysts exhibited comparable developmental potential that was superior to that of either morulae or early blastocysts. However, expanding or expanded blastocysts derived from 7-cell or 8-cell embryos were developmentally superior to those derived from other cleavage stages, regardless of fragmentation or asymmetry. Collectively, these findings further understanding of morphological predictors of viability, thereby improving the ability to select the most viable embryo for transfer.

Adult↗

Embryo selection by the first cleavage parameter between 25 and 27 hours after ICSI.

PURPOSE: The objective of this study was to assess first embryo cleavage (FEC) 25-27 h after intracytoplasmic sperm injection (ICSI) as a parameter for the embryo selection process. METHODS: From January 1998 to December 1999, a total of 670 patients were subjected to the ICSI programme at the Centre for Human Reproduction, Sinhá Junqueira Maternity Foundation, and the FEC parameter was evaluated in three situations. RESULTS: In the first, a total of 300 zygotes were analyzed on the basis of a score (16-18 h after ICSI) and observed for the presence or absence of FEC (25-27 h after ICSI). A significant (p < 0.02) presence of FEC was observed in zygotes with a score of 15 (ideal score). In the second, a total of 200 patients were selected and divided into two groups matched for age and laboratory performance. Group I (n = 100) was subjected to transfer of embryos with the absence of FEC only (since in this cycle no embryos with FEC were detected within 25-27 h after ICSI) and Group II (n = 100) was subjected to transfer of embryos with the presence of FEC only. The age of Group I patients (33.8 +/- 4.2 years) did not differ significantly (p = 0.50) from that of Group II patients (33.5 +/- 4.3 years). The number of embryos transferred was similar (p = 0.07) for Group I (2.7 +/- 1.1) and Group II (2.9 +/- 0.88). In Group II, the 17.5% implantation rate was significantly higher (p < 0.01) than the 5.9% rate obtained for Group I. The pregnancy rate for Group II was significantly higher (p < 0.01) (33%) than that for Group I (12%). The incidence of abortion was 16.6% in Group I as compared with 6% in Group II. In the third situation, we observed the frequency of embryos with FEC in 36 patients whose implantation rate was 100% (ideal result) and obtained a value of 82%. CONCLUSIONS: The data suggest that the presence of the FEC parameter that was evaluated 25-27 h after ICSI could be used to select embryos with a higher implantation power. The data reported here may justify routine analysis of embryos with FEC for the process of embryo selection after ICSI.

Adult↗

Predictors of success after embryo transfer: experience from a single provider.

OBJECTIVE: Our goal was to examine the variables present at the time of embryo transfer and to determine their effects on the clinical pregnancy rate. STUDY DESIGN: All fresh and frozen embryo transfers during a 3-year period in a university-based in vitro fertilization program were examined. Female age, previous in vitro fertilization attempt, diagnosis, embryo number and quality, transfer technique, and presence of a clinical pregnancy were recorded for each couple. Logistic regression analyses were performed both univariately and multivariately to determine the association between a clinical pregnancy and the independent variables. RESULTS: All transfers during the study period were included in the analysis. The four primary diagnoses were pelvic or tubal disease, male factor infertility, unexplained infertility, and endometriosis. The 46 frozen embryo transfers had a clinical pregnancy rate similar to that among the 159 fresh embryo transfers and were therefore included in the analysis. One variable was found to significantly affect the outcome, the number of high-grade embryos placed. The presence of a previous failed embryo transfer tended to lower the success rate for future attempts; however, this result did not reach statistical significance. The catheter type and the transfer difficulty did not affect outcome. CONCLUSION: The two most important variables for predicting a clinical pregnancy are a first-time transfer and the number of high-grade embryos placed. Neither the type of embryo transfer catheter used nor the diagnosis affected outcome. In this small sample difficult embryo transfers did not diminish the chance for a successful outcome.

Adult↗

The influence of the depth of embryo replacement into the uterine cavity on implantation rates after IVF: a controlled, ultrasound-guided study.

BACKGROUND: Traditionally, embryo transfer after IVF has been performed blindly and placing the embryos approximately 1 cm below the fundal endometrial surface. However, it has been suggested that transferring embryos rather lower in the uterine cavity or high in the uterus may improve implantation rates. Nevertheless, there has not yet been a controlled trial to prove this theory. This prospective randomized study investigates the influence of the depth of embryo replacement on the implantation rate after embryo transfer carried out under transabdominal ultrasound guidance. METHODS: A total of 180 consecutive patients undergoing ultrasound-guided embryo transfer were randomized to three study groups according to the distance between the tip of the catheter and the uterine fundus at the moment of the embryo deposition in the lumen of the endometrial cavity: group 1: 10 +/- 1.5 mm; group 2: 15 +/- 1.5 mm; group 3: 20 +/- 1.5 mm. RESULTS: There was equal distribution between all three study groups regarding the main demographic and baseline characteristics of the patients, ovarian response, oocyte retrieval and IVF outcome, as well as the characteristics of embryo transfer and luteal phase support. The position of the catheter tip in relation to the fundal endometrial surface in groups 1 (10.2 +/- 0.9 mm), 2 (14.6 +/- 0.7 mm) and 3 (19.3 +/- 0.8 mm) was significantly different. Implantation rate was significantly higher (P < 0.05) in groups 2 (31.3%) and 3 (33.3%) compared with group 1 (20.6%). CONCLUSIONS: The depth of the embryo replacement into the uterine cavity may influence implantation rates, and thus it should be considered as an additional procedure among factors recently proposed as associated with successful embryo transfer after IVF.

Adult↗

Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer.

BACKGROUND: The respective advantages of day 3 and day 5 embryo transfer are a matter of debate. Previous comparisons did not include pronuclear stage zygote scoring and cumulative success rates (fresh and cryopreserved embryos). METHODS: Patients were randomized prospectively for day 3 or day 5 embryo transfer. Day 3 embryos were selected for transfer and cryopreservation by using combined evaluation at the pronuclear and cleavage stages. RESULTS: There was no difference between day 3 and day 5 fresh embryo transfers as to the rates of pregnancy (58 versus 62%), clinical pregnancy (56 versus 58%), delivery (50 versus 48%), implantation (35 versus 38%) and birth (33 versus 36%) rates. The corresponding values for cryopreserved embryo transfers were also similar. However, day 3 embryo transfer compared favourably with day 5 transfer when the pregnancy (90 versus 66%), clinical pregnancy (85 versus 62%) and delivery (77 versus 52%) rates were calculated per oocyte recovery attempt. CONCLUSIONS: With a selected population of good prognosis patients and our embryo selection criteria, the implantation potential of day 3 and day 5 embryos is equal. Per oocyte recovery attempt, day 3 transfer is more clinically efficient than day 5 transfer, but at least one transfer of cryopreserved embryos is necessary to manifest this superiority.

Adult↗

High outcome predictability after IVF using a combined score for zygote and embryo morphology and growth rate.

BACKGROUND: A scoring system has been developed to determine preimplantation embryo quality, and used to select embryos for transfer into the uterus of patients. METHODS AND RESULTS: The system was used to study early embryo development and to test whether these scores alone can accurately predict IVF outcome. Following zygote and embryo scores through early development, the data showed that a top quality zygote does not necessarily indicate that the resulting embryo will be top quality after in-vitro culture. The embryo quality score can change dramatically when embryos are cultured to day 2 or 3 post-fertilization. Pregnancy rates and implantation rates were compared with the cumulative and separated zygote and embryo scores. Analysis of the predictability of scoring systems suggested that morphological scores alone are relatively unpredictive of IVF outcome. When weighted for in-vitro growth rate, scores were highly predictive, more so than the rate of development alone. CONCLUSIONS: These data suggested that a combination of in-vitro growth rate and morphological analysis both of zygotes and embryos was highly indicative of outcome after IVF. The results can be adopted to the single embryo transfer approach to IVF.

Blastocyst↗

Single top quality embryo transfer as a model for prediction of early pregnancy outcome.

BACKGROUND: Single embryo transfer (particularly of a top quality embryo) is an excellent model to correlate embryo quality in terms of morphological criteria to early pregnancy. We investigated whether this model could provide us with more information on what happens after implantation in the first trimester of pregnancy. METHODS: The outcome of 370 consecutive single top quality embryo transfers in patients younger than 38 years was analysed for pregnancy and first-trimester pregnancy loss (FTPL) before 13 weeks of gestation. Analysis was done on each cohort of embryos from which the transferred top quality embryo was selected. Serum HCG levels were measured on day 8 and day 12 after day 3 embryo transfer. The HCG index was calculated as the level of HCG on day 12/HCG on day 8. RESULTS: The pregnancy rate after single top quality embryo transfer was 51.9%. This was independent of the patients' age. FTPL, however, appeared to be age dependent: 15.4% for the whole group, 9% in patients younger than 30 years and 19% in patients above 30 years. The pregnancy rate was 50% in IVF cycles and 52% in ICSI cycles; FTPL was 19% in IVF cycles and 10% in ICSI cycles. Multiple regression analysis showed that these differences originated from age differences between both populations rather than from technique-related factors. An HCG level >or=45 IU/l on day 12 was predictive for ongoing pregnancy with 75.6% sensitivity and 100% specificity; an HCG index >or=3.5 similarly predicted ongoing pregnancy with 72.3% sensitivity and 100% specificity. CONCLUSIONS: These data show that embryo selection for transfer on day 3 can be used as an excellent tool for prediction of pregnancy but not for prediction of FTPL. The pregnancy rate of a single top quality embryo is not related to age, whereas FTPL is age dependent.

Abortion, Spontaneous↗

Number of embryos for transfer after IVF and ICSI: a Cochrane review.

BACKGROUND: The most common complication of IVF is multiple pregnancy, which occurs in 25% of pregnancies following the transfer of two embryos. Single embryo transfer can minimize twin pregnancies but could also lower live birth rates. Our aim was to perform a systematic review of randomized trials to determine the effectiveness of single versus double embryo transfer. METHODS: Cochrane Collaboration review methods were followed. Randomized controlled trials comparing single and double embryo transfers were identified by searching Medline, EMBASE and the Cochrane register of controlled trials. Contents of specialist journals and proceedings from meetings of relevant societies were hand searched. Data were pooled with Rev Man software using the Peto-modified Mantel-Hanzel method. RESULTS: Pooled results from four trials indicate that although double embryo transfer leads to a higher live birth rate per woman [odds ratio (OR) 1.94, 95% confidence interval (CI) 1.47-2.55] in a fresh IVF cycle, comparable results are obtained by subsequent transfer of a frozen embryo (OR 1.19, 95% CI 0.87-1.62). The multiple pregnancy rate is significantly higher (OR 62.83, 95% CI 8.52-463.57) after double embryo transfer. CONCLUSIONS: Single embryo transfer significantly reduces the risk of multiple pregnancy, but also decreases the chance of live birth in a fresh IVF cycle. Subsequent replacement of a single frozen embryo achieves a live birth rate comparable with double embryo transfer.

Adult↗

A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen-thawed embryo transfers.

BACKGROUND: Assisted hatching (AH) in fresh embryo transfer (ET) could be associated with increased implantation rates. However, very few prospective randomized studies have specifically addressed the issue of AH during frozen-thawed embryo transfers (FET) cycles, those that have reported controversial results. The aim of this study was to evaluate the benefit of an enzymatic zona pellucida treatment of frozen-thawed embryos before transfer. METHODS: This was a prospective study including 125 non-donor FET cycles from 125 infertile couples. FETs were randomly allocated into AH group (n = 61, embryos pretreated with pronase 5 IU/ml for 1 min at 37 degrees C) or control group (n = 64, untreated embryos). Zona pellucida thickness was measured for each transferred embryo. The main outcome parameters were clinical pregnancy and implantation rates. RESULTS: The two groups were comparable regarding mean women's age, duration and indications of infertility, IVF outcome after fresh ETs, numbers and quality of fresh and frozen embryos, frozen-thawed embryo survival rates and blastomeres survival indexes. Despite a statistically significant decrease of zona pellucida thickness after pronase treatment [(mean +/- SD) 18.5 +/- 2.25 versus 14.5 +/- 2.75 microm; P < 0.0001], implantation (9.6 versus 9.2%) and clinical pregnancy rates (18.0 versus 17.2%) were not statistically different after FETs, with a similar mean number of embryos transferred between AH and control groups, respectively. CONCLUSION: Within the constraints of our protocol, partial enzymatic digestion of zona pellucida by pronase was not related with any benefit of the FET outcome especially concerning the implantation ability of frozen-thawed embryos.

Adult↗

Ultrasound derived indices of follicular blood flow before HCG administration and the prediction of oocyte recovery and preimplantation embryo quality.

The principal aim of the study was to relate ultrasound-derived indices of blood flow in individual follicles on the day of, but before, the administration of human chorionic gonadotrophin (HCG) to the subsequent recovery of oocytes and the production of preimplantation embryos. Data were obtained from 21 women (aged 29-43 years) with bilateral tubal occlusion, who were undergoing treatment by in-vitro fertilization (IVF) and embryo transfer. Transvaginal ultrasonography with colour Doppler imaging and pulsed Doppler spectral analysis were used to measure follicular volume and derive indices of blood flow. The end-points for each follicle were the volume, peak systolic velocity (PSV), pulsatility index (PI), and the recovery or non-recovery of an oocyte, the subsequent production or non-production of a preimplantation embryo and the morphological grade of each embryo. A total of 94 follicles were studied; 74 oocytes were recovered (79%) and 40 embryos (33 grade I or II) were produced. There were four clinical pregnancies (pregnancy rate 25.0% per transfer, 19.0% per patient). There was a significant correlation between whether or not follicular blood flow was detected and whether or not an oocyte was recovered (P < 0.05, chi 2 test). The values for volume and PI were not clinically useful. The PSV (cm/s, mean +/- SD) was higher in follicles that were associated with the production of an embryo (12.7 +/- 5.9) compared with those that were not (8.5 +/- 5.0; P < 0.05, Student's t-test). The probability of producing a grade I or grade II embryo was 75% if the PSV was > or = 10 cm/s. The corresponding value was 40% if the PSV was < 10 cm/s and 24% if blood flow was not detected (i.e. PSV < 3 cm/s). There was a significant increase (P < 0.05, Student's t-test) in the PSV before aspiration in those follicles associated with the subsequent production of an embryo. We conclude that the value for PSV, before the administration of HCG, can be used to identify follicles with a high probability of producing an oocyte and a high grade preimplantation embryo. The information may also be used to time the administration of HCG to achieve the optimum number and quality of embryos for patient management.

Adult↗

Cryopreservation of human embryos and oocytes.

The success rate of human embryo cryopreservation depends on technical and embryonic parameters. First of all, the cryoprotectant can affect embryo survival as we found by comparing two freeze-thaw procedures using propanediol (PROH) (1.5 mol) alone or with sucrose (0.1 mol). Embryo survival was significantly enhanced with sucrose (62 versus 32%). Embryo quality is another major parameter involved in the success of freezing; the rates of positive survival were found to be 67% for morphologically normal embryos versus 49% for embryos with fragments (P less than 0.001). The efficiency of embryo cryopreservation in an IVF programme could be estimated in 1986: a woman with extra embryos, stored after transfer of 3-4 fresh embryos (16% of all cycles), can expect a 22% pregnancy rate per transfer of fresh embryos and a 32% pregnancy rate per collection after transfer of the stored eggs. A comparative study of the cryopreservability of immature or mature oocytes was performed in humans. Human oocytes have a low survival rate (36%) whatever the cryopreservation protocol or the initial maturation stage. Immature human oocytes could survive freezing and thawing, mature and be fertilized in vitro, but with a very low efficiency.

Embryo Transfer↗

Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova.

Almost 30 years after the first successful in vitro fertilization (IVF) in golden hamsters (Mesocricetus auratus), we report that IVF hamster embryos can develop in a chemically defined, protein-free culture medium into morulae and blastocysts, and produce normal offspring after transfer to recipients. When examined 96 h post-insemination, 82% (160/200) of IVF ova had cleaved to at least 2 cells, 55% (97/200) had developed beyond the 4-cell stage, and 22% (38/200) had developed into morulae/blastocysts. In vitro development of IVF embryos to greater than or equal to 8 cells was absolutely dependent on hypotaurine. Twenty living offspring were produced from transfer of IVF embryos to recipients, with an overall success rate of 5% and 17% for oviductal (2-cell) and uterine (8-cell/morulae) transfers, respectively. In vivo-fertilized pronucleate embryos collected 3 h after egg activation were less able to develop in vitro than embryos collected only 6 h later, revealing a critical influence of the oviduct within the first hours of embryo development. Hypotaurine partly compensated for the decreased oviductal exposure of early 1-cell embryos. Establishment of a key role for hypotaurine in hamster embryo development, support of IVF embryos to morula/blastocyst stages in vitro, and production of living offspring after IVF embryo transfer are significant steps towards the goal of obtaining comparative data on preimplantation embryogenesis.

Animals↗

Evidence of damage in cryopreserved and fresh bovine embryos using the Tunel technique.

The objective of the present study was to evaluate the quality of bovine embryos cryopreserved in different years in Chiapas, Mexico. The embryos were obtained from a government institution (FIMEGEN) dedicated to promoting embryo transfer among dual-purpose cattle farmers. Forty-three embryos frozen in 1988, 1989, 2000 and 2002 were analysed with the Tunel technique to detect programmed cell death (apoptosis). Eleven fresh embryos were used as controls. Analysis of variance was used in embryos stored in the different years with averages tested using Tukey's test. Student's t-test was employed to compare fresh and frozen cells. Embryos with shorter storage time presented a lower number (p < 0.001) of Tunel-positive cells compared with embryos stored for longer time. On the contrary, when comparing the number of apoptotic cells between frozen and fresh embryos a higher number of positive cells (p < 0.05) were found in the former. The present results suggest that the cryopreservation per se caused damage that compromises the viability of the embryo. Another explanation for the lower pregnancy rate found in the tropics could be irreversible damage caused by poor storage technique in these large operations.

Animals↗

Development of reconstituted embryos derived from somatic cell nuclei in the rabbit.

Production of cloned laboratory animals is helpful in the establishment of medical models. In this study, we examined to produce reconstituted embryos derived from somatic cell nuclei, and to establish embryonic stem (ES) cell lines from the embryo in rabbits. Metaphase II (M-II) oocytes from superovulated rabbit were used as nuclear recipients. Nuclear donor cells were fibroblasts collected from a Dutch Beleted rabbit. The M-II chromosome and the 1st polar body were aspirated, and a fibroblast was inserted into the perivitelline space of the enucleated oocyte. The pairs were electrofused for cell membrane fusion using a cell fusion apparatus, and reconstituted embryos were produced. The embryos were activated and cultured in modified HTF medium and DMEM. The embryos developed to the blastocyst stage were removed their zona pellucida, and they were cultured on the feeder cell layer. As a result of having observed development of reconstituted embryos, 21.2% of the embryos were developed to the blastocyst stage. In the embryos cultured on the feeder cells, the adhesion on feeder cells was observed. We obtained inner cell mass (ICM) colony derived from reconstituted embryos. At present, we are investigating to establish the ES cell lines derived from the embryos reconstituted by nuclear transfer.

Adenine↗