Effect of Different Concentrations of Recombinant Leukemia Inhibitory Factor on Different Development Stage of Mouse Embryo In Vitro

Purpose: To assess the influence of different concentrationsof recombinant human leukemia inhibitory factor (LIF) onthe in vitro development of mouse embryos. Methods: The 2- to 4-cell embryos of CB6F1 mice werecultured in the human tubal fluid (HTF) media containingdifferent concentrations of LIF. Mouse embryos were dividedinto seven groups: (1) HTF; (2) 1500 IU/ml LIF; (3) 1000IU/ml LIF; (4) 750 IU/ml LIF; (5) 500 IU/ml LIF; (6) 250IU/ml LIF; (7) 125 IU/ml LIF. The embryonic numbers ofdifferent stages including 5–8 cell, 9–16 cell, morula, blastocyst,and hatching blastocyst were recorded. Results: The percentage of early embryo stage (2-cellembryos to 6- to 16-cell stages) in all groups were nonsignificantlydifferent. There were higher formation rates of preimplantationembryos (morula to hatching blastocyst) ingroups 2, 3, 4, and 5 than in groups 1, 6 and 7. Conclusions: LIF has positive effects on preimplantationembryo development and has nonsignificant influence onthe early embryo development. The lowest concentration ofLIF which could provide the optimal embryo developmentis 500 IU/ml.     

Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos

Purpose: We reported previously on the use of coculture with cumulus cells in insemination medium for the development of human embryos in vitro. Here we describe a prospective trial to determine if this procedure has a significant beneficial effect. Methods: On the day after insemination, zygotes were randomized for culture in either a fresh drop of medium without (– cum) or were left in their insemination drop with (+ cum) cumulus cells. Embryos with the best morphological quality were replaced on the third day of development at the eight-cell stage. The remaining embryos were cultured for a further 3 days and cryopreserved if they reached the fully expanded blastocyst (FEB) stage. Three different culture media were used over the period of this study. Results: In 11 patients, supernumerary embryos were available only for continued culture in + cum and three patients had embryos cultured in only – cum. Thirty-nine other patients had embryos assigned to both + cum and – cum treatments. In the + cum group, 98 blastocysts developed from 216 embryos cultured for 6 days (45%), and this was significantly greater (P<0.01) than the 48 blastocysts from 156 embryos (31%) developing in the absence of cumulus cells. In basal HTF medium (HTF medium with EDTA and glutamine) and basal XI HTF medium (similar to basal HTF but devoid of glucose and phosphate), culture of embryos with cumulus cells produced significantly more FEBs than in the absence of cumulus cells. There was no significant difference between the two culture treatments when regular HTF medium was used. Preliminary results indicate that pronectin-coated dishes provide a good substratum for cumulus cell attachment and embryo development. Conclusions: The culture of human embryos with their cumulus cells in insemination drops of medium produces a significantly greater proportion of FEBs than when the zygotes are transferred to fresh culture drops devoid of cumulus cells. This is the first report of a significantly higher blastocyst rate with coculture in which a real comparison has been made between two culture treatments which differ only in the presence or absence of homologous cumulus cells in insemination drops.Presented in part at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction. Vienna, Austria. April 3–7, 1995.     

Mouse oocytes injected with cryopreserved round spermatids can develop into normal offspring

Purpose: This study was performed to determine whether frozen-thawed mouse round spermatids can fertilize oocytes and contribute to normal embryo development. Methods: Freshly collected mouse testicular cells were frozen in PBS containing 7.5% glycerol and 7.5% fetal bovine serum. After thawing and removal of the cryoprotectants, round spermatids were selected and injected individually into mature oocytes which had been previously activate with Sr ²⁺ -containing Ca ²⁺ -free medium. Results: After thawing, 75–85% of testicular cells were alive. About 90% of the oocytes were fertilized by intracytoplasmic injection of frozen-thawed round spermatids; 11% (17/150) of embryos transferred to foster mothers developed into normal offspring. Conclusions: Mouse round spermatids can be cryopreserved for production of normal offspring.     

Pregnancy achieved with pronuclear-stage embryos that were cryopreserved and thawed twice: A case report

Purpose: Our purpose was to determine if pronuclear-stage embryos (2PN) could be thawed, then frozen again with subsequent survival and cleavage after thawing. Methods: A simplified cryopreservation protocol was used in which a slow cooling program is started at the seeding temperature of –6°C in an alcohol-bath controlled-rate freezer. 1,2-Propanediol (1.5 M) was added to embryos before cooling. A fast thawing technique at room temperature was used. The cryoprotectant was removed in one step using a I M sucrose solution. Results: Three months after refreezing, the three 2PN embryos were thawed and all three cleaved after 24 hr in culture. Following embryo transfer a pregnancy was achieved and a healthy full-term baby girl was born. Conclusions: This is the third case reported of successful pregnancies after transfer of human embryos that were frozen twice before transfer and the first case where the second freeze occurred at the pronuclear stage. This is also the first successful refreezing of human embryos using a simplified freezing and thawing technique with one-step addition and removal of cryoprotectant.     

Clinical experiences of ICSI-ET thawing cycles with embryos cryopreserved at different developmental stages

Purpose: The aim of our study was to analyze factors including survival, implantation and pregnancy rate, patients’ age and BMI, abortions and extra uterine pregnancies that might influence the outcome of ICSI-ET thawing cycles.Methods: A total of 147 cycles with embryos cryopreserved at different developmental stages were retrospectively evaluated.Results: No difference was found in the survival, implantation and pregnancy rates of embryos cryopreserved on Day 2–3 and 5. However, in the pregnant group significantly higher implantation rate was observed with Day 5 blastocysts then with Day 2 or 3 early embryos. We found no difference in the number of abortions and extra uterine pregnancies between fresh and frozen ICSI-ET cycles. Higher BMI was found in the pregnant than in the non-pregnant group. However, the age of patient had no effect on the results.Conclusions: Developmental stage of embryo and patients’ BMI influences the success of ICSI-ET thawing cycles.     

Experience with the cryopreservation of human embryos using the mouse as a model to establish successful techniques

Mouse embryos at the one-, two-, and eight-cell stages have been used to optimize the conditions for cryopreservation of human oocytes and embryos. For storage in glass vials using 1.5 M dimethyl sulfoxide (DMSO) as a cryoprotectant and slow cooling (0.3°C/min), phosphate-buffered medium was superior to Hepes-buffered medium. Termination of slow cooling at –80°C before transfer to liquid nitrogen with subsequent slow thawing (–8°C/min) resulted in more embryos surviving than when cooling terminated at –40°C and rapid thawing (–500°C/min) was employed. Dilution of DMSO upon thawing with medium containing 0.5M sucrose gave higher embryo survival rates than a stepwise (0.25 M decrements) dilution. Using these techniques, three pregnancies were established upon the transfer of 11 frozenthawed embryos to seven patients. Rates of embryo survival using the simpler cryopreservation technique of ice-free vitrification in 0.25-ml straws have been disappointing.     

Relationship of the Human Cumulus-Free Oocyte Maturational Profile with In Vitro Outcome Parameters After Intracytoplasmic Sperm Injection

Purpose: We investigated whether the human oocyte maturational profile at the removal of cumulus/corona cells affects the fertilization rate and subsequent embryo quality after intracytoplasmic sperm injection. Methods: A total of 1011 oocytes from 150 cycles was included in this retrospective analysis. Cumulus-free oocytes that were in prophase or metaphase I of meiosis at the removal of cumulus/corona cells were incubated in vitro until they reached metaphase II (in vitro-matured oocytes) and were then immediately injected with a single spermatozoa. Oocytes that were in metaphase II at the removal of cumulus/corona cells (MII oocytes) received sperm injection after 3–4 hr of preinjection incubation. Results: The fertilization rate of the MII oocytes was significantly higher than that of in vitro-matured oocytes (81 vs 62%; P < 0.001). The cleavage rates were similar in the two groups (MII oocytes, 94%; in vitro-matured oocytes, 91%). However, MII oocytes had significantly higher percentages of good-quality embryos (grade 1–3 embryos, 87 vs 58%, P < 0.001) and embryos with high cumulative embryo scores (score 10–32 embryos, 62 vs 33%, P < 0.001). The mean cumulative embryo score of MII oocytes after fertilization was also higher than that of in vitro-matured oocytes (12.1 ± 3.8 vs 8.8 ± 3.4; P = 0.014). Conclusions: MII oocytes that extruded the first polar body at the removal of cumulus/corona cells had better fertilization rates and embryo morphology than in vitro-matured oocytes that extruded the first polar body following the removal of cumulus/corona cells and in vitro culture.     

Clinical evaluation of frozen/thawed embryo transfer following transport of oocytes and embryos

Background and Aims  We evaluated the efficacy of the transport oocyte/embryo frozen/thawed embryo transfer method, in which oocytes or embryos were transported from satellite clinics to the main assisted reproductive technology (ART) center, and surplus embryos were placed in cryopreservation. Methods  We evaluated 41 cycles in 34 patients in the transport oocyte group (TO group). In the TO group the oocytes were collected at the satellite clinics, transported to the main ART center and underwentin vitro fertilization or intracytoplasmic sperm injection. Surplus embryos were used for frozen/thawed embryo transfer. We also evaluated 17 cycles in 10 patients in the transport embryo group (TE group), where surplus embryos were transported to the main ART center and used for frozen/thawed embryo transfer; and 189 cycles in 134 patients in the center group (C group), where surplus embryos collected at the same time at the main ART center were used for frozen/thawed embryo transfer. Oocytes were transported from satellite clinics in HEPES buffered human tubal fluid (HTF) culture medium, and embryos in 30% synthetic serum substitute + HEPES buffered HTF, using a portable incubator we devised. Results  The proportions of undamaged embryos after freeze/ thawing were 47% for the C group, 46% for the TO group, and 46% for the TE group. The numbers of embryos transferred were 2.0 ± 0.7 for the C group, 2.0 ± 0.6 for the TO group, and 2.2 ± 0.4 for the TE group. The rate of embryo transfer was 63% for the C group, 68% for the TO group, and 76% for the TE group. Pregnancy rates per patient were 16% for the C group, 24% for the TO group, and 40% for the TE group. The embryo survival rates (number of embryos with ≥50% viable blastomeres/total number of embryos) were 55% for the C group, 60% for the TO group, and 54% for the TE group. No significant differences were seen between the C group and either the TO or TE groups in any of these parameters. Conclusions  Favorable results were achieved with the transport oocyte/embryo frozen/thawed embryo transfer method, and it is suitable for widespread clinical application.     

Clinical Assisted Reproduction: The Prognostic Significance of Day 3 Embryo Cleavage Stage on Subsequent Blastocyst Development in a Sequential Culture System

Purpose: To determine prognostic significance of blastomere number on Day 3 of culture upon subsequent blastocyst (BL) development. Methods: A retrospective analysis was conducted in 37 IVF subjects undergoing standard protocols and BL transfer after sequential embryo culture in P1 and BL media. Results: Of Day 3 embryos containing 7 or more blastomeres, 68.9% (186/270) developed into BL compared to embryos containing 4–6 blastomeres, 38.1% (56/147), P < 0.0001. The majority of BL, 68.9% (168/244), were observed on Day 5. Extended Day 6 culture represented 31.1% (76/244) of all BLs. Conclusions: The observation of 7 or more blastomeres on Day 3 yielded a significantly greater likelihood of BL development. Embryos containing 4–6 blastomeres are still relatively likely to progress to a BL. Extended culture to Day 6 still yields a significant proportion of BL. Cell cleavage stage on Day 3 appears to be a useful prognostic indicator of subsequent BL development.     

A Simplified Coculture System Using Homologous, Attached Cumulus Tissue Results in Improved Human Embryo Morphology and Pregnancy Rates During In Vitro Fertilization

Purpose: This study was undertaken to evaluate simplified methods of human embryo coculture using either attached or nonattached autologous cumulus tissue. Methods: Eight hundred one zygotes were cultured for 48 hr in a prospective, randomized trial comparing culture of embryos either with intact cumulus tissue, with cumulus tissue added to the droplet of culture medium, or without any cumulus tissue. In a follow-up study, embryo quality, pregnancy rates, and implantation rates were compared in 120 consecutive patients undergoing in vitro fertilization with a coculture system using cumulus tissue compared to a cohort of 127 patients undergoing IVF immediately preceding the institution of the coculture protocol. Results: Embryo morphology was significantly improved (P < 0.05) following culture with attached cumulus tissue (5.61 ± 0.29) and culture with added cumulus tissue (4.72 ± 0.31) compared to that of embryos grown in culture medium without cumulus tissue (3.95 ± 0.26). The clinical pregnancy rate improved from 39.4% (50/127) to 49.2% (59/120) following institution of a system of coculture with attached cumulus tissue. Conclusions: These data indicate that a simple coculture system using autologous cumulus tissue can result in improved embryo morphology, implantation rates, and clinical pregnancy rates during in vitro fertilization. This coculture system is simple, is non-labor intensive, and eliminates many of the risks which may be present in other embryo coculture systems.     

Improvement of Development of Vitrified Two-Cell Mouse Embryos by Vero Cell Coculture

Purpose: The purpose of this study was to determine the developmental potential of two-cell mouse embryos resulting from vitrification could increased using monolayer of Vero cells. Methods: Two-cell mouse embryos were divided into vitrified and nonvitrified groups. Embryos in the vitrified group were frozen with a combination of 10% ethylene glycol, 30% ficoll, and 0.5% M sucrose (EFS10) as cryoprotectants, and thawed rapidly with 0.5 M sucrose. The survived embryos were cultured either with Vero cells monolayer or in T6 medium. Accordingly the embryos of the nonvitrified group were also cultured. The rates of the development in all the groups were daily determined and statistically compared. At the end of the cultivation period, several expanded blastocysts from each group were stained with ethidium bromide and the mean number of the blastomers were counted and statistically compared. Results: After 4 days of culture, the developmental potential of vitrified-thawed embryos were significantly reduced in Vero cell-free medium, and the mean cell number of embryos reaching the expanded blastocyst stage were also lower than that of nonvitrified embryos. With exception of last day of culture, Vero cell coculture, resulted in a significant increase in the rate of development of vitrified-thawed embryos as well as improved the mean cell number of expanded blastocysts. On the other hand, the mean cell number of expanded blastocysts of nonvitrified group was significantly improved in coculture group. However, the rate of embryo development except for the first day of culture was similar to that of medium alone. Conclusions: The developmental potential of vitrified-thawed embryos appears to be retarded in conventional medium and Vero cell monolayer is capable to eliminate the postthaw deleterious effect of vitrification during the first 3 days of cultivation, but not for a longer period.     

Human preimplantation embryo development in vitro: a morphological assessment of sibling zygotes cultured in a single medium or in sequential media

Abstract

A comparison was made of the development of human zygotes in either a one-step (Global^® medium) or two-step culture system (Quinn's Advantage^®). A total of 257 normally fertilized 2PN zygotes from 28 patients were used in the study. The study was broken down into two parts: the first concerned the development of embryos from Days 1 to 3 in Global^® medium and Quinn's Advantage^® cleavage medium; the second consisted of a comparison of the development of embryos from Day 3 to 5/6 in Global^® medium and Quinn's Advantage^® blastocyst medium. There were no significant differences between the two culture media with respect to embryo quality throughout the preimplantation phase of human embryo development as determined by the extent and variability of the cell counts, fragmentation, and nucleation. A difference was noted in the blastomere symmetry of Day 2 embryos in the two media, but was no longer apparent on examination of Day 3 embryos. No differences were noted in the rates of blastocyst development, inner cell mass (ICM), and trophectoderm (TE) scores in the two culture media. Finally, no significant differences were noted with either the proportion of blastocysts chosen for transfer or cryopreservation (vitrification). The findings support the view that two-step sequential media protocols are sufficient but not necessary to support the complete in vitro development of human preimplantation embryos.     

Effects of platelet activating factor on mouse embryo implantation in vitro

Purpose Our purpose was to assess the role of platelet activating factor (PAF) in embryo implantation, we examined the effects of PAF and a PAF antagonist on the in vitro implantation of mouse embryos, using an in vitro embryo culture system in the absence of the endometrium.Methods BDF1 mouse pronuclear-stage embryos were cultured until they developed to the two-cell, the four- to eight-cell, or the morula stage in the absence of PAF or its antagonist CV6209. The medium was then changed and supplemented with PAF or CV6209 at various concentrations. We also examined the reversible effects of PAF addition to the media containing the PAF antagonist.Results The addition of PAF to the culture from the two-cell stage significantly (P <0.05) increased the rates of embryo implantation in vitro (control, 69.8%; 10 ^–10 MPAF, 90.1%; 10 ^–9 MPAF, 95.5%). Similarly, the addition of PAF to the cultures from the four- to eight-cell and morula stage also significantly (P <0.05) increased their rates of implantation in vitro. In contrast, the addition of CV6209 to the culture significantly (P <0.01) decreased the rates of embryo implantation in vitro. CV6209 also decreased the rate of blastocyst formation. The degree of inhibition by CV6209 decreased with the advancing stage of embryos. The addition of PAF to media containing CV6209 reversed the inhibition and restored the implantation rate in vitro.Conclusions These results suggest that PAF may act directly on the mouse embryo and favor its implantation like an autocrine activating factor, irrespective of the presence or absence of the endometrium.     

Effect of anordrin on the development of mouse preimplantation embryos in vitro

Purpose: The in vitro effect of anordrin and anordiol on the development of mouse two-cell embryos was studied. Method: Female mice were primed with gonadotropins for superovulation and caged with male mice. Preimplantation embryos, at the two-cell stage, were recovered from the oviducts at 40 hr post-hCG. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of anordrin for 3, 12, 24, and 80 hr and then grown in the anordrin-free culture medium and assessed for the formation of total and hatching blastocysts at 80 hr. In the second experiment, two-cell embryos were grown in culture medium containing different concentrations of anordiol and assessed for the formation of total and hatching blastocysts at 80 hr in vitro. Results: Exposure of two-cell embryos to anordrin concentrations of 2.5–7.5µg/ml for 12 hr, 2.5–5.0µg/ml for 24 hr, and 2.5µg/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 2.5–7.5µg/ml for 12 hr, 1.0–2.5µg/ml for 24 hr, and 1.0µg/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts, in a exposure time-dependent and dose-dependent manner. Exposure of two-cell embryos to anordiol concentrations of 15–25µg/ml for 80 hr caused significant inhibition of the formation of total blastocysts and to 15–20µg/ml for 80 hr caused significant inhibition of the formation of hatching blastocysts in a dose-dependent manner. Conclusion: Anordrin and its metabolite anordiol inhibit the development of two-cell embryos in vitro.Presented orally at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994, Abstract No. O-176.     

Comparison of Pregnancy Outcome of Pronuclear- and Multicellular-Stage Frozen–Thawed Embryo Transfers

Purpose: Our purpose was to determine if supernumerary embryos generated by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) should be frozen (using 1,2-propanediol) at the pronuclear or multicellular stage. Methods: The study was a retrospective analysis conducted at the Dubai Gynaecology & Fertility Centre of the Department of Health & Medical Services, Dubai, U.A.E. One hundred forty-one women undergoing frozen–thawed embryo replacement cycles with IVF generated embryos and 84 women undergoing the same with ICSI generated embryos. Results: Supernumerary, IVF-generated embryos frozen at the multicellular stage had a significantly higher rate of survival on thawing (73.9%) than embryos frozen at the pronuclear stage (64.4%). The morphological grades of the embryos in the two groups were similar, but a significantly higher pregnancy rate was obtained with embryos frozen at the multicellular stage (22.8%) than with pronuclear-stage embryos (14.8%). Similarly, with ICSI-generated embryos, significantly higher survival was seen with multicellular-stage frozen embryos (74.8%) than pronuclear-stage embryos (64.4%). The morphological grades of the embryos and pregnancy outcomes of the two groups were similar. Conclusions: Supernumerary embryos generated by IVF and ICSI should be frozen at the multicellular stage so as to allow selection of the best embryos for transfer and embryo freezing of only robust embryos.     

Successful Use of a Laser for Human Embryo Biopsy in Preimplantation Genetic Diagnosis: Report of Two Cases

Purpose: The use of Tyrode's acid to drill the zona pellucida for embryo biopsy is the most widely used methodology in preimplantation genetic diagnosis. Instead of this, we propose the use of a 1.48-m diode noncontact laser, which is quicker, simpler, and safer. Methods: The laser beam was tangentially guided to the zona pellucida of the embryo. Depending on zona pellucida measurement, two to four consecutive shots of 8–22 msec were necessary to drill the zona pellucida of the 13 embryos biopsied for two patients (hemophilia carriers). Results: Female embryos were replaced into the uterus of the patients (1.5 embryos/replacement). One single pregnancy was established (33.3% implantation rate). Coculture of untransferable embryos showed a blastocyst rate of 66.7% (4/6) for male embryos and 25% (1/4) for abnormal ones. Conclusions: These results demonstrate the safety and usefulness of laser methodology in preimplantation genetic diagnosis.     

Comparison of four media types during 3-day human IVF embryo culture

The aim of this study was to compare the effectiveness of human tubal fluid (HTF), G1.2, Sage Cleavage and Life Global media for IVF outcome during 3-day culture of human embryos. A three-phase auto-controlled study was conducted in which IVF outcome was compared between (1) HTF and G1.2, (2) HTF and Cleavage, and (3) Cleavage and Life Global. In phase 1, no differences in embryo quality were observed between HTF and G1.2. However, embryos derived from intracytoplasmic sperm injection (ICSI) displayed significantly improved quality when grown in HTF versus G1.2. No differences in pregnancy and implantation rates were observed in cases where embryos transferred were grown exclusively in HTF or G1.2 media. In phase 2, embryo quality was significantly improved for embryos cultured in Cleavage versus HTF media (P < 0.001). However, pregnancy, implantation and spontaneous abortion rates were similar between the two media. In phase 3, there were no differences in embryo quality, pregnancy, implantation, and spontaneous abortion rates between Cleavage and Life Global media. Overall, the data indicate that Life Global and Cleavage media yield similar results in a 3-day IVF culture programme. Cleavage medium is superior to HTF, as evidenced by significantly improved embryo quality (P < 0.001). Meanwhile, HTF medium is superior to G1.2 for ICSI cases.     

Pregnancies after transfer of ultrarapidly frozen human embryos

Purpose: We report our experience of freezing human embryos using an ultrarapid freezing method. Methods: The patients were superovulated. Oocytes were inseminated and cultured in HTF + 10% serum. A maximum of three embryos was transferred and the rest of the embryos were frozen ultrarapidly after a 3-min equilibration period in PB1 + 3.5 MDMSO + 0.25 Msucrose. Embryos were thawed in a 37°C water bath for 6 sec, then cultured in PB1 + 20% serum for 10 min. The surviving embryos were transferred into patients on the same day of thawing. Results: Sixty-three embryos were thawed. of which 52 embryos (83%) survived with at least one intact blastomere. Nineteen frozen-thawed embryo transfers were made. The mean embryos per transfer was 2.7. Three pregnancies (16%/transfer) were established. One miscarriage occurred in the eighth week of pregnancy. Two pregnancies went to term and three healthy infants were born. Conclusions: The present data demonstrate that ultrarapid freezing is a method worth consideration in the area of human embryo freezing.     

Lower embryonic loss rates among twin gestations following assisted reproduction

Purpose: To determine whether maternal age and number of transferred embryos influence early pregnancy losses in twin pregnancies compared to singletons following IVF/ICSI.Methods: We compared the pregnancy loss rates in singleton (n = 549) and twin (n = 252) gestations, stratified by maternal age (≤35 and > 35 years) and the number of transferred embryos (1–3 and 4–9).Results: Loss rates of singleton pregnancies were significantly higher than that in twins (OR 3.0, 95% CI 1.9, 4.9), especially among singletons conceived after transfer of 4–9 embryos (OR 5.0, 95% CI 2.2, 11.9). Younger mothers of twins had lower loss rates (OR 0.3, 95% CI 0.1, 0.9).Conclusion: Twins have a significantly reduced spontaneous miscarriage rate compared with singletons following IVF/ICSI. Higher implantation rates per cycle (i.e., development of twins rather than one live embryo) may represent a better capacity of the uterus for early embryonic development.     

Enhanced cryosurvival of bovine blastocysts produced in vitro in serum-free medium

Purpose : Culture systems affect the development of IVP embryos and consequently their cryosurvival potential. The viability of postthawed bovine IVP embryos developed from IVM/IVC medium in the presence or absence of serum was compared. Methods : Cumulus-oocyte complexes were matured in IVM medium supplemented with or without serum. Some oocytes were evaluated for nuclear maturation status and others were inseminated with semen. Presumptive zygotes were cultured in IVC medium supplemented with or without serum for 9 days. Blastocysts were cryopreserved with 1.5 M ethylene glycol in PBS. Results : No difference was observed in the nuclear maturation status and cleavage rates in both groups, but significantly (P < 0.05) higher in blastocyst rates in the serum-supplemented group. After freezing, survival of blastocysts was higher in the serum-free group. At 36 h culture after thawing, blastocysts developed without serum had significantly (P < 0.05) higher cell number than those cultured with serum. Conclusions : We conclude that serum-free culture system enhances the viability of frozen-thawed bovine embryos.