Use of coculture with cumulus cells in insemination medium in human in vitro fertilization (IVF)

Purpose In an initial trial, 16 of 33 (48%) bipronuclear human zygotes left in culture in the insemination drop from which they had originated developed to fully expanded blastocysts.Results This method was subsequently used for all supernumerary embryos judged unsuitable for replacement or cryopreservation on Day 1, 2, or 3 of development. Over a 4-year period, embryos reaching the fully expanded blastocyst stage were cryopreserved. Of 113 such blastocysts thawed, 81 survived (72%), and upon transfer to 52 patients, 8 clinical pregnancies were established (15%), of which 6 were live births. Subsequently, following modification of some culture parameters, 60 patients had 296 supernumerary embryos cultured for 6 days; 43 of these patients (72%) had 148 embryos (50%) that cavitated and 134 (45%) of these cavitating embryos were judged to be fully expanded blastocycts; 125 (42%) of these embryos were cryopreserved.Conclusion The blastocyst formation rate is similar to that reported by others using conventional culture procedures or coculture on Vero or other cell types. I conclude that cumulus cells are a ready source of feeder cells for the coculture of human embryos.Presented in part at the 8th World Congress on in Vitro Fertilization and Alternate Assisted Reproduction, Kyoto, Japan, September 12–15, 1993.     

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.     

Culture with Matrigel Inhibits Development of Mouse Zygotes

Purpose: It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F ₁ hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a two-cell block similar to the developmental blocks of other species. Methods: Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. Results: Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F ₁ -derived zygotes. Conclusions: Matrigel may be a deleterious addition to embryo culture or coculture systems.     

Clinical Assisted Reproduction: Prolonged Culture of Human Cryopreserved Embryos with Recombinant Human Leukemia Inhibitory Factor

Purpose: To evaluate the efficiency of recombinant humanleukemia inhibitory factor (LIF) in the prolonged culture ofhuman cryopreserved-thawing embryos. Methods: After thawing, all embryos were divided into fourgroups: (1) Human tubal fluid (HTF), (2) HTF + LIF, (3)M3^TH medium, and (4) M3^TH medium plus LIF. Followingprolonged culture, embryo development in each groupwas compared. Results: In embryo development from about the 2– to 4–cellto 9– to 16–cell stage, there were nonsignificant differencesbetween each group. There was lower morula formation ratein group 1 (6.9%) than those in other groups (23.2%, 19.7%,23.1%). The lower blastocyst formation in group 1 and 3(0%, 0%) than those in group 2 and 4 (11.0%, 12.8%)were noted. Conclusions: LIF is beneficial for preimplantation embryos.LIF does not influence the early embryo development.LIF-supplemented HTF provided a similar culture environmentfor thawing embryos as LIF-supplemented M3^TH medium.Supported by China Medical College Hospital, Taiwan     

Effects of medium composition on murine and human blastocyst formation and hatching rate

Purpose Murine two-cell embryos (n =5573) were cultured for 96 hr in human tubal fluid (HTF) medium (n =2709) or alpha modification of minimum essential medium (MEM; n =2864) through the hatched blastocyst stage from mid-1990 to mid-1991. An additional 373 embryos were cultured in MEM or HTF with 0, 1, 5, or 10 ng/ml E. coli endotoxin. A total of 17 patients had supernumerary embryos simultaneously cultured in HTF (n =48) or MEM (n =61). Additionally, pregnancy rates were compared for July to December 1990, when MEM was used as growth medium, and for July to December 1989, when HTF was used. Results Blastocyst formation was higher (P <0.001) for murine embryos cultured in MEM (blasts = 95%) compared to HTF (blasts = 70%). When cultured with endotoxin, blastocyst formation was higher (P <0.01) for embryos cultured in MEM compared with HTF for controls and at each endotoxin level. No difference in human blastocyst development was observed in HTF and MEM. However, more MEM-cultured blastocysts were cryopreserved (P <0.05). There also was a lower spontaneous abortion rate and a higher multiple gestation rate when embryos were cultured in MEM. Conclusion Thus, MEM may result in healthier blastocyst development, especially when culture conditions are substandard, although this is not an acceptable substitution for meticulous technique.Portions of these data were presented at the 44th Annual Meeting of the American Fertility Society, Orlando, Florida, 1991.     

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.     

Effect of chilling on the development of in vitro produced bovine embryos at various cleavage stages

Purpose: Bovine embryos and zygotes are known to be sensitive to “temperature shock” when cooled to temperatures near 0°C. The effect of chilling on in vitro derived embryos at various cleavage stages was investigated. Methods: Cumulus-oocyte-complexes (COCs) were matured in IVM medium with serum. Presumptive zygotes were cultured in serum free in vitro culture (IVC) medium. Embryos were used as chilled or control samples at the 2-cell, 4-cell, 8-cell, morula, and blastocyst stages. Embryos in 0.2 mL PBS in plastic straws were cooled rapidly in ethanol baths at 0°C for 30 min. Embryo viability was assessed by in vitro development. Results: The percentage of control embryos that hatched as blastocysts increased the later stage at which they were selected. Relative proportion of embryos increased from 28% to 48% to 68% when chilled at the 8-cell, morula or blastocyst stages. Conclusions: IVF-produced embryos are differentially susceptible to cooling injury. Cell counts made of those blastocysts formed from chilled embryos indicated subtle effects of chilling.     

Randomized Autocontrolled Comparison of the Embryo Culture Performance of Nunc and Falcon Petri Dishes

Purpose: Our purpose was to compare the embryo culture performance of two types of petri dishes (Nunc and Falcon). Methods: Mouse zygotes were cultured up to the expanded blastocyst stage in both types of dishes. The oocytes from 50 in vitro fertilization cycles were randomly divided between the two types of dishes. Fertilization, cleavage, and embryo quality were compared. Oocytes from another 50 cycles were all cultured at random in either type of dish. Pregnancy and implantation rates were compared between the two types. Results: Of 91 mouse zygotes, 81 cleaved to two-cell-stage embryos, and 64 became expanded blastocysts in Falcon dishes; of 99 zygotes, 81 cleaved to two-cell-stage embryos and 66 became expanded blastocysts in Nunc dishes. Of 248 oocyte–cumulus complexes (OCC), 145 fertilized in Falcon dishes, and of 269 OCC, 175 fertilized in Nunc dishes. The high quality embryo ratio was 51 out of 118 in Falcon dishes, not different from that in Nunc dishes, 58 out of 139. In Falcon dishes 72 out of 118 embryos were at least at the four-cell stage after 45 hr, versus 70 out of 139 in Nunc dishes. Twenty-three clinical pregnancies were obtained in the first 50 cycles with sibling oocytes. In the second group, with randomization of the cycles between Nunc and Falcon, 8 pregnancies were obtained in the Nunc and 10 in the Falcon dishes. The implantation rate in this second group of 50 cycles was 9 out of 61 in Falcon and 11 out of 57 in Nunc dishes. Conclusions: No differences were observed.     

Peptides extracted from vero cell cultures overcome the blastocyst block of mouse embryos in a serum-free medium

Purpose The aim of this work was to evaluate the effect of a Vero cell coculture system on the development of mouse embryos. Methods Mouse embryos were randomly divided and cultured in human tubal fluid (HTF) medium with/without Vero cell monolayers, conditioned medium (CM) obtained from Vero cell cultures, and HTF medium supplemented with peptides extracted from CM. The concentrated CM was examined by SDS/PAGE. Results The development of mouse embryos was blocked at the blastocyst stage in pure HTF medium (1.4% hatching at day 5). This “blastocyst block≓ was overcome by coculture with Vero cell monolayers (48.1% hatching at day 5; 1.4 vs 48.1%; P<0.001). CM and the addition of 5% fetal bovine serum (24.1 and 34.9% hatching, respectively, at day 5) were also able to enhance the process of hatching. In the other experiment, the addition of peptides extracted from Vero cell cultures also overcame the blastocyst block (12.5%) compared with pure HTF medium (2.1%) (P<0.05). Electrophoretic separation revealed several classes of polypeptides consistently secreted into CM obtained from Vero cell cultures. Most peptides occurred in the M_r range between 6.5 kd and 35.9 kd. Conclusion A developmental block (blastocyst block) of mouse embryos in a serum- and protein-free medium (HTF) was discovered in this study. This block was effectively overcome by HTF plus serum and coculture with Vero cell monolayers and also by the peptides extracted from Vero cell-conditioned medium. We speculate that certain factors secreted or converted by Vero cells may be critical in hatching of mouse embryos. Further study of these factors may be helpful in delineating its mechanism.     

Animal Experimentation: Vero Cells, But Not Oviductal Cells, Increase the Hatching Frequency and Total Cell Count of Mouse Blastocysts Partly by Changing Energy Substrate Concentrations in Culture Medium

Purpose: To investigate the embryotrophic mechanisms of Vero and oviductal cells coculture. Methods: Mouse embryos were cultured in Chatot, Ziomek, and Bavister medium (CZB), in modified CZB media (MM) with nutrient concentrations adjusted to that found in conditioned media after different periods of Vero cells or oviductal cells culture, in reconstituted medium (RM) containing the purified >100-kDa components of Vero cell conditioned medium that had been reconstituted with CZB medium, and cocultured with Vero cells with an interposing membrane. Results: The blastulation rate was not different among embryos cultured in different Vero-cell–derived MMs. Nine-hour Vero-cell-derived MM significantly increased the total cell number and hatching frequency of the embryos. There was no difference in these parameters with oviductal-cell–derived MMs. The RM of Vero cells did not possess embryotrophic activity. The presence of a porous membrane between Vero cells and embryos did not affect the embryotrophic activity of coculture. Conclusions: Vero cells, but not oviductal cells, improved mouse embryo development partly by modifying the energy substrate concentration in culture medium.     

Pregnancy resulting from transfer of repeat vitrified blastocysts produced by in-vitro matured oocytes in patient with polycystic ovary syndrome

This report describes a live birth produced from repeat vitrification and thawing of blastocysts derived from in-vitro matured (IVM) oocytes in a woman with polycystic ovarian syndrome. Immature oocyte retrieval was performed on day 12 of her induced menstrual cycle. The patient was administered 10,000 IU of human chorionic gonadotrophin s. c. 36 h before immature oocyte retrieval. A total of 47 immature oocytes were collected. Following IVM of these immature oocytes, 76.6% (36/47) become mature (at metaphase II stage). Thirty oocytes (30/36, 86.1%) were normally fertilized following insemination by intracytoplasmic sperm injection. The fertilized zygotes (two-pronuclear stage) were co-cultured with cumulus cells in YS medium supplemented with 10% human follicular fluid. On day 5 after insemination, three blastocysts were transferred. Unfortunately, fresh embryo transfer did not result in pregnancy. The remaining 10 embryos developed to the expanded blastocyst stage. These remaining blastocysts were vitrified with electron microscope grids following artificial shrinkage. Three months later, three blastocysts were thawed due to a clinical error. Consequently, the embryos were revitrified. After a week, the three blastocysts were warmed again. Two of them developed to hatched blastocysts. Following transfer, a full-term pregnancy resulted in the delivery of healthy twins.     

Effect of Vero Cell Coculture on the Development of Frozen–Thawed Two-Cell Mouse Embryos

Purpose: Our purpose was to evaluate the beneficial effects of long-term coculture of Vero cells on the development of frozen–thawed two-cell mouse embryos. Methods: Two-cell mouse embryos were frozen slowly with 1,2-propandiol and sucrose as cryoprotectants and thawed rapidly, followed by stepwise dilution. Vero cells were cultured in drops of RPMI 1640 to establish monolayers. Frozen–thawed embryos were cultured alone (control) or cocultured with Vero cells. The rate of development in both groups was compared. Results: After 4 days of culture, significantly more embryos in coculture were developed to expanded blastocysts (61 vs 37% for controls; P 0.0001). In addition, on the fifth day of cultivation, more embryos in coculture showed the potential of hatching from the zona pellucida (26 vs 7% in controls; P 0.0001). The rate of degeneration in coculture was also much lower than in controls (6 and 15%, respectively). Conclusions: Coculture of cryopreserved preimplantation-stage embryos with Vero cells seems to be a useful tool to eliminate the postthaw deleterious effect of freezing and also to obtain better-quality embryos appropriate for transfer.     

Coculture of mouse embryos with cryopreserved human oviduct epithelial cells

Purpose The effect of coculturing mouse embryos with cryopreserved human oviduct epithelial cells was investigated. The cryopreserved cells in Cellbanker were thawed and cultured in Richard D. Goldsby culture medium to establish monolayers. Two-cell-stage mouse embryos were cultured alone (control group) or cocultured with monolayers established from cryopreserved cells (cryopreserved coculture group) or from fresh cells (fresh coculture group). The rates of embryo development and the qualities of the blastocysts in the three groups were compared.Results The two coculture groups had significantly higher blastocyst development rates (cryopreserved coculture group, 81.6%; fresh coculture group, 82.2%) than the control group (63.1%). The two coculture groups had significantly more blastomeres (cryopreserved coculture group, 108.3 ± 25.9; fresh coculture group, 108.4 ± 25.1) than the control group (87.7 ± 31.9).Conclusion The method of cryopreservation of human oviduct epithelial cells using Cellbanker is simpler than conventional cryopreservation methods. These cryopreserved human oviduct epithelial cells may provide a constant supply of cells for coculture for in vitro fertilization and embryo transfer.     

The Well-of-the-Well system: an efficient approach to improve embryo development

Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P < 0.05). In a separate study, also in human, a total of 48 patients with a cumulative 214 unsuccessful previous IVF cycles were selected for the trials. In subsequent intracytoplasmic sperm injection cycles, oocytes/embryos were cultured individually in the WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.     

Factors affecting the success of human blastocyst development and pregnancy following in vitro fertilization and embryo transfer

Objective: To determine the factors affecting blastocyst development and pregnancy after IVF and ET.

Design: Retrospective analysis of data arising from a clinical trial.

Setting: Private in vitro fertilization clinic.

Patient(s): Fifty-six patients aged ≤40 years, undergoing IVF procedures for infertility, recruited specifically for blastocyst transfer.

Intervention(s): All zygotes were cultured to days 5 or 6 after insemination, and one to four of the most advanced blastocysts were transferred to the patient’s uterus.

Main Outcome Measure(s): Development of zygotes to blastocysts in vitro and pregnancy and implantation rates after ET.

Result(s): Fifty-one percent of all zygotes developed to blastocysts. Significant positive correlation between the number of blastocysts formed was observed with the number of oocytes, pronuclear zygotes, and eight-cell embryos formed. There was a negative correlation with male factor infertility. By day 5 or 6, 93% of the patients had at least one blastocysts, and the clinical pregnancy rate per transfer was 43% and the implantation per embryo transferred was 25%. No other clinical factor significantly affected the number of blastocysts formed, pregnancy rate, or implantation rate.

Conclusion(s): The numbers of oocytes, zygotes, and normally developing embryos in culture significantly affects the production of blastocysts in vitro. Male infertility significantly reduces blastocyst production. The number and the quality of the blastocysts transferred significantly influences clinical pregnancy rate.     

Impact of oxygen concentration on embryonic development of mouse zygotes

The aim of the present study was to examine the effect of culture under 5 and 20% oxygen on the development, differentiation and viability of zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development. First, zygotes collected in a common pool were cultured in 20% O2 for 0, 23, 46 and 95 h. Zygotes and in-vivo-produced embryos at the 2-cell and 8-cell stages of development were then cultured in 5 or 20% O2. The proportion of embryos reaching the compaction and blastocyst stages of development did not differ between groups regardless of the period of time embryos were cultured in 20% O2 or the stage at beginning of culture. Duration of culture under 20% O2 had a significant effect on total number of blastocyst cells. A stage-specific effect was observed on total and trophectoderm cell numbers in blastocysts resulting from the culture of zygotes and in-vivo-produced embryos under 20% O2. ICM and percent ICM development was significantly decreased by culture in 20% O2 at all stages examined. Oxygen concentration had no effect on implantation rate and fetal weights upon embryo transfer. However, transfer of zygotes grown to the blastocyst stage in 20% O2 resulted in a dramatic decrease in fetal development per blastocyst and fetal development per implantation. These results demonstrate that culture of F1 mouse zygotes in 20% O2 compromises the developmental potential of resultant blastocysts, which appear to be normal on morphological assessment.     

The development of fertilized human ova to the blastocyst stage in KSOM(AA) medium: is a two-step protocol necessary?

Current protocols for the culture of human zygotes to blastocysts use two-step sequential media systems. The efficacy of a one-step system involving potassium simplex optimized medium (KSOM(AA)) has been investigated. In study 1, development of zygotes from days 1 to 3 in KSOM(AA) was compared with that for medium P-1. In study 2, embryos were cultured from days 1 to 3 in P-1 followed by culture from days 3 to 5 either in KSOM(AA) or medium CCM. In study 3, the ability of KSOM(AA) to support development of embryos from days 1 to 5, without medium renewal, was compared with the sequential media system P-1-->CCM. The cell numbers and fragmentation scores of day 3 embryos were distributed similarly following culture in KSOM(AA) or P-1. Significantly more KSOM(AA) embryos exhibited cytoplasmic pitting. Blastocyst formation rates were not significantly different whether embryos were cultured in the P-1-->KSOM(AA) or the P-1-->CCM systems, or when cultured from days 1 to 5 in KSOM(AA) without medium renewal or in P-1-->CCM. Five babies have been born from nine blastocysts transferred after extended culture in KSOM(AA). A one-step protocol involving KSOM(AA) can be used successfully to culture human zygotes to the blastocyst stage.     

Cytogenetic analysis of human embryos and embryonic stem cells derived from monopronuclear zygotes

Purpose

By comparing the chromosomal constitution among the arrested cleavage-stage embryos, blastocysts and human embryonic stem cells (hESCs) which are all derived from monopronuclear (1PN) zygotes, it is aimed to determine whether chromosomally normal embryos can be reliably selected by blastocyst culture.

Methods

After 1PN zygotes are sequentially cultured for 5 days, the blastocysts and arrested cleavage-stage embryos were analyzed by fluorescence in situ hybridization (FISH) with probes for chromosomes 18, X and Y; G-banding analysis was adopted to analyze the karyotype of 1PN hESCs.

Results

The diploid rate of blastocysts was 74.6%, which was significantly (P?<?0.001) higher than that of arrested cleavage-stage embryos (31.6%), and the diploid rate of hESCs was 97.0%, which was significantly (P < 0.01) higher than that of blastocysts; the haploid embryos were excluded by blastocyst culture; nevertheless, there still existed such chromosomal abnormalities as mosaic and monosomic in blastocysts and trisomy in hESCs.

Conclusions

Blastocyst culture is an effective method to select against chromosomal abnormalities, especially the haploids in 1PN embryos; however, development to the blastocyst stage is not a reliable marker for mosaicism or aneuploidy.     

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.     

Effects of type and state of co-culture cells on in-vitro development of porcine oocytes matured and fertilized in vitro

Purpose: The present study was to investigate the impact of type and state of co-culture cells on developmental competence of porcine oocytes matured and fertilized in vitro.Methods: Porcine zygotes were co-cultured with granulosa cells (GCs) (Group 1) or porcine oviductal epithelial cells (pOECs) at follicular stage (Group 2), ovulation stage (Group 3) or corpus luteum (CL) stage (Group 4) or cultured in a medium without co-culture cells (control group).Results: The proportion of oocytes developed to 2-cell stage embryos in Group 2 was similar to that in control group, but significantly (p < 0.05) lower than that in Groups 1, 3 and 4. The proportions of oocytes developed to ≥4-cell stage embryos in Groups 3 and 4 were significantly (p < 0.05) higher than that in Groups 1 and 2. At 144 h after insemination, 12.0, 14.8 and 20.0% of oocytes developed to blastocysts in Groups 1, 3 and 4, respectively. However, no embryos in control group developed beyond 4-cell stage and no embryos in Group 2 developed to blastocyst stage.Conclusion: As compared with GCs and pOECs at follicular stage, the pOECs at ovulation and CL stages had a better competence to support porcine embryo development under in vitro conditions.