Coculture with homologous oviductal cells improved the implantation of human embryos—A prospective randomized control trial

Purpose: The efficacy of homologous oviductal cell coculture on the success of a humanin vitro fertilization program was investigated in a prospective randomized control clinical trial. Methods and Results: One hundred eighty-one couples were randomized into the control and the coculture groups. Pronu-clear-stage zygotes were either cultured in Earles' balanced salt solution supplemented with 15% preovulatory serum (control) or cultured with human oviductal cells (coculture) for 24 hr before embryo transfer. There was no difference in the age of the patients, indication for treatment, number of oocyte retrieved or fertilized, or number of embryo replaced between the two groups. The pregnancy rates per transfer for the control and the coculture group were 12.8 and 19.3%, respectively. The number of viable fetus was significantly higher (P<0.01, chi-square test) in the coculture group (25/264) than in the control group (8/262). The coculture group also showed a higher multiple pregnancy rate, lower abortion rate, and more spare embryos suitable for cryopreservation.     

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.     

Human oviductal cells reduce the incidence of apoptosis in cocultured mouse embryos

Objective: To investigate the effect of human oviductal cell coculture on the incidence of apoptosis in mouse embryos.

Design: Experimental laboratory study.

Setting: University gynecology unit.

Patient(s): Fallopian tubes were obtained from patients undergoing hysterectomy.

Intervention(s): Mouse embryos were cocultured with human oviductal cells.

Main Outcome Measure(s): Blastocyst development, allocation of inner cell mass (ICM) and trophectoderm (TE) in blastocyst, and apoptosis in embryos.

Results: Oviductal cells significantly enhanced the blastulation (38%) and hatching rate (22%) of the cocultured zygotes. The corresponding values in medium alone culture were 21% and 9%, respectively. The cocultured embryos also had higher blastomere count at blastocyst stage (P<0.005). This was due to increase in both the cell count of ICM (P<0.05) and TE (P<0.001). Coculture reduced the incidence of apoptosis in the cultured morula and blastocyst from 38% and 48% to 16% (P<0.001) and 27% (P<0.05), respectively. The number of apoptotic blastomeres per morula (1.5 ± 0.6; P<0.005) and blastocyst (2.3 ± 0.7; P<0.005) after coculture was also significantly lower than that of the corresponding control (morula, 2.1 ± 0.8; blastocyst, 3.5 ± 1.1).

Conclusion(s): Human oviductal cells improved mouse embryo development partly by decreasing the incidence of apoptosis.     

Successful freezing of unfertilized mouse oocytes and effect of cocultures in oviducts on development of in vitro fertilized embryos after thawing

Purpose To establish a freeze-thawing method for unfertilized oocytes with a high success rate, we examined several conditions for freeze-thawing. The effects of EDTA and cocultures in oviducts on the development of embryos fertilized in vitro after thawing were also studied. Results In the first experiment, unfertilized oocytes that were frozen in 1.5 Mdimethylsulfoxide (DMSO) supplemented with 0.2 Msucrose by a slow freeze-thawing method showed the best results (fertilization rate, 71.9%; blastocyst rate per frozen oocyte, 18.8%). The proportion of embryos that developed to blastocysts was significantly higher when DMSO was added at 4‡C than at room temperature (39.4 vs 19.4%; P<0.01). The addition of EDTA (10 ΜM)to the culture medium did not promote embryo development after fertilization in vitro. However, the rate of development of in vitro fertilized embryos to blastocysts after thawing was significantly higher when the embryos were cultured in oviducts in vitro than the rates in control cultures and those cultured with EDTA (blastocyst rate from fertilized oocytes, 71.4 vs 51.0 and 52.8%, respectively; P<0.01). Conclusion Unfertilized mouse oocytes can be cryopreserved successfully by a slow freeze-thawing method with the addition of 1.5 MDMSO and 0.2 Msucrose at low temperatures, and coculture with oviducts enhances the development of embryos that are fertilized in vitro after thawing.     

Coculture of human spermatozoa with reproductive tract cell monolayers can enhance sperm functions better than coculture with vero cell monolayers

Purpose: In order to develop a better system for support of human sperm function in vitro, we conducted studies to evaluate whether reproductive tract cells are better than non-reproductive tract cells as an adjunt in that regard. Methods: Human spermatozoa were cocultured with Vero cells, with human oviduct cells and endometrial cells, and without cells (control) for either 1, 4, or 24 hr. Sperm motility was then analyzed with a computer-aided sperm analyzer (CASA-Hamiliton Thron, HTM IVOS Motility Analyzer). Aliquots of spermatozoa incubated for 24 hr were also stained with Hoechst 33258 and FITC-PNA to evaluate the status of acrosome in live cells. Results: Significant differences (P<0.05) between the oviduct cell and the control groups after 24 hr were evident in the curvilinear velocity (VCL) (81.4±13.4 vs 60.0±14.1 µm/sec) and amplitude of lateral head displacement (ALH) (5.2±0.6 vs 4.1±0.5 µm). The incidence of acrosome reaction of live sperm was significantly higher in the endometrial cell group than in the controls (25.4±9.9 vs 6.6±2.4%;P<0.001). Conclusions: Coculture with human reproductive tract cells seems to improve some functional parameters of human spermatozoa. Coincubation with such cell lines, especially oviduct cells, might be a feasible approach to optimization of human spermatozoa for assisted fertilization using subfertile or frozen-thawed samples. We think coincubating human spermatozoa with a human reproductive tract cell line, especially oviduct cells, might be a feasible approach in preparing human spermatozoa for assisted fertilization in subfertile and frozen-thawed semen samples.     

人分泌早期子宫内膜共培养体系对早期鼠胚凋亡的影响

目的研究人分泌早期子宫内膜共培养体系对早期鼠胚凋亡的影响。方法将2一细胞期鼠胚与子宫内膜(A冻融、B传代)共培养以及单一培养液培养(C组)。72h后对桑椹期鼠胚用TUNNEL法进行凋亡检测。结果共培养组鼠胚的凋亡率明显低于单一培养组，且不受内膜细胞传代及冻融的影响；共培养组优质胚胎数均高于对照组，而共培养组之间无明显差异。结论共培养人分泌早期子宫内膜能减少早期鼠胚的凋亡，提高胚胎体外发育的质量，延长体外培养时间。     

Improvement in blastocyst hatching of mouse embryos cocultured with human placental cells

Purpose Although the hatching of embryos is an important phenomenon, the mechanism of hatching remains controversial. Therefore, we attempted to develop a new coculture system with human placental cells to investigate the hatching of mouse embryos.Results In our new system there was no difference in development from the two-cell stage to blastocysts between embryos cultured with a T6 medium and embryos cocultured with human placental cells at 1 × 10⁵, 5 × 10⁵, and 1 × 10⁶ cells/ml. However, the hatching-rate cell number increased significantly in embryos cocultured with placental cells compared to embryos cultured without placental cells. [³H]Thymidine uptake did not show any significant difference from the beginning of in vitro culture to the hatching stage between the coculture group and the control group. Nevertheless, the [³H]uridine uptake was significantly different in the two groups, measuring 2167 ± 532 cpm/10 embryos in the coculture group and 804 ± 86 cpm/10 embryos in the control group at 114 hr after human chorionic gonadotropin injection (P < 0.01).Conclusion These results therefore seem to indicate that the hatching of blastocysts depends on the protein synthesis of the embryos and not on DNA duplications.     

Effects of human follicular fluid on spermatozoa that have been cocultured with human oviductal cells

Objective: To investigate the sequential effects of human oviductal cells and human follicular fluid (hFF) on various sperm functions.Design: Laboratory experimental study.Setting: University gynecology unit.Patient(s): Fallopian tubes were from patients undergoing tubal ligation or hysterectomy. Semen was from men attending the subfertility clinics.Intervention(s): Spermatozoa were treated with [1] 6 hours in Earle’s balanced salt solution (EBSS-BSA; control); [2] 5 hours in EBSS-BSA and 1 hour with hFF (hFF); [3] 5 hours with oviductal cells and 1 hour in EBSS-BSA (coculture); and [4] 5 hours with oviductal cells and 1 hour with hFF (sequential).Main Outcome Measure(s): Motility, acrosome reaction, zona binding, and oocyte fusion.Result(s): Groups II and III spermatozoa had similar motility and were better than that of group I. Group IV displayed higher motility parameters than the other groups. Human follicular fluid induced acrosome reaction. The incidence of acrosome reaction in group IV was significantly lower than that in group II. Group III did not affect the acrosome reaction. Spermatozoa in groups II–IV had lower zona binding capacity than those in group I. Human follicular fluid stimulated oocyte penetration, whereas oviductal cells suppressed this effect of hFF.Conclusion(s): Oviductal cells maintained the fertilizing capacity of spermatozoa, whereas hFF facilitated the fertilization process of oviductal spermatozoa.     

Sequential observation of mitochondrial distribution in mouse oocytes and embryos

Objective The purpose of this study was to elucidate changes in the distribution of mitochondria through the cell cycle.Materials and Methods Mouse oocytes and embryos were recovered sequentially from mice and stained with the vital fluorescent mitochondrial stain rhodamine 123. Mitochondrial staining pattern were classified into three types: aggregation (Ag), homogeneous (H), and perinuclear accumulation (PA).Results Sequential observations revealed that mitochondria of oocytes and embryos grown in vivo translocated in the cytoplasm during the cell cycle, showing the H pattern be fore human chorionic gonadotropin (hCG) administration, the PA pattern 8–9 hr post-hCG, the H pattern again 10–14 hr post-hCG, and the PA pattern again 24 and 31–32 hr post-hCG following fertilization. In the twocell stage, the Ag pattern was shown 35 hr post-hCG, the H pattern was observed 40 hr post-hCG, and the PA pattern was found 48 hr post-hCG. In the embryos cultured in vitro and showing developmental block, mitochondrial translocation was shown to be inhibited after they aggregated in the early two-cell stage (35 hr post-hCG). Moreover, the translocation of mitochondria was restored by the addition of superoxide dismutase or thioredoxin to the culture medium. Both of these enzymes have already been shown to have the ability to overcome developmental block.Conclusion The present study revealed that mitochondria translocated in the cell cycle and suggested that there is a close relationship between mitochondrial translocation and developmental arrest.     

Human follicular fluid and mouse cumulus cells act synergistically to enhance preimplantation mouse Balb/cJ embryo development

Problem The development of preimplantation mammalian embryos in vitro is less than optimal. Follicular fluid and cumulus cells have both been used, independently, to improve preimplantation embryo quality in culture.Method To determine the ability of mouse cumulus cell coculture in the presence of human follicular fluid to support preimplantation mouse Balb/cJ embryo development in vitro.Results Culture of preimplantation mouse Balb/cJ embryos independently in human follicular fluid or on mouse cumulus cells had no significant affect on blastocyst development or total cell number per blastocyst. The coculture of mouse Balb/cJ preimplantation-stage embryos on mouse cumulus cells in the presence of human follicular fluid significantly (P<0.07) improved blastocyst development and the total number of cells per blastocyst.Conclusions Cumulus cells and follicular fluid have a positive synergistic affect on preimplantation mouse Balb/cJ embryo development and formation in vitro.     

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.     

人输卵管胚胎营养因子对着床前鼠胚胎基因表达的影响

目的探讨人输卵管上皮细胞分泌的胚胎营养因子（ETF）对着床前鼠胚胎基因表达的影响。方法采用液相色谱技术从体外培养的人输卵管上皮细胞系OE-E6／E7的培养液中分离出ETF。加入培养液后培养鼠胚胎。采用差异显示RT．PCR技术比较ETF培养的鼠胚胎（培养组）与普通培养液（未加入ETF）培养的鼠胚胎（对照组）问的基因表达差异。将差异表达片段分离后,进行扩增、克隆、测序和分析。结果培养组胚胎与对照组胚胎共分离克隆了8个差异表达基因,即：RABgeranylgeranyl转移酶、DEAD-box蛋白50、RNA结合结构域蛋白27、二磷酸甲羟戊酸盐脱羧酶、丝氨酸／精氨酸重复基质1、肿瘤差异表达基因2（TDE2）、T细胞受体α／8、胚胎发育因子1。结论人输卵管上皮细胞分泌的ETF对着床前鼠胚胎的基因表达有影响。     

The effects of coculture with autologous cryopreserved endometrial cells on human in vitro fertilization and early embryo morphology: A randomized study

Purpose: The aim of this study was to examine the influence of endometrial cells on the fertilization rate and early embryonic morphology following routine in vitro fertilization (IVF). Cryopreservation with subsequent thawing allowed the use of autologous somatic cells, thus minimizing the risk of transmission of infective agents. Interpatient variability was eliminated by randomizing oocytes from each cycle into the control or coculture group. Results: Two hundred ninety-four oocytes from 24 IVF cycles (21 patients) were included in the study (145 coculture and 149 control). The normal fertilization rate of control oocytes (56.4%) was not significantly different from that of oocytes cocultured with endometrial cells (61.4%). The mean number of blastomeres in cocultured embryos (3.65) was not significantly different from the number in control embryos (3.46) 2 days after insemination, but the proportion of embryos with minimal or no fragmentation was significantly higher in the coculture group [34/84 (40.5%) vs.17/80 (21.3%);P<0.01]. Conclusions: The inclusion of cryopreserved autologous endometrial cells in routine clinical IVF procedures does not influence fertilization or the early cleavage rate but may reduce the extent of embryo fragmentation during the early cleavage divisions.     

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.     

Effect of coculture on subsequent survival and implantation of cryopreserved human embryos

Purpose This retrospective analysis was designed to assess the performance of human embryos following cryopreservation based on whether they were originally developed in standard culture medium (65 cycles, 223 embryos) or cocultured on partial monolayers of bovine oviductal epithelial cells (63 cycles, 198 embryos). Embryo cryosurvival and implantation were compared between the study group and the contemporaneously matched controls.Results During a 2-year period when no factors of the cryopreservation program were altered, 63 transfers of 159 surviving thawed control cleavage-stage embryos (71.3% survival) that were 54% intact gave rise to 11 viable pregnancies (17.5%/ET), to yield an implantation rate of 6.9% per embryo. Sixty-three transfers of 147 thawed cocultured embryos (74.2% survival) that were 61% intact gave rise to 17 viable pregnancies (27%/ET), which gave an implantation rate of 13.6% per embryo that was significantly higher than the control group (P< 0.05).Conclusion Coculture of embryos prior to cryopreservation does not appear to improve cryosurvival; however, it does improve implantation postthaw compared with embryos following standard culture prior to cryopreservation.Presented at the IXth World Congress on In Vitro Fertilization and Alternate Assisted Reproduction, Vienna, Austria, April 3–7, 1995.     

Cytogenetic and cryobiology of human cocultured embryos: A 3-year experience

Purpose Coculture, which allows good-quality human blastocysts with good yields to be obtained, has been designed mainly to select the best embryos for transfers.Methods In a first attempt during coculture, we have studied by fluorescent in situhybridization the chromosomic content of the in vitroblocked embryos, using centromeric probes for chromosomes 1, 12, and 18. Close to 37% of the arrested embryos show aneuploidymosaicism.Results Freezing cocultured blastocysts gives good recovery rates after transfer (83%). The ongoing pregnancy rates per transfer (19%) are high, and the implantation rate per embryo is 13%. This compares favorably with freezing at an early stage.Conclusions We observed that the quality of the endometrium is always the limiting step, as first of all we observed wide variations according to the hormonal preparation of the patients. Moreover the implantation per embryo in the pregnant patients is very high (57%), indicating that most of the losses are directly related to the receptivity of the endometrium.Presented at the 50th Annual Meeting of the American Fertility Society, San Antonio, Texas, November 5–10, 1994.     

Effect of removal of cumulus cells from one-cell mouse embryos on in vitro development

The effects of the removal of cumulus cells from fertilized mouse oocytes (one-cell embryos) and the presence of streptomycin in culture medium on in vitro development were studied. Ham's F-10 medium with (0.075 g/liter) or without streptomycin was supplemented with human serum (15%). Cumulus-intact embryos were harvested from oviducts after mice were superovulated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Hyaluronidase (300 IU/ml) was used to remove the cumuli. Embryos were cultured (i) with cumulus/without streptomycin (n=238), (ii) with cumulus/with streptomycin (n=185), (iii) without cumulus/with streptomycin (n=210), and (iv) without cumulus/without streptomycin (n=218). Embryonic development was assessed 24, 96, and 120 hr after initiation of culture. Percentage two cells and percentage small or expanded blastocysts were not different (P>0.05) among experimental groups. Percentages hatched blastocysts for the four groups were (i) 36±8 and 54±7, (ii) 35±8 and 55±6, (iii) 19±5 and 42±6, (iv) 23±5 and 47±5 at 96 and 120 hr, respectively. Percentages all (small, expanded, and hatched combined) blastocysts were (i) 74±5 and 74±5, (ii) 74±9 and 72±5, (iii) 56±6 and 63±5, and (iv) 61±5 and 63±5 at 96 and 120 hr, respectively. A greater (P<0.05) percentage of embryos developed to blastocysts and hatched by 96 and 120 hr, when they were cultured with the cumulus intact. There was no effect of streptomycin on embryonic development. Cumulus cells may act as nurse cells during a critical stage of the development of the embryo.     

Development of two-cell mouse embryos in protein-free and protein-supplemented media

Two-cell mouse embryos from CFW (Swiss-Webster) mice were cultured to the blastocyst stage in Ham's F10, Whittingham's T6, or human tubal fluid medium. Media were used without any protein supplements or were supplemented with human maternal serum, human fetal cord serum, or human serum albumin. Blastocysts were transferred to modified Eagle's basal medium supplemented with 10% fetal bovine serum for postblastocyst development. Blastocyst and postblastocyst development was depressed among embryos cultured during the preimplantation stage in protein-free Whittingham's T6 and human tubal fluid media compared with embryos cultured in protein-free Ham's F10 medium. This advantage of Ham's F10 disappeared when amino acids and vitamins were added to the other two media. Whittingham's T6 and human tubal fluid supplemented with human serum albumin, human maternal serum, or human fetal cord serum also supported excellent embryo development. When supplermented with protein, Ham's F10 was the poorest of the media in supporting embryo development. Although these results suggest that Ham's F10 is not the best medium for culture of mouse embryos, there is need for caution in extrapolating results from the mouse to the human