1 Department of Obstetrics and Gynecology, Nagoya University School of Medicine, 2 Maternity and Perinatal Care Center, Nagoya University Hospital and 3 Department of Anatomy, Nagoya University School of Medicine, Nagoya, Japan
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Abstract |
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Key words: co-culture/human blastocyst/in-vitro fertilization/leukaemia inhibitory factor/oviduct
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Introduction |
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Two different approaches have been used to improve the culture conditions for blastocyst stage transfer (Ménézo et al., 1998a). One is the co-culture of human embryos with monolayer human and animal cells; the other is the use of sequential culture media (combination of two different media using before/after genomic activation). A beneficial effect of human oviduct cell co-culture on blastocyst formation has been reported (Bongso et al., 1989b
). Similar results were also obtained from the co-culture study using bovine oviduct cells (Wiemer et al., 1993
). Positive effects on the development of human embryos using other commercially available cell lines, such as Vero cells (African green monkey kidney cells) and Buffalo rat liver (BRL) cells, have also been reported (Ménézo et al., 1990
; Olivennes et al., 1994
; Hu et al., 1997
). In contrast, it has been shown (Desai et al., 1997
) that there is an advantage in using a commercial globulin-enriched protein supplement (synthetic serum substitute, SSS), during extended culture of human embryos. Others (Gardner et al., 1998
) reported that their original sequential serum-free culture media can support >50% blastocyst development. Recently, clinical use of the serum-free blastocyst media has been widely accepted on a large scale (Ménézo et al., 1998b
). Apparently, the co-culture systems will be obsolete clinically early in the 21st century.
The potential role of the Fallopian tube in the early events of pregnancy is still being neglected somewhat. It is probable that the oviduct cells secrete beneficial substances for the development of preimplantation embryos, keeping them concentrated near the cell surface by cilia and microvilli. However, human oviductal cells are not constantly available, and the cultured cells are usually non-ciliated due to a failure of ciliated cells to adhere and survive, or due to processes of deciliation or dedifferentiation in vitro. It appears that there have been no stable human oviductal cell lines useful for the co-culture study of the embryos to the blastocyst stage.
The aim of the present study was to establish a ciliated cell line that could provide a valuable resource for the development of improved IVF culture media, rather than for the direct use in co-culture. Thus, human Fallopian tubal epithelial cells in primary culture were transfected with a plasmid vector carrying simian virus 40 (SV40) large T antigen. The origin-defective SV40T antigen plasmid has been used to immortalize various normal cells with fewer changes of differentiation characteristics by our group (Ishida et al., 1995) and by other investigators (Stoner et al., 1991
; Bodine et al., 1996
). Here, the establishment and characterization of a ciliated human oviductal cell line, named as NT/T-S, is presented. Data from our preliminary co-culture study have supported the beneficial effect of NT/T-S cells on the significant growth of the surplus embryos with fragments from our IVFembryo transfer programme up to the expanded blastocyst stage. Conditioned media of NT/T-S and some other reference cell cultures have been analysed to show that various cytokines/growth factors, including leukaemia inhibitory factor (LIF), were secreted from the NT/T-S cells and tubal epithelial cells in primary culture.
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Materials and methods |
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Human oviduct cell culture and transfection
Fresh Fallopian tubes were perfused with 0.5% collagenase (Wako, Osaka, Japan) in phosphate-buffered saline (PBS) for 10 min. The epithelium was then trimmed off with scissors and incubated (with shaking) in 0.25% trypsin (Life Technologies, Rockville, MD, USA) with 1 mg/ml EDTA (Life Technologies) in PBS at 37°C for 30 min. The mixture was then centrifuged at 50 g for 3 min, and the pelleted cells were suspended in Eagle's minimum essential medium (MEM; Sigma, St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Life Technologies) and plated onto type I collagen-coated plastic dishes.
On day 3 or 4 of the primary culture, cells were transfected by calcium phosphate precipitation with pMK16-SV40T(ori) as previously described (Ishida et al., 1995). Briefly, cells were incubated with the plasmid for 12 h at 37°C and then washed with MEM/10% FCS. On the next day, almost all the cells were dead. A few days later, expanding colonies were observed on the dishes. Each colony was picked up and subcloned to be cultured in an atmosphere of 5% CO2 in air at 37°C. The cell line, named as NT/T-S, was immortalized without crisis and the cells were cultured for 2 or more years. The medium was replaced every 2 or 3 days. Subculture was performed every 7 or 8 days by detaching the cells in 0.05% trypsinEDTA solution and by transferring to new culture flasks.
Cultured cells were observed with an IMT-2 phase-contrast microscope (Olympus, Tokyo, Japan).
Antibodies and double labelling
Anti-human cytokeratin-7 and anti-SV40 T antigen were purchased from Cymbus Bioscience (Southampton, Hampshire, UK) and Oncogene Science (Manhasset, NY, USA) respectively. For the double labelling experiment, NT/T-S cells were grown on the Chamber-TekTM chamber slide (Miles, Naperville, IL, USA). The cells were stained with the HistostainTM-double staining kit (Zymed Laboratories, South San Francisco, CA, USA) according to the manufacturer's instructions.
Transmission and scanning electron microscopy
For transmission electron microscopy (TEM), NT/T-S cells cultured on a plastic dish for 36 days or Fallopian tube tissues were pre-fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l phosphate buffer (pH 7.4) (also called Karnovsky's fixative) at 4°C for 2 days. They were post-fixed with 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections were electron-stained and observed with a Hitachi H-7100 electron microscope.
Some cells from the same passage were grown on a glass coverslip placed on a plastic dish for 36 days for scanning electron microscopy (SEM). Cells were fixed in Karnovsky's fixative at 4°C for 15 min. The coverslips were dehydrated through a series of graded alcohols and critical point-dried. The mounted coverslips were coated in a SEM E5000 coating unit (Osmium Plasma Coater 80N; Nippon Laser and Electronics Lab., Nagoya, Japan) and examined in a Hitachi S-800 scanning electron microscope.
Source of surplus embryos
A total of 133 surplus embryos from 52 patients admitted for IVF at Nagoya University Hospital was used in this study. Studies to use surplus embryos were previously approved by the Institutional Ethics Committee. The stimulation protocol was described elsewhere (Nakamura et al., 1992). In summary, the gonadotrophin-releasing hormone (GnRH) agonist, nafarelin acetate (NasanylTM, Yamanouchi Pharmaceutical, Tokyo, Japan) was used in combination with FSH (FertinormTM-P, Serono, Tokyo, Japan, or human menopausal gonadotrophin (HMG `Nikken'TM, Nikken Chemicals, Tokyo, Japan) to stimulate multiple follicular development. Follicular growth was monitored by transvaginal sonography, together with serial measurement of the serum oestradiol concentrations for the controlled stimulation. When the dominant follicle was judged to be mature (>18 mm diameter), 5000 IU of human chorionic gonadotrophin (HCG; GonatropinTM, Teikoku Hormone Mfg, Tokyo, Japan) was given to induce final oocyte maturation. Ultrasound-guided oocyte retrieval was performed 35 h after HCG administration. Insemination was performed ~4 h after oocyte retrieval. Oocytes and embryos were cultured in MEM supplemented with 10% synthetic serum substitute (SSS; Irvine Scientific, Santa Ana, CA, USA) in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 37°C. On day 2, up to three embryos were transferred, and the embryos at the 4- to 8-cell stage were cryopreserved if the fragments of the embryonic surface were <10%. Therefore, the surplus embryos at the 4- to 8-cell stage, in which fragments occupied
10% of the surface area, were used (with informed written consent) in this study.
Co-culture
The surplus embryos were (co-)cultured in 6-well plates (Falcon) for an additional 69 days in MEM supplemented with 10% FCS. The wells, in which NT/T-S cells were semi-confluent, were used for co-culture. The medium was replaced on the day before co-culture was started. Co-cultured embryos were moved to another well on every second day when the monolayer cells reached confluence. As a co-culture control, NFL/T cells were co-cultured with some embryos in the same manner as NT/T-S co-culture. As a cell-free control, some other embryos were cultured in the co-culture medium (MEM/10% FCS) alone. NFL/T cells are the human fetal liver epithelial immortalized cell line that was established after transfection with the SV40 T antigen plasmid (Ishida et al., 1995). Surplus embryos were randomly allocated to each group. The co-culture experiments were performed in an atmosphere of 5% CO2 in air at 37°C. The embryos were not transferred to the uterus after the experiments because the safety of the SV40 T antigen plasmid is unclear.
Assay for cytokines/growth factors in the conditioned media
Conditioned medium (0.125 ml/cm2 dish) for 48 h cell culture was assayed for various cytokines/growth factors. Cells were cultured in the same condition as that for the co-culture. The conditioned media of the following cells were tested: NT/T-S cells, NFL/T cells, human tubal epithelial cells in primary culture (TEC) and human tubal fibroblasts in tertiary culture (TSC). The conditioned media for TEC and TSC were collected from the independent cultures from different patients' tissues. The control medium (MEM/10% FCS) was also assayed. Each sample was assayed for interleukin (IL)-1ß, IL-6, LIF, transforming growth factor ß1 (TGF-ß1) and basic fibroblast growth factor (bFGF) with sandwich enzyme immunoassay (EIA; R&D Systems, Minneapolis, MN, USA), for insulin-like growth factor-I (IGF-I) using an immunoradiometric assay (IRMA; Bayer Medical, Tokyo, Japan), and for IL-4, IL-8 and hepatocyte growth factor (HGF) using an enzyme-linked immunosorbent assay (ELISA; BioSource International, Camarillo, CA, USA; TORAY, Tokyo, Japan, and Otsuka Pharmaceutical, Tokyo, Japan respectively). Sensitivity and intra- and inter-assay coefficients of variation for each assay are shown in Table II.
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Results |
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In order to confirm the physiological role of these factors on embryonic development in human Fallopian tubes, the conditioned medium of the primary culture of human tubal epithelial cells (TEC) and that of the tertiary culture of tubal fibroblasts (TSC) was also studied. IL-6, LIF, IL-8, TGF-ß1, bFGF and HGF were detected in at least one of the conditioned media (Table II). The higher concentrations of LIF and IL-8 in TEC culture were similar to those in NT/T-S culture.
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Discussion |
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NT/T-S cells retained morphological features associated with tubal epithelial cells in situ in that they showed CK7, microvilli and cilia. Cilia, although as short as microvilli, were clearly observed in sectional profiles using TEM. However, SEMwhich mainly detects surface morphologycould not distinguish cilia from microvilli. The reason why cilia were observed with TEM, but not with SEM, in the cells from the same passage is unknown. It is most likely that the culture on a glass coverslip for SEM is unsuitable for the production of cilia (Comer et al., 1998). Since the ciliation observed with TEM was detectable only at higher magnification (Figure 4
), ciliation may require a much longer time, in addition to oestrogen stimulation in vitro (Comer et al., 1998
). The cells of 300 PD were used in the current study, and therefore the cells from other passages were not analysed. Cilia could be constantly observed independently from the passages thus far. There was no evidence for the presence of secretory-type cells at present. It has been proposed (Comer et al., 1998
) that tubal ciliated cells are derived by differentiation from cells of a secretory-like phenotype. Thus, a secretory-like phenotype could be induced in NT/T-S cells by changing the culture conditions. Though this topic is very interesting, further investigations are desirable in which various immunocytochemical markers can be obtained.
Beneficial effects of embryo co-culture with various vertebrate cell monolayers have been reported in many studies (for a review, see Plachot, 1996). In particular, human embryo co-culture with Vero cells (African green monkey kidney cells) was used in IVF programmes and gave excellent pregnancy rates in some institutions (Olivennes et al., 1994; Schillaci et al., 1994
). Thus, cells from the human genital tract are not essential for human embryo co-culture. However, the final goal of the co-culture should not be the clinical use, but the elucidation of physiologically important substances for culture media from fertilization to blastocyst. Thus, a study using human oviductal cells may provide ideal progress in this respect.
The problems associated with the use of cells from human Fallopian tubes for co-culture are as follows: (i) human Fallopian tube tissues are not always available; (ii) isolation of the epithelial cells from the tubal tissues is technically difficult; (iii) the number of cells prepared for co-culture is variable at each opportunity; and (iv) tubal epithelial cells, grown in monolayer culture, lose morphological features associated with the epithelium in situ, such as cilia. The new cell line, NT/T-S, may overcome these problems. This is the first report on the establishment of a human oviductal immortalized cell line which has shown a positive effect on the development of human embryo by co-culture.
In the preliminary co-culture experiments using surplus embryos at the 4- to 8-cell stage, the positive effect of co-culture on blastocyst formation was significant only when the fragments of the embryo were <30% of the surface area. This result is comparable with our IVF data, in which the implantation rate was extremely low when only embryos with >30% fragmentation rate were transferred to the uterus.
The NT/T-S co-culture data also showed a positive effect on the formation of the expanded blastocysts, but did not show any significant effect on hatching rate. It has been reported (Bongso et al., 1994) that 63% and 40% of embryos expanded and hatched in the sequential oviductalendometrial co-culture system, compared with 41% and 9% in the oviductal system alone respectively.
The effect of co-culture between two cell lines immortalized by the same origin-defective SV40 T antigen plasmid was compared. One of the reasons for this experimental design was to eliminate the beneficial effect of co-culture itself, such as prevention of free radical production, detoxification of the culture medium and fall in oxygen tension at the embryo surface. Another reason was to omit the effect of the possible transformation of the feeder cells. Although the co-culture study was small scale, and thus preliminary, it may be suggested that cytokines/growth factors predominantly secreted by NT/T-S cells are important. It may be also suggested that the surface morphology of NT/T-S cells, such as cilia and microvilli, is related to a favourable result.
It was not possible to compare the NT/T-S co-culture with the sequential culture media due to the depletion of surplus embryos. In the current study, the medium for co-culture was MEM/10% FCS, which was used as the NT/T-S culture medium. Moreover, the cell-free control group was tested in an atmosphere of 5% CO2 in air based on a scientific comparison with the study group. Therefore, clinical interest may arise as to how beneficial the NT/T-S co-culture might be in comparison with commercial blastocyst media in an atmosphere of 5% CO2, 5% O2 and 90% N2. Provided that enough embryos of the same quality are obtained, the two groups should be directly comparable.
Based on the results of the present study, LIF appears to have a promising role in embryo development. It has been reported (Kauma and Matt, 1995) that co-culture cells that express LIF enhance mouse blastocyst development in vitro when compared to co-culture cells that do not express LIF. Others (Arici et al., 1997
) found LIF expression in human follicular fluid. Furthermore, mRNA for LIF receptor ß and gp130 have been demonstrated in human embryos (van Eijk et al., 1996
). Recently, a positive effect of recombinant LIF on preimplantation mouse blastocyst growth, differentiation and hatching was reported (Tsai et al., 1999
). It is likely that not only LIF, but also other cytokines/growth factors secreted by NT/T-S cells might support the development of the embryos, both directly and indirectly.
Ciliated epithelial cells are predominant in the Fallopian tubal epithelium in situ, the other type of epithelial cells being the secretory-type. It has been suggested (Comer et al., 1998) that tubal ciliated cells are derived by differentiation from cells of a secretory-like phenotype. It is known that in vitro tubal epithelial cells lose morphological features associated with the epithelium in situ, including cilia. It is unclear whether the absence of ciliated cells in primary cultures is due to a failure of such cells to adhere and survive, or due to processes of deciliation or dedifferentiation in vitro. The ciliated cells are thought to be involved in gamete and embryo transportation, and whether they have any direct effect on embryo development is unknown. However, it is probable that the secreted cytokines/growth factors are retained and concentrated near the apical cell surface due to the presence of microvilli and cilia. The implication of microvilli and cilia for embryo growth should be further investigated using NT/T-S cells.
Therefore, the newly established NT/T-S cells may be excellent tools for clarification of the physiological roles of human Fallopian tube on the development of preimplantation embryos. The surface morphology of the Fallopian tube, such as cilia and microvilli, together with the various substances secreted from the tubal cells, may play an important role in the early events of pregnancy.
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Acknowledgments |
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Notes |
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References |
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Bodine, P.V., Vernon, S.K. and Komm, B.S. (1996) Establishment and hormonal regulation of a conditionally transformed preosteocytic cell line from adult human bone. Endocrinology, 137, 45924604.[Abstract]
Bongso, A., Ng, S.C., Sathananthan, H. et al. (1989a) Establishment of human ampullary cell cultures. Hum. Reprod., 4, 486494.[Abstract]
Bongso, A., Soon-Chye, N., Sathananthan, H. et al. (1989b) Improved quality of human embryos when co-cultured with human ampullary cells. Hum. Reprod., 4, 706713.[Abstract]
Bongso, A., Fong, C.Y., Ng, S.C. et al. (1994) Human embryonic behavior in a sequential human oviduct-endometrial coculture system. Fertil. Steril., 61, 976978.[ISI][Medline]
Comer, M.T., Leese, H.J. and Southgate, J. (1998) Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium. Hum. Reprod., 13, 31143120.[Abstract]
Desai, N., Kinzer, D., Loeb, A. et al. (1997) Use of synthetic serum substitute and -minimum essential medium for the extended culture of human embryos to the blastocyst stage. Hum. Reprod., 12, 328335.[Abstract]
Gardner, D.K., Phil, D., Vella, P. et al. (1998) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril., 69, 8488.[ISI][Medline]
Hu, Y.X., Maxson, W.S., Eager, S. et al. (1997) Co-culture of human embryos using Buffalo rat liver (BRL) cells for women with decreased prognosis in IVF. Am. J. Obstet. Gynecol., 157, 358363.
Ishida, T., Ando, H., Nomura, S. et al. (1995) Establishment and characterization of human fetal liver epithelial cell line transfected with SV40 T antigen. Proc. Soc. Exp. Biol. Med., 209, 251256.[Abstract]
Kauma, S.W. and Matt, D.W. (1995) Coculture cells that express leukemia inhibitory factor (LIF) enhance mouse blastocyst development in vitro. J. Assist. Reprod. Genet., 12, 153156.[ISI][Medline]
Kondo, I., Suganuma, N., Ando, T. et al. (1996) Clinical factors for successful cryopreserved-thawed embryo transfer. J. Assist. Reprod. Genet., 13, 201206.[ISI][Medline]
Menezo, Y., Veiga, A. and Benkhalifa, M. (1998a) Improved methods for blastocyst formation and culture. Hum. Reprod., 13 (Suppl. 4), 256265.[Medline]
Menezo, Y.J., Hamamah, S., Hazout, A. et al. (1998b) Time to switch from co-culture to sequential defined media for transfer at the blastocyst stage. Hum. Reprod., 13, 20432044.
Menezo, Y.J., Guerin, J.F. and Czyba, J.C. (1990) Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells. Biol. Reprod., 42, 301306.[Abstract]
Nakamura, K., Oosawa, M., Kondou, I. et al. (1992) Menotropin stimulation after prolonged gonadotropin releasing hormone agonist pretreatment for in vitro fertilization in patients with endometriosis. J. Assist. Reprod. Genet., 9, 113117.[ISI][Medline]
Olivennes, F., Hazout, A., Lelaidier, C. et al. (1994) Four indications for embryo transfer at the blastocyst stage. Hum. Reprod., 9, 23672373.[Abstract]
Plachot, M. (1996) Co-culture of embryos and feeder cells. Hum. Reprod., 11 (Suppl. 1), 3542; discussion 5135.
Schillaci, R., Ciriminna, R. and Cefalu, E. (1994) Vero cell effect on in-vitro human blastocyst development: preliminary results. Hum. Reprod., 9, 11311135.[Abstract]
Stoner, G.D., Kaighn, M.E., Reddel, R.R. et al. (1991) Establishment and characterization of SV40 T-antigen immortalized human esophageal epithelial cells. Cancer Res., 51, 365371.[Abstract]
Tsai, H., Chang, C., Hsieh, Y. et al. (1999) Recombinant human leukemia inhibitory factor enhances the development of preimplantation mouse embryo in vitro. Fertil. Steril., 71, 722725.[ISI][Medline]
van Eijk, M.J., Mandelbaum, J., Salat-Baroux, J. et al. (1996) Expression of leukemia inhibitory factor receptor subunits LIFR beta and gp130 in human oocytes and preimplantation embryos. Mol. Hum. Reprod., 2, 335360.[Abstract]
Wiemer, K.E., Hoffman, D.I., Maxson, W.S. et al. (1993) Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells. Hum. Reprod., 8, 97101.[Abstract]
Submitted on January 10, 2000; accepted on April 7, 2000.